Marc Coosemans

Trypanosoma brucei spp. development in the tsetse fly: characterization of the post-mesocyclic stages in the foregut and proboscis

Post-mesocyclic development of Trypanosoma brucei in the tsetse fly in its migration from midgut to salivary glands, was revisited by sequential microdissection, morphometry and DNA-cytofluorometry. This development started by day 6 after the infective feed, with passage of mesocyclic midgut trypomastigotes through proventriculus and upward migration along foregut and proboscis to the salivary gland ducts. Kinetics of salivary gland infection showed that colonization of the salivary glands by epimastigotes occurred only during the time-limited presence of this developmental phase in the foregut and proboscis. Post-mesocyclic trypanosomes in the foregut and proboscis were pleomorphic, with 4 morphological stages in various constant proportions and present all through from proventriculus up to the salivary gland ducts: 67% long trypomastigotes, 27% long epimastigotes, 4% long epimastigotes undergoing asymmetric cell division and 2% short epimastigotes. Measurements of DNA content demonstrated a predominant tetraploidy for 67% of these trypanosomes, the remainder consisting of the homogeneous diploid short epimastigotes and some long epimastigotes. According to the experimental data, the following sequence of trypanosome differentiation in the foregut and proboscis is proposed as the most obvious hypothesis. Incoming mesocyclic trypomastigotes (2N) from the ectoperitrophic anterior midgut start to replicate DNA to a 4N level, are arrested at this point, and differentiate into the long epimastigote (4N) which give rise, by an asymmetric cell division, to 2 unequal, diploid daughter cells: a long, probably dead-end long epimastigote and a short epimastigote. The latter is responsible for the epimastigote colonization of the salivary glands if launched at the vicinity of the gland epithelium by the asymmetric dividing epimastigote.

Behavioural heterogeneity of Anopheles species in ecologically different localities in Southeast Asia: a challenge for vector control.

In Southeast Asia the biodiversity of Anopheles species in the domestic environment is very high. Only few species are considered major vectors throughout the region, whereas the vector status of other species varies from area to area. Often it is difficult to identify an Anopheles species as a malaria vector in areas with low malaria incidence. The behaviour of Anopheles species largely determines their vector status, and insights into their behaviour are essential to evaluate the appropriateness of vector control measures. This study was conducted in six ecologically different localities in Southeast Asia to rank the different Anopheles species in terms of anthropophily and endophagy in order to estimate their current epidemiological importance. Concurrently, the biting and resting behaviour of the vectors was analysed to evaluate the appropriateness of insecticide‐impregnated bed nets and residual house spraying in vector control. Anopheles dirus A was highly anthropophilic at all sites where it occurred. By contrast, the degree of anthropophily exhibited by An. minimus A depended on availability of cattle. Anopheles campestris, An. nimpe, An. sinensis, An. maculatus, An. aconitus showed a high degree of anthropophily in certain villages, indicating their potential of participating in malaria transmission, although the actual incidence of malaria in the study villages can be fully explained by transmission of the major vectors (An. dirus A, An. minimus A and An. sundaicus). Late biting of An. minimus A and biting activity throughout the night of An. sundaicus favour bed nets as a control method for these species, whilst exophilic and outdoor biting in combination with early feeding behaviour of An. dirus A will make both insecticide‐impregnated bed nets and indoor residual spraying less suitable for controlling this species. Spatial variation in biting and resting behaviour was observed within almost all Anopheles species. These heterogeneities may result in the differences in epidemiological importance and in response to vector control of Anopheles species in different areas. Moreover, environmental changes and changes in human practice are expected to influence the behaviour, hence the role of the different species in malaria transmission. The effect of environmental changes on vector behaviour should be followed up carefully.

