Molecular incidence and clearance of Plasmodium falciparum infection.

BACKGROUND: Although the epidemiology of malaria has been based primarily on microscopy and rapid diagnostic tests, molecular methods are necessary to understand the complexity of natural infection in regions where transmission is intense and simultaneous infection with multiple parasite genotypes is common such as sub-Saharan Africa. METHODS: To compare microscopic and molecular estimates of the incidence and clearance of Plasmodium falciparum infection, we followed 80 children monthly for 1 year in the village of Bancoumana in Mali. RESULTS AND DISCUSSION: Similar seasonal patterns were observed with both methods (rainy season peak, dry season nadir), although molecular methods detected more infections than microscopy (571 vs 331 in 906 specimens), more new infections (311 vs 104 during 829 person-months) and spontaneous clearance events (317 vs 116) and found higher incidence (0.38 vs 0.13 new genotypes/person/month, p < 0.001) and spontaneous clearance rates (0.38 vs 0.14 genotypes cleared/person/month, p < 0.001). These differences were greatest for persistently-infected subjects in whom neither new infections nor the clearance of old infections could be detected by microscopy (0.71 new infections and 0.73 cleared infections per month using molecular methods vs 0.000 by microscopy, p < 0.001). CONCLUSIONS: Molecular methods provide information about genetic diversity, the intensity of transmission and spontaneous clearance in the absence of drug treatment that cannot be obtained by microscopy. They will be necessary to evaluate the efficacy of vaccines, drugs and other control strategies for diseases such as malaria in which simultaneous infection with more than one organism (genotype) is common.

Evaluation and optimization of membrane feeding compared to direct feeding as an assay for infectivity.

BACKGROUND: Malaria parasite infectivity to mosquitoes has been measured in a variety of ways and setting, includind direct feeds of and/or membrane feeding blood collected from randomly selected or gametocytemic volunteers. Anopheles gambiae s.l is the main vector responsible of Plasmodium falciparum transmission in Bancoumana and represents about 90% of the laboratory findings, whereas Plasmodium malariae and Plasmodium ovale together represent only 10%. MATERIALS AND METHODS: Between August 1996 and December 1998, direct and membrane feeding methods were compared for the infectivity of children and adolescent gametocyte carriers to anopheline mosquitoes in the village of Bancoumana in Mali. Gametocyte carriers were recruited twice a month through a screening of members of 30 families using Giemsa-stained thick blood smears. F1 generation mosquitoes issued from individual female wild mosquitoes from Bancoumana were reared in a controlled insectary conditions and fed 5% sugar solution in the laboratory in Bamako, until the feeding day when they are starved 12 hours before the feeding experiment. These F1 generation mosquitoes were divided in two groups, one group fed directly on gametocyte carriers and the other fed using membrane feeding method. RESULTS: Results from 372 Plasmodium falciparum gametocyte carriers showed that children aged 4-9 years were more infectious than adolescents (p = 0.039), especially during the rainy season. Data from 35 carriers showed that mosquitoes which were used for direct feeding were about 1.5 times more likely to feed (p < 0.001) and two times more likely to become infected, if they fed (p < 0.001), than were those which were used for membrane feeding. Overall, infectivity was about three-times higher for direct feeding than for membrane feeding (p < 0.001). CONCLUSION: Although intensity of infectivity was lower for membrane feeding, it could be a surrogate to direct feeding for evaluating transmission-blocking activity of candidate malaria vaccines. An optimization of the method for future trials would involve using about three-times more mosquitoes than would be used for direct feeding.

Spatial distribution of the chromosomal forms of anopheles gambiae in Mali.

BACKGROUND: Maps of the distribution of malaria vectors are useful tools for stratification of malaria risk and for selective vector control strategies. Although the distribution of members of the Anopheles gambiae complex is well documented in Africa, a continuous map of the spatial distribution of the chromosomal forms of An. gambiae s.s. is not yet available at country level to support control efforts. METHODS: Bayesian geostatistical methods were used to produce continuous maps of the spatial distribution of the chromosomal forms of An. gambiae s.s. (Mopti, Bamako, Savanna and their hybrids/recombinants) based on their relative frequencies in relation to climatic and environmental factors in Mali. RESULTS: The maps clearly show that each chromosomal form favours a particular defined eco-climatic zone. The Mopti form prefers the dryer northern Savanna and Sahel and the flooded/irrigated areas of the inner delta of the Niger River. The Savanna form favours the Sudan savanna areas, particularly the South and South-Eastern parts of the country (Kayes and Sikasso regions). The Bamako form has a strong preference for specific environmental conditions and it is confined to the Sudan savanna areas around urban Bamako and the Western part of Sikasso region. The hybrids/recombinants favour the Western part of the country (Kayes region) bordering the Republic of Guinea Conakry. CONCLUSION: The maps provide valuable information for selective vector control in Mali (insecticide resistance management) and may serve as a decision support tool for the basis for future malaria control strategies including genetically manipulated mosquitoes.

Pathway to Deployment of Gene Drive Mosquitoes as a Potential Biocontrol Tool for Elimination of Malaria in Sub-Saharan Africa: Recommendations of a Scientific Working Group†.

Gene drive technology offers the promise for a high-impact, cost-effective, and durable method to control malaria transmission that would make a significant contribution to elimination. Gene drive systems, such as those based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein, have the potential to spread beneficial traits through interbreeding populations of malaria mosquitoes. However, the characteristics of this technology have raised concerns that necessitate careful consideration of the product development pathway. A multidisciplinary working group considered the implications of low-threshold gene drive systems on the development pathway described in the World Health Organization Guidance Framework for testing genetically modified (GM) mosquitoes, focusing on reduction of malaria transmission by Anopheles gambiae s.l. mosquitoes in Africa as a case study. The group developed recommendations for the safe and ethical testing of gene drive mosquitoes, drawing on prior experience with other vector control tools, GM organisms, and biocontrol agents. These recommendations are organized according to a testing plan that seeks to maximize safety by incrementally increasing the degree of human and environmental exposure to the investigational product. As with biocontrol agents, emphasis is placed on safety evaluation at the end of physically confined laboratory testing as a major decision point for whether to enter field testing. Progression through the testing pathway is based on fulfillment of safety and efficacy criteria, and is subject to regulatory and ethical approvals, as well as social acceptance. The working group identified several resources that were considered important to support responsible field testing of gene drive mosquitoes.

Spatial analysis of malaria transmission parameters in the rice cultivation area of Office du Niger, Mali.

The effects of rice growth environment on malaria transmission, taking into account spatial correlation, were assessed in the Office du Niger, Mali. Between April 1999 to January 2001, 8 quarterly entomologic surveys were conducted in 18 villages in 3 agricultural zones. Vector densities in sleeping houses were related to rice crop, rice development stages, vegetation abundance, water state, and seasons. They were high throughout the rice-growing seasons, increased as the rice crop developed, and decreased as vegetation became abundant. They also showed large spatial correlations (up to 30.6 km). The vectorial capacity exhibited both seasonal and village-to-village variation. Parity and the human blood index were weakly related to adult densities and showed low spatial correlations (up to 3.4 km), which suggested that small area variation in malaria transmission results mainly from variations in vector-human contact. Control strategies in rice cultivation areas should pay attention to this local variation.

Year-to-year variation in the age-specific incidence of clinical malaria in two potential vaccine testing sites in Mali with different levels of malaria transmission intensity.

To explore the feasibility of field sites for malaria vaccine trials, we conducted a prospective study of clinical malaria incidence during two consecutive transmission seasons in children and young adults living in two areas of Mali with different entomologic inoculation rates (EIRs). Approximately 200 subjects (3 months to 2 years of age) were enrolled per site and followed weekly. Malaria smears were performed monthly in all participants and when symptoms or signs of malaria were present. In Sotuba (annual EIR < 15 infective bites per person), the incidence of clinical malaria was comparable across all age groups but varied significantly between the 2 years. In contrast, in Donéguébougou (annual EIR > 100 infective bites per person), incidence rates decreased significantly with increasing age but remained stable between years. Our results suggest that, although the age distribution of clinical malaria depends on transmission intensity, the total burden of disease may be similar or higher in settings of low transmission.

Genetic differentiation between the BAMAKO and SAVANNA chromosomal forms of Anopheles gambiae as indicated by amplified fragment length polymorphism analysis.

The main vector of malaria in sub-Saharan Africa, Anopheles gambiae, is subdivided into five chromosomal forms. Three of them (i.e., BAMAKO, SAVANNA, and MOPTI) are found in sympatry in Mali, where MOPTI can be distinguished from the other two forms based on differences in the ribosomal DNA locus. However, no molecular markers are available to distinguish BAMAKO from SAVANNA. We examined the banding patterns of 139 amplified fragment length polymorphism primer combinations in an attempt to identify diagnostic differences between SAVANNA and BAMAKO. Despite screening > 10,000 bands, no diagnostic differences were found. However, additional AFLP analyses indicated that BAMAKO is genetically differentiated from SAVANNA, with a significant Phi(st) value of 0.072. This could indicate that gene flow between these forms is restricted in at least some portion of the genome and the lack of identifiable fixed differences between the two forms is probably due to their recent origin.

Water quality and immatures of the M and S forms of Anopheles gambiae s.s. and An. arabiensis in a Malian village.

INTRODUCTION: The associations between the immatures of Anopheles gambiae s.s. (Diptera: Culicidae), its M and S forms, and Anopheles arabiensis among and within larval breeding habitats in Banambani, Mali were investigated under varying conditions of water quality and rainfall. The intent was to elucidate on niche partitioning of these taxa. METHODS: Immatures of An. arabiensis, An. gambiae s.s., and its M and S forms were sampled every alternate day for a month in mid-rainy season from three sampling sites in each of the larval breeding habitats (rock pools, swamp, and puddles). Water quality was characterized by alkalinity, conductivity, dissolved oxygen (D.O.), nitrate, orthophosphate, pH, temperature, total dissolved solids (TDS), and turbidity. A type 3 analysis of the GENMOD model was used to examine the associations between the proportional frequencies of young (first and second instar larvae) and old (third and fourth instar larvae and pupae) or total immatures of species or forms among sampling sites within and among larval breeding habitats during a category of rainfall as influenced by water quality. RESULTS: Of the 4,174 immatures sampled, 1,300 were molecularly identified to species and forms. Significant association between the proportional frequencies of young larvae of An. arabiensis, An. gambiae s.s., its M and S forms was found among sampling sites within habitats but not among larval breeding habitats. The proportional frequencies of young larvae of M and S forms varied daily perhaps due to recruitment, mortality, and dispersal within habitats. Conductivity and TDS had significant effects when the proportional frequencies of young larvae of M and S forms among sampling sites within habitats were significantly associated. Alkalinity, D.O., orthophosphate, pH, nitrate, temperature and turbidity had no effects on niche partitioning of species and forms among sampling sites within habitats. Rainfall did not affect the frequencies of these immatures. CONCLUSION: Conductivity and TDS have significant effects on niche partitioning of young larvae of M and S forms among sampling sites within habitats in Banambani, Mali.

Dynamics of antigenemia and transmission intensity of Wuchereria bancrofti following cessation of mass drug administration in a formerly highly endemic region of Mali.

BACKGROUND: After seven annual rounds of mass drug administration (MDA) in six Malian villages highly endemic for Wuchereria bancrofti (overall prevalence rate of 42.7%), treatment was discontinued in 2008. Surveillance was performed over the ensuing 5 years to detect recrudescence. METHODS: Circulating filarial antigen (CFA) was measured using immunochromatographic card tests (ICT) and Og4C3 ELISA in 6-7 year-olds. Antibody to the W. bancrofti infective larval stage (L3) antigen, Wb123, was tested in the same population in 2012. Microfilaraemia was assessed in ICT-positive subjects. Anopheles gambiae complex specimens were collected monthly using human landing catch (HLC) and pyrethrum spray catch (PSC). Anopheles gambiae complex infection with W. bancrofti was determined by dissection and reverse transcriptase polymerase chain reaction (RT-PCR) of mosquito pools. RESULTS: Annual CFA prevalence rates using ICT in children increased over time from 0% (0/289) in 2009 to 2.7% (8/301) in 2011, 3.9% (11/285) in 2012 and 4.5% (14/309) in 2013 (trend χ 2 = 11.85, df =3, P = 0.0006). Wb123 antibody positivity rates in 2013 were similar to the CFA prevalence by ELISA (5/285). Although two W. bancrofti-infected Anopheles were observed by dissection among 12,951 mosquitoes collected by HLC, none had L3 larvae when tested by L3-specific RT-PCR. No positive pools were detected among the mosquitoes collected by pyrethrum spray catch. Whereas ICT in