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Background: Malaria "hotspots" have been proposed as potential intervention units for targeted malaria elimination. Little is known about hotspot formation and stability in settings outside sub-Saharan Africa., Methods: Clustering of Plasmodium infections at the household and hotspot level was assessed over 2 years in 3 villages in eastern Cambodia. Social and spatial autocorrelation statistics were calculated to assess clustering of malaria risk, and logistic regression was used to assess the effect of living in a malaria hotspot compared to living in a malaria-positive household in the first year of the study on risk of malaria infection in the second year., Results: The crude prevalence of Plasmodium infection was 8.4% in 2016 and 3.6% in 2017. Living in a hotspot in 2016 did not predict Plasmodium risk at the individual or household level in 2017 overall, but living in a Plasmodium-positive household in 2016 strongly predicted living in a Plasmodium-positive household in 2017 (Risk Ratio, 5.00 [95% confidence interval, 2.09-11.96], P < .0001). There was no consistent evidence that malaria risk clustered in groups of socially connected individuals from different households., Conclusions: Malaria risk clustered more clearly in households than in hotspots over 2 years. Household-based strategies should be prioritized in malaria elimination programs in this region., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Malaria elimination needs a concentration of activities towards identification of residual transmission foci and intensification of efforts to eliminate the last few infections, located in so-called 'malaria hotspots'. Previous work on characterizing malaria transmission hotspots has mainly focused on falciparum malaria and especially on symptomatic cases, while the malaria reservoir is expected to be mainly concentrated in the asymptomatic human population when transmission is low. For Plasmodium vivax, there has been less effort in identifying transmission hotspots. The main aim of this study was to uncover micro-epidemiological mechanisms of clustering of malaria infections at a sub-village level, based on geographical or behavioural features., Methods: A cross-sectional survey was performed in three villages within the highest malaria endemic province of Cambodia. The survey took place in the dry season, when the malaria reservoir is expected to be low and residing in the asymptomatic part of the population. Village and field locations of households were georeferenced, blood samples were taken from as many residents as possible and a short questionnaire probing for individual risk factors was taken. Asymptomatic malaria carriers were detected by PCR, and geographical clustering analysis (SaTScan) as well as risk factor analysis were performed., Results: A total of 1540 out of 1792 (86%) individuals were sampled. Plasmodial DNA was detected in 129 individuals (8.4%). P. vivax was most prevalent (5.5%) followed by Plasmodium malariae (2.1%) and Plasmodium falciparum (1.6%). Mixed infection occurred in 12 individuals. In two out of three villages geographical clustering of high and low malaria infection risk was clearly present. Cluster location and risk factors associated with the infection differed between the parasite species. Age was an important risk factor for the combined Plasmodium infections, while watching television at evenings was associated with increased odds of P. vivax infections [OR (CI): 1.86 (0.95-3.64)] and bed net use was associated with reduced odds of P. falciparum infections [OR (CI): 0.25 (0.077-0.80)]., Conclusions: Clusters of malaria carriers were malaria species specific and often located remotely, outside village centres. As such, at micro-epidemiological level, malaria is not a single disease. Further unravelling the micro-epidemiology of malaria can enable programme managers to define the interventions likely to contribute to halt transmission in a particular hotspot location.

In the present review, we aim to provide a general introduction to different facets of the arms race between pathogens and their hosts/environment, emphasizing its evolutionary aspects. We focus on vector-borne parasitic protozoa, which have to adapt to both invertebrate and vertebrate hosts. Using Leishmania, Trypanosoma and Plasmodium as main models, we review successively (i) the adaptations and counter-adaptations of parasites and their invertebrate host, (ii) the adaptations and counter-adaptations of parasites and their vertebrate host and (iii) the impact of human interventions (chemotherapy, vaccination, vector control and environmental changes) on these adaptations. We conclude by discussing the practical impact this knowledge can have on translational research and public health., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Malaria incidence worldwide has steadily declined over the past decades. Consequently, increasingly more countries will proceed from control to elimination. The malaria distribution in low incidence settings appears patchy, and local transmission hotspots are a continuous source of infection. In this study, species-specific clusters and associated risk factors were identified based on malaria prevalence data collected in the north-east of Cambodia. In addition, Plasmodium falciparum genetic diversity, population structure and gene flows were studied., Method: In 2012, blood samples from 5793 randomly selected individuals living in 117 villages were collected from Ratanakiri province, Cambodia. Malariometric data of each participant were simultaneously accumulated using a standard questionnaire. A two-step PCR allowed for species-specific detection of malaria parasites, and SNP-genotyping of P. falciparum was performed. SaTScan was used to determine species-specific areas of elevated risk to infection, and univariate and multivariate risk analyses were carried out., Result: PCR diagnosis found 368 positive individuals (6.4%) for malaria parasites, of which 22% contained mixed species infections. The occurrence of these co-infections was more frequent than expected. Specific areas with elevated risk of infection were detected for all Plasmodium species. The clusters for Falciparum, Vivax and Ovale malaria appeared in the north of the province along the main river, while the cluster for Malariae malaria was situated elsewhere. The relative risk to be a malaria parasite carrier within clusters along the river was twice that outside the area. The main risk factor associated with three out of four malaria species was overnight stay in the plot hut, a human behaviour associated with indigenous farming. Haplotypes did not show clear geographical population structure, but pairwise Fst value comparison indicated higher parasite flow along the river., Discussion: Spatial aggregation of malaria parasite carriers, and the identification of malaria species-specific risk factors provide key insights in malaria epidemiology in low transmission settings, which can guide targeted supplementary interventions. Consequently, future malaria programmes in the province should implement additional specific policies targeting households staying overnight at their farms outside the village, in addition to migrants and forest workers.