The Anopheles dirus complex: spatial distribution and environmental drivers

BackgroundThe Anopheles dirus complex includes efficient malaria vectors of the Asian forested zone. Studies suggest ecological and biological differences between the species of the complex but variations within species suggest possible environmental influences. Behavioural variation might determine vector capacity and adaptation to changing environment. It is thus necessary to clarify the species distributions and the influences of environment on behavioural heterogeneity.MethodsA literature review highlights variation between species, influences of environmental drivers, and consequences on vector status and control. The localisation of collection sites from the literature and from a recent project (MALVECASIA) produces detailed species distributions maps. These facilitate species identification and analysis of environmental influences.ResultsThe maps give a good overview of species distributions. If species status partly explains behavioural heterogeneity, occurrence and vectorial status, some environmental drivers have at least the same importance. Those include rainfall, temperature, humidity, shade, soil type, water chemistry and moon phase. Most factors are probably constantly favourable in forest. Biological specificities, behaviour and high human-vector contact in the forest can explain the association of this complex with high malaria prevalence, multi-drug resistant Plasmodium falciparum and partial control failure of forest malaria in Southeast Asia.ConclusionEnvironmental and human factors seem better than species specificities at explaining behavioural heterogeneity. Although forest seems essential for mosquito survival, adaptations to orchards and wells have been recorded. Understanding the relationship between landscape components and mosquito population is a priority in foreseeing the influence of land-cover changes on malaria occurrence and in shaping control strategies for the future.

Detection of the East and West African kdr mutation in Anopheles gambiae and Anopheles arabiensis from Uganda using a new assay based on FRET/Melt Curve analysis

BackgroundAppropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide use in malaria control programmes. The malaria vectors Anopheles gambiae s.s. and Anopheles arabiensis have developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, better known as knockdown resistance (kdr). In An. gambiae s.s. and An. arabiensis, two different substitutions in the para-type sodium channel, a L1014F substitution common in West Africa and a L1014S replacement found in Kenya, are linked with kdr. Two different allele-specific polymerase chain reactions (AS-PCR) are needed to detect these known kdr mutations. However, these AS-PCR assays rely on a single nucleotide polymorphism mismatch, which can result in unreliable results.MethodsHere, a new assay for the detection of knockdown resistance in An. gambiae s.s. and An. arabiensis based on Fluorescence Resonance Energy Transfer/Melt Curve analysis (FRET/MCA) is presented and compared with the existing assays.ResultsThe new FRET/MCA method has the important advantage of detecting both kdr alleles in one assay. Moreover, results show that the FRET/MCA is more reliable and more sensitive than the existing AS-PCR assays and is able to detect new genotypes. By using this technique, the presence of the East African kdr mutation (L1014S) is shown for the first time in An. arabiensis specimens from Uganda. In addition, a new kdr genotype is reported in An. gambiae s.s. from Uganda, where four An. gambiaes.s. mosquitoes possess both, the West (L1014F) and East (L1014S) African kdr allele, simultaneously.ConclusionThe presence of both kdr mutations in the same geographical region shows the necessity of a reliable assay that enables to detect both mutations in one single assay. Hence, this new assay based on FRET/MCA will improve the screening of the kdr frequencies in An. gambiae s.s. and An. arabiensis.

Bionomics, taxonomy, and distribution of the major malaria vector taxa of {Anopheles} subgenus Cellia in Southeast Asia: an updated review

There is high diversity of Anopheles mosquitoes in Southeast Asia and the main vectors of malaria belong to complexes or groups of species that are difficult or impossible to distinguish due to overlapping morphological characteristics. Recent advances in molecular systematics have provided simple and reliable methods for unambiguous species identification. This review summarizes the latest information on the seven taxonomic groups that include principal malaria vectors in Southeast Asia, i.e. the Minimus, Fluviatilis, Culicifacies, Dirus, Leucosphyrus, and Sundaicus Complexes, and the Maculatus Group. Main issues still to be resolved are highlighted. The growing knowledge on malaria vectors in Southeast Asia has implications for vector control programs, the success of which is highly dependant on precise information about the biology and behavior of the vector species. Acquisition of this information, and consequently the application of appropriate, sustainable control measures, depends on our ability to accurately identify the specific vectors.

Residual Transmission of Malaria: An Old Issue for New Approaches

Malaria is one of the most serious vector-borne diseases, affecting millions of people mainly in the tropics. Recently, a substantial decline in malaria incidence has been observed all over the world. Vector control is one of the key elements in achieving this world-wide malaria decline, with scaling up of Insecticide Treated Nets (ITNs) and the expansion of Indoor Residual Spraying (IRS) programmes contributing significantly. Besides the personal protec‐ tion, ITNs confer a community protection when wide coverage is assured, meaning that unprotected persons benefit from the large scale intervention [1]. IRS is only meaningful when applied at a large coverage. In the 2011 World Malaria Report [2], the percentage of households owning at least one ITN in sub-Saharan Africa is estimated to have risen from 3% in 2000 to 50% in 2011 while the percentage protected by indoor residual spraying (IRS) rose from less than 5% in 2005 to 11% in 2010. Household surveys indicate that 96% of persons with access to an ITN within the household actually use it [2]. Although these numbers might overestimate the real ITN use, they show that in recent years, several vector control measures were scaled up substantially. Despite these large increases in coverage, a widely held view is that with the currently available tools, namely vector control tools, intermittent preventive treatment, and early diagnosis and treatment, much greater gains could be achieved, including elimination from a number of countries and regions [3].

Multiple Insecticide Resistance: An Impediment to Insecticide-Based Malaria Vector Control Program

Background Indoor Residual Spraying (IRS), insecticide-treated nets (ITNs) and long-lasting insecticidal nets (LLINs) are key components in malaria prevention and control strategy. However, the development of resistance by mosquitoes to insecticides recommended for IRS and/or ITNs/LLINs would affect insecticide-based malaria vector control. We assessed the susceptibility levels of Anopheles arabiensis to insecticides used in malaria control, characterized basic mechanisms underlying resistance, and evaluated the role of public health use of insecticides in resistance selection. Methodology/Principal findings Susceptibility status of An. arabiensis was assessed using WHO bioassay tests to DDT, permethrin, deltamethrin, malathion and propoxur in Ethiopia from August to September 2009. Mosquito specimens were screened for knockdown resistance (kdr) and insensitive acetylcholinesterase (ace-1R) mutations using AS-PCR and PCR-RFLP, respectively. DDT residues level in soil from human dwellings and the surrounding environment were determined by Gas Chromatography with Electron Capture Detector. An. arabiensis was resistant to DDT, permethrin, deltamethrin and malathion, but susceptible to propoxur. The West African kdr allele was found in 280 specimens out of 284 with a frequency ranged from 95% to 100%. Ace-1R mutation was not detected in all specimens scored for the allele. Moreover, DDT residues were found in soil samples from human dwellings but not in the surrounding environment. Conclusion The observed multiple-resistance coupled with the occurrence of high kdr frequency in populations of An. arabiensis could profoundly affect the malaria vector control programme in Ethiopia. This needs an urgent call for implementing rational resistance management strategies and integrated vector control intervention.

Reductions in malaria and anaemia case and death burden at hospitals following scale-up of malaria control in Zanzibar, 1999-2008

BackgroundIn Zanzibar, the Ministry of Health and partners accelerated malaria control from September 2003 onwards. The impact of the scale-up of insecticide-treated nets (ITN), indoor-residual spraying (IRS) and artemisinin-combination therapy (ACT) combined on malaria burden was assessed at six out of seven in-patient health facilities.MethodsNumbers of outpatient and inpatient cases and deaths were compared between 2008 and the pre-intervention period 1999-2003. Reductions were estimated by segmented log-linear regression, adjusting the effect size for time trends during the pre-intervention period.ResultsIn 2008, for all age groups combined, malaria deaths had fallen by an estimated 90% (95% confidence interval 55-98%)(p < 0.025), malaria in-patient cases by 78% (48-90%), and parasitologically-confirmed malaria out-patient cases by 99.5% (92-99.9%). Anaemia in-patient cases decreased by 87% (57-96%); anaemia deaths and out-patient cases declined without reaching statistical significance due to small numbers. Reductions were similar for children under-five and older ages. Among under-fives, the proportion of all-cause deaths due to malaria fell from 46% in 1999-2003 to 12% in 2008 (p < 0.01) and that for anaemia from 26% to 4% (p < 0.01). Cases and deaths due to other causes fluctuated or increased over 1999-2008, without consistent difference in the trend before and after 2003.ConclusionsScaling-up effective malaria interventions reduced malaria-related burden at health facilities by over 75% within 5 years. In high-malaria settings, intensified malaria control can substantially contribute to reaching the Millennium Development Goal 4 target of reducing under-five mortality by two-thirds between 1990 and 2015.

False positive circumsporozoite protein ELISA: a challenge for the estimation of the entomological inoculation rate of malaria and for vector incrimination

BackgroundThe entomological inoculation rate (EIR) is an important indicator in estimating malaria transmission and the impact of vector control. To assess the EIR, the enzyme-linked immunosorbent assay (ELISA) to detect the circumsporozoite protein (CSP) is increasingly used. However, several studies have reported false positive results in this ELISA. The false positive results could lead to an overestimation of the EIR. The aim of present study was to estimate the level of false positivity among different anopheline species in Cambodia and Vietnam and to check for the presence of other parasites that might interact with the anti-CSP monoclonal antibodies.MethodsMosquitoes collected in Cambodia and Vietnam were identified and tested for the presence of sporozoites in head and thorax by using CSP-ELISA. ELISA positive samples were confirmed by a Plasmodium specific PCR. False positive mosquitoes were checked by PCR for the presence of parasites belonging to the Haemosporidia, Trypanosomatidae, Piroplasmida, and Haemogregarines. The heat-stability and the presence of the cross-reacting antigen in the abdomen of the mosquitoes were also checked.ResultsSpecimens (N = 16,160) of seven anopheline species were tested by CSP-ELISA for Plasmodium falciparum and Plasmodium vivax (Pv210 and Pv247). Two new vector species were identified for the region: Anopheles pampanai (P. vivax) and Anopheles barbirostris (Plasmodium malariae). In 88% (155/176) of the mosquitoes found positive with the P. falciparum CSP-ELISA, the presence of Plasmodium sporozoites could not be confirmed by PCR. This percentage was much lower (28% or 5/18) for P. vivax CSP-ELISAs. False positive CSP-ELISA results were associated with zoophilic mosquito species. None of the targeted parasites could be detected in these CSP-ELISA false positive mosquitoes. The ELISA reacting antigen of P. falciparum was heat-stable in CSP-ELISA true positive specimens, but not in the false positives. The heat-unstable cross-reacting antigen is mainly present in head and thorax and almost absent in the abdomens (4 out of 147) of the false positive specimens.ConclusionThe CSP-ELISA can considerably overestimate the EIR, particularly for P. falciparum and for zoophilic species. The heat-unstable cross-reacting antigen in false positives remains unknown. Therefore it is highly recommended to confirm all positive CSP-ELISA results, either by re-analysing the heated ELISA lysate (100°C, 10 min), or by performing Plasmodium specific PCR followed if possible by sequencing of the amplicons for Plasmodium species determination.

Malaria in central Vietnam: analysis of risk factors by multivariate analysis and classification tree models

BackgroundIn Central Vietnam, forest malaria remains difficult to control due to the complex interactions between human, vector and environmental factors.MethodsPrior to a community-based intervention to assess the efficacy of long-lasting insecticidal hammocks, a complete census (18,646 individuals) and a baseline cross-sectional survey for determining malaria prevalence and related risk factors were carried out. Multivariate analysis using survey logistic regression was combined to a classification tree model (CART) to better define the relative importance and inter-relations between the different risk factors.ResultsThe study population was mostly from the Ra-glai ethnic group (88%), with both low education and socio-economic status and engaged mainly in forest activities (58%). The multivariate analysis confirmed forest activity, bed net use, ethnicity, age and education as risk factors for malaria infections, but could not handle multiple interactions. The CART analysis showed that the most important risk factor for malaria was the wealth category, the wealthiest group being much less infected (8.9%) than the lower and medium wealth category (16.6%). In the former, forest activity and bed net use were the most determinant risk factors for malaria, while in the lower and medium wealth category, insecticide treated nets were most important, although the latter were less protective among Ra-glai people.ConclusionThe combination of CART and multivariate analysis constitute a novel analytical approach, providing an accurate and dynamic picture of the main risk factors for malaria infection. Results show that the control of forest malaria remains an extremely complex task that has to address poverty-related risk factors such as education, ethnicity and housing conditions.