Vincent Sluydts

An innovative tool for moving malaria PCR detection of parasite reservoir into the field

BackgroundTo 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.MethodsDNA 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).ResultsThe 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 24–48xa0hours 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 <24xa0hours 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%.ConclusionsThe 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.

Spatial clustering and risk factors of malaria infections in Ratanakiri Province, Cambodia

BackgroundMalaria 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.MethodIn 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.ResultPCR 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.DiscussionSpatial 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.

Re-imagining malaria: heterogeneity of human and mosquito behaviour in relation to residual malaria transmission in Cambodia.

BackgroundIn certain regions in Southeast Asia, where malaria is reduced to forested regions populated by ethnic minorities dependent on slash-and-burn agriculture, malaria vector populations have developed a propensity to feed early and outdoors, limiting the effectiveness of long-lasting insecticide-treated nets (LLIN) and indoor residual spraying (IRS). The interplay between heterogeneous human, as well as mosquito behaviour, radically challenges malaria control in such residual transmission contexts. This study examines human behavioural patterns in relation to the vector behaviour.MethodsThe anthropological research used a sequential mixed-methods study design in which quantitative survey research methods were used to complement findings from qualitative ethnographic research. The qualitative research existed of in-depth interviews and participant observation. For the entomological research, indoor and outdoor human landing collections were performed. All research was conducted in selected villages in Ratanakiri province, Cambodia.ResultsVariability in human behaviour resulted in variable exposure to outdoor and early biting vectors: (i) indigenous people were found to commute between farms in the forest, where malaria exposure is higher, and village homes; (ii) the indoor/outdoor biting distinction was less clear in forest housing often completely or partly open to the outside; (iii) reported sleeping times varied according to the context of economic activities, impacting on the proportion of infections that could be accounted for by early or nighttime biting; (iv) protection by LLINs may not be as high as self-reported survey data indicate, as observations showed around 40% (non-treated) market net use while (v) unprotected evening resting and deep forest activities impacted further on the suboptimal use of LLINs.ConclusionsThe heterogeneity of human behaviour and the variation of vector densities and biting behaviours may lead to a considerable proportion of exposure occurring during times that people are assumed to be protected by the distributed LLINs. Additional efforts in improving LLIN use during times when people are resting in the evening and during the night might still have an impact on further reducing malaria transmission in Cambodia.

Survival and maturation rates of the African rodent, Mastomys natalensis: density‐dependence and rainfall

Survival and maturation rates of female Mastomys natalensis were analysed based on a ten-year monthly capture-recapture data set. We investigated whether direct and delayed density dependent and independent (rainfall) variables accounted for the considerable variation in demographic traits. It was estimated that seasonal and annual covariates accounted for respectively 29 and 26% of the total variation in maturation rates and respectively 17 and 11% of the variation in survival rates. Explaining the between-year differences in maturation rates with annual past rainfall or density did not improve the model fit. On the other hand we showed that maturation rates were correlated negatively with density the previous month and positively to cumulative rainfall over the past three months. Survival estimates of both adults and subadults varied seasonally, with higher estimates during the increase phase (dry season). The subadults were characterised by a very high survival rate (> 0.95) during this phase. In the decrease phase only minor differences were found between survival rates of subadults and adults. We found that 39% of the between-year variation in survival can be explained by accumulated rainfall over the past year.

Field Evaluation of Picaridin Repellents Reveals Differences in Repellent Sensitivity between Southeast Asian Vectors of Malaria and Arboviruses

Scaling up of insecticide treated nets has contributed to a substantial malaria decline. However, some malaria vectors, and most arbovirus vectors, bite outdoors and in the early evening. Therefore, topically applied insect repellents may provide crucial additional protection against mosquito-borne pathogens. Among topical repellents, DEET is the most commonly used, followed by others such as picaridin. The protective efficacy of two formulated picaridin repellents against mosquito bites, including arbovirus and malaria vectors, was evaluated in a field study in Cambodia. Over a period of two years, human landing collections were performed on repellent treated persons, with rotation to account for the effect of collection place, time and individual collector. Based on a total of 4996 mosquitoes collected on negative control persons, the overall five hour protection rate was 97.4% [95%CI: 97.1–97.8%], not decreasing over time. Picaridin 20% performed equally well as DEET 20% and better than picaridin 10%. Repellents performed better against Mansonia and Culex spp. as compared to aedines and anophelines. A lower performance was observed against Aedes albopictus as compared to Aedes aegypti, and against Anopheles barbirostris as compared to several vector species. Parity rates were higher in vectors collected on repellent treated person as compared to control persons. As such, field evaluation shows that repellents can provide additional personal protection against early and outdoor biting malaria and arbovirus vectors, with excellent protection up to five hours after application. The heterogeneity in repellent sensitivity between mosquito genera and vector species could however impact the efficacy of repellents in public health programs. Considering its excellent performance and potential to protect against early and outdoor biting vectors, as well as its higher acceptability as compared to DEET, picaridin is an appropriate product to evaluate the epidemiological impact of large scale use of topical repellents on arthropod borne diseases.