Background: To achieve the goal of malaria elimination in low transmission areas such as in Cambodia, new, inexpensive, high-throughput diagnostic tools for identifying very low parasite densities in asymptomatic carriers are required. This will enable a switch from passive to active malaria case detection in the field., Methods: DNA extraction and real-time PCR assays were implemented in an "in-house" designed mobile laboratory allowing implementation of a robust, sensitive and rapid malaria diagnostic strategy in the field. This tool was employed in a survey organized in the context of the MalaResT project (NCT01663831)., Results: The real-time PCR screening and species identification assays were performed in the mobile laboratory between October and November 2012, in Rattanakiri Province, to screen approximately 5,000 individuals in less than four weeks and treat parasite carriers within 24-48 hours after sample collection. An average of 240 clinical samples (and 40 quality control samples) was tested every day, six/seven days per week. Some 97.7% of the results were available <24 hours after the collection. A total of 4.9% were positive for malaria. Plasmodium vivax was present in 61.1% of the positive samples, Plasmodium falciparum in 45.9%, Plasmodium malariae in 7.0% and Plasmodium ovale in 2.0%., Conclusions: The operational success of this diagnostic set-up proved that molecular testing and subsequent treatment is logistically achievable in field settings. This will allow the detection of clusters of asymptomatic carriers and to provide useful epidemiological information. Fast results will be of great help for staff in the field to track and treat asymptomatic parasitaemic cases. The concept of the mobile laboratory could be extended to other countries for the molecular detection of malaria or other pathogens, or to culture vivax parasites, which does not support long-time delay between sample collection and culture.

Background: Despite progress in malaria control, malaria remains an important public health concern in Cambodia, mostly linked to forested areas. Large-scale vector control interventions in Cambodia are based on the free distribution of long-lasting insecticidal nets (LLINs), targeting indoor- and late-biting malaria vectors only. The present study evaluated the vector density, early biting activity and malaria transmission of outdoor-biting malaria vectors in two forested regions in Cambodia., Methods: In 2005 two entomological surveys were conducted in 12 villages and their related forest plots in the east and west of Cambodia. Mosquitoes were collected outdoors by human landing collections and subjected to enzyme-linked immunosorbent assay (ELISA) to detect Plasmodium sporozoites after morphological identification. Blood samples were collected in the same villages for serological analyses. Collected data were analysed by the classification and regression tree (CART) method and linear regression analysis., Results: A total of 11,826 anophelines were recorded landing in 787 man-night collections. The majority (82.9%) were the known primary and secondary vectors. Most of the variability in vector densities and early biting rates was explained by geographical factors, mainly at village level. Vector densities were similar between forest and village sites. Based on ELISA results, 29% out of 17 Plasmodium-positive bites occurred before sleeping time, and 65% in the forest plots. The entomological inoculation rates of survey 1 were important predictors of the respective seroconversion rates in survey 2, whereas the mosquito densities were not., Discussion: In Cambodia, outdoor malaria transmission in villages and forest plots is important. In this context, deforestation might result in lower densities of the primary vectors, but also in higher densities of secondary vectors invading deforested areas. Moreover, higher accessibility of the forest could result in a higher man-vector contact. Therefore, additional vector control measures should be developed to target outdoor- and early-biting vectors.

Background: Cambodia reduced malaria incidence by more than 75% between 2000 and 2015, a target of the Millennium Development Goal 6. The Cambodian Government aims to eliminate all forms of malaria by 2025. The country's malaria incidence is highly variable at provincial level, but less is known at village level. This study used passive case detection (PCD) data at village level in Ratanakiri Province from 2010 to 2014 to describe incidence trends and identify high-risk areas of malaria to be primarily targeted towards malaria elimination. Methods: In 2010, the Cambodian malaria programme created a Malaria Information System (MIS) to capture malaria information at village level through PCD by village malaria workers and health facilities. The MIS data of Ratanakiri Province 2010-2014 were used to calculate annual incidence rates by Plasmodium species at province and commune levels. For estimating the trend at provincial level only villages reporting each year were selected. The communal incidences and the number of cases per village were visualized on a map per Plasmodium species and per year. Analysis of spatial clustering of village malaria cases by Plasmodium species was performed by year. Results: Overall, malaria annual incidence rates per 1000 inhabitants decreased from 86 (2010) to 30 (2014). Falciparum incidence decreased (by 79% in 2014 compared to 2010; CI 95% 76-82%) more rapidly than vivax incidence (by 19% in 2014 compared to 2010; CI 95% 5-32%). There were ten to 16 significant spatial clusters each year. Big clusters tended to extend along the Cambodian-Vietnamese border and along the Sesan River. Three clusters appeared throughout all years (2010-2014): one with 21 villages appeared each year, the second shrunk progressively from 2012 to 2014 and the third was split into two smaller clusters in 2013 and 2014. Conclusion: The decline of malaria burden can be attributed to intensive malaria control activities implemented in the areas: distribution of a long-lasting insecticidal net per person and early diagnosis and prompt treatment. Dihydro-artemisinin piperaquine was the only first-line treatment for all malaria cases. No radical treatment with primaquine was provided for Plasmodium vivax cases, which could explain the slow decrease of P. vivax due to relapses. To achieve malaria elimination by 2025, priority should be given to the control of stable malaria clusters appearing over time. [ABSTRACT FROM AUTHOR]

Background: Serological markers for exposure to different Plasmodium species have recently been used in multiplex immunoassays based on the Luminex technology. However, interpretation of the assay results requires consideration of the half-life of specific antibodies against these markers. Therefore, the aim of the present study was to document the half-life of malaria specific serological makers, as well as assessing the sensitivity of these markers to pick up recent changes in malaria exposure. Methods: A recently developed multiplex immunoassay was used to measure the intensity of antibody (Ab) responses against 19 different Plasmodium specific antigens, covering different human malaria parasites and two vector saliva antigens. Therefore, 8439 blood samples from five cross-sectional surveys in Ratanakiri, Cambodia, were analysed. These involve a random selection from two selected surveys, and an additional set of blood samples of individuals that were randomly re-sampled three, four or five times. A generalized estimating equation model and linear regression models were fitted on log transformed antibody intensity data. Results: Results showed that most (17/21) Ab-responses are higher in PCR positive than PCR negative individuals. Furthermore, these antibody-responses follow the same upward trend within each age group. Estimation of the half-lives showed differences between serological markers that reflect short-(seasonal) and long-term (year round) transmission trends. Ab levels declined significantly together with a decrease of PCR prevalence in a group of malaria endemic villages. Conclusion: For Plasmodium falciparum, antibodies against LSA3.RE, GLURP and Pf.GLURP.R2 are most likely to be a reflexion of recent (range from 6 to 8 months) exposure in the Mekong Subregion. PvEBP is the only Plasmodium vivax Ag responding reasonably well, in spite of an estimated Ab half-life of more than 1 year. The use of Ab intensity data rather dichotomizing the continuous Ab-titre data (positive vs negative) will lead to an improved approach for serological surveillance. [ABSTRACT FROM AUTHOR]

Background: Epidemiological surveillance is a key activity in malaria control and elimination in low-transmission and pre-elimination settings. Hence, sensitive tools for estimating malaria force of infection are crucial. Serological markers might provide additional information in estimating force of infection in low-endemic areas along with classical parasite detection methods. Serological markers can be used to estimate recent, past or present malaria exposure, depending on the used markers and their half-life. Methods: An assay based on 14 Plasmodium-specific peptides, one peptide specific for Anopheles gambiae saliva protein and five Plasmodium-specific recombinant proteins was developed for the MAGPIX system, assessed for its performance, and applied on blood spots from 2000 individuals collected in the Ratanakiri Province, Cambodia. Results: A significant correlation for the use of 1000 and 2000 beads/antigen/well as well as for the monoplex versus multiplex assay was observed for all antigens (p < 0.05). For the majority of antigens, antigen-coupled beads were stable for at least 2 months. The assay was very reproducible with limited intercoupling, interplate and intraplate variability (mean RSD <15 %). Estimating seroconversion and seroreversion per antigen using reversible catalytic models and models allowing two seroconversion rates showed higher seroconversion rates in adults. Conclusion: The multiplex bead-based immunoassay was successfully implemented and analysis of field blood samples shows that changes detected in force of malaria infection vary according to the serological markers used. Multivariate analysis of the antibody responses and insights into the half-life of antibodies are crucial for improving the interpretation of these results and for identifying the most useful serological markers of past and recent malaria infection. [ABSTRACT FROM AUTHOR]