Injections, cocktails and diviners: therapeutic flexibility in the context of malaria elimination and drug resistance in Northeast Cambodia

Background Adherence to effective malaria medication is extremely important in the context of Cambodia’s elimination targets and drug resistance containment. Although the public sector health facilities are accessible to the local ethnic minorities of Ratanakiri province (Northeast Cambodia), their illness itineraries often lead them to private pharmacies selling “cocktails” and artemether injections, or to local diviners prescribing animal sacrifices to appease the spirits. Methods The research design consisted of a mixed methods study, combining qualitative (in-depth interviews and participant observation) and quantitative methods (household and cross-sectional survey). Results Three broad options for malaria treatment were identified: i) the public sector; ii) the private sector; iii) traditional treatment based on divination and ceremonial sacrifice. Treatment choice was influenced by the availability of treatment and provider, perceived side effects and efficacy of treatments, perceived etiology of symptoms, and patient-health provider encounters. Moreover, treatment paths proved to be highly flexible, changing mostly in relation to the perceived efficacy of a chosen treatment. Conclusions Despite good availability of anti-malarial treatment in the public health sector, attendance remained low due to both structural and human behavioral factors. The common use and under-dosage of anti-malaria monotherapy in the private sector (single-dose injections, single-day drug cocktails) represents a threat not only for individual case management, but also for the regional plan of drug resistance containment and malaria elimination.

Efficacy of topical mosquito repellent (picaridin) plus long-lasting insecticidal nets versus long-lasting insecticidal nets alone for control of malaria: a cluster randomised controlled trial

BACKGROUNDnAlthough effective topical repellents provide personal protection against malaria, whether mass use of topical repellents in addition to long-lasting insecticidal nets can contribute to a further decline of malaria is not known, particularly in areas where outdoor transmission occurs. We aimed to assess the epidemiological efficacy of a highly effective topical repellent in addition to long-lasting insecticidal nets in reducing malaria prevalence in this setting.nnnMETHODSnA cluster randomised controlled trial was done in the 117 most endemic villages in Ratanakiri province, Cambodia, to assess the efficacy of topical repellents in addition to long-lasting insecticidal nets in controlling malaria in a low-endemic setting. We did a pre-trial assessment of village accessibility and excluded four villages because of their inaccessibility during the rainy season. Another 25 villages were grouped because of their proximity to each other, resulting in 98 study clusters (comprising either a single village or multiple neighbouring villages). Clusters were randomly assigned (1:1) to either a control (long-lasting insecticidal nets) or intervention (long-lasting insecticidal nets plus topical repellent) study group after a restricted randomisation. All clusters received one long-lasting insecticidal net per individual, whereas those in the intervention group also received safe and effective topical repellents (picaridin KBR3023, SC Johnson, Racine, WI, USA), along with instruction and promotion of its daily use. Cross-sectional surveys of 65 randomly selected individuals per cluster were done at the beginning and end of the malaria transmission season in 2012 and 2013. The primary outcome was Plasmodium species-specific prevalence in participants obtained by real-time PCR, assessed in the intention-to-treat population. Complete safety analysis data will be published seperately; any ad-hoc adverse events are reported here. This trial is registered with ClinicalTrials.gov, number NCT01663831.nnnFINDINGSnOf the 98 clusters that villages were split into, 49 were assigned to the control group and 49 were assigned to the intervention group. Despite having a successful distribution system, the daily use of repellents was suboptimum. No post-intervention differences in PCR plasmodium prevalence were observed between study groups in 2012 (4·91% in the control group vs 4·86% in the intervention group; adjusted odds ratio [aOR] 1·01 [95% CI 0·60-1·70]; p=0·975) or in 2013 (2·96% in the control group vs 3·85% in the intervention group; aOR 1·31 [0·81-2·11]; p=0·266). Similar results were obtained according to Plasmodium species (1·33% of participants in the intervention group vs 1·10% in the intervention group were infected with Plasmodium falciparum; aOR 0·83 [0·44-1·56]; p=0·561; and 1·85% in the control group vs 2·67% in the intervention group were infected with Plasmodium vivax; aOR 1·51 [0·88-2·57]; p=0·133). 41 adverse event notifications from nine villages were received, of which 33 were classified as adverse reactions (11 of these 33 were cases of repellent abuse through oral ingestion, either accidental or not). All participants with adverse reactions fully recovered and 17 were advised to permanently stop using the repellent.nnnINTERPRETATIONnMass distribution of highly effective topical repellents in resource-sufficient conditions did not contribute to a further decline in malaria endemicity in a pre-elimination setting in the Greater Mekong subregion. Daily compliance and appropriate use of the repellents remains the main obstacle.nnnFUNDINGnBill & Melinda Gates Foundation.

Factors influencing the use of topical repellents: implications for the effectiveness of malaria elimination strategies

In Cambodia, despite an impressive decline in prevalence over the last 10 years, malaria is still a public health problem in some parts of the country. This is partly due to vectors that bite early and outdoors reducing the effectiveness of measures such as Long-Lasting Insecticidal Nets. Repellents have been suggested as an additional control measure in such settings. As part of a cluster-randomized trial on the effectiveness of topical repellents in controlling malaria infections at community level, a mixed-methods study assessed user rates and determinants of use. Repellents were made widely available and Picaridin repellent reduced 97% of mosquito bites. However, despite high acceptability, daily use was observed to be low (8%) and did not correspond to the reported use in surveys (around 70%). The levels of use aimed for by the trial were never reached as the population used it variably across place (forest, farms and villages) and time (seasons), or in alternative applications (spraying on insects, on bed nets, etc.). These findings show the key role of human behavior in the effectiveness of malaria preventive measures, questioning whether malaria in low endemic settings can be reduced substantially by introducing measures without researching and optimizing community involvement strategies.

Characterizing Types of Human Mobility to Inform Differential and Targeted Malaria Elimination Strategies in Northeast Cambodia.

Human population movements currently challenge malaria elimination in low transmission foci in the Greater Mekong Subregion. Using a mixed-methods design, combining ethnography (nu2009=u2009410 interviews), malariometric data (nu2009=u20094996) and population surveys (nu2009=u2009824 indigenous populations; nu2009=u2009704 Khmer migrants) malaria vulnerability among different types of mobile populations was researched in the remote province of Ratanakiri, Cambodia. Different structural types of human mobility were identified, showing differential risk and vulnerability. Among local indigenous populations, access to malaria testing and treatment through the VMW-system and LLIN coverage was high but control strategies failed to account for forest farmers’ prolonged stays at forest farms/fields (61% during rainy season), increasing their exposure (pu2009=u20090.002). The Khmer migrants, with low acquired immunity, active on plantations and mines, represented a fundamentally different group not reached by LLIN-distribution campaigns since they were largely unregistered (79%) and unaware of the local VMW-system (95%) due to poor social integration. Khmer migrants therefore require control strategies including active detection, registration and immediate access to malaria prevention and control tools from which they are currently excluded. In conclusion, different types of mobility require different malaria elimination strategies. Targeting mobility without an in-depth understanding of malaria risk in each group challenges further progress towards elimination.

Uncovering the secret lives of sewer rats (Rattus norvegicus): movements, distribution and population dynamics revealed by a capture–mark–recapture study

Abstract Context.In many parts of the world, brown rats have colonised sewer systems and the rat populations in sewers are often thought to be a source of problems with rats on the surface. The management of sewer rat populations is usually performed with little, if any, knowledge of the dynamics and behaviour of these populations. Aims.The study aimed to uncover the population dynamics, movements and distribution of rats in populations living in sewers. Methods.We studied rats in a capture–mark–recapture study in two adjacent but separate sewer systems in suburban Copenhagen, Denmark, covering a surface area of 30u2009Ha. Multiple-capture live traps were placed in manholes in public sewers; animals were marked individually and data were collected every fourth week for a period of almost 3 years. Key results.In total, 332 individual rats were trapped. Approximately one-third were only caught once, half of these were juveniles. The median time an individual was present in the sewers was 4 months with maxima up to 22 months. Abundance ranged from 2 to 44 animals per sewer system and the dynamics showed no regular fluctuations. Rats were distributed throughout the study area, regardless of sewer dimensions. Juvenile rats were captured more in the smaller and drier sewer sections. Activity areas of rats of both sexes overlapped partially, and observations of bite wounds were rare, suggesting a high degree of tolerance. The average maximal distance covered by rats was ∼200u2009m for both sexes. Day-to-day movements took place over shorter distances. There were no movements of rats between sewer systems and no sewer rats were observed to be active on the surface. The rat populations seemed to be substructured in smaller groups. Conclusions.Sewer rat populations vary irregularly in abundance. Pipe dimensions are not important for the presence of rats but breeding seems concentrated in smaller and drier pipes. Most individuals are resident and live in small groups that are active in a limited area only, but still there is a fair group of dispersing rats. Implications. Sewer rat management should be spread out spatially in a sewer system since most rats move over short areas only. Recolonisation by dispersing rats must be taken into account if sewer systems are connected. It is likely that targeting smaller and drier pipes will affect reproductive success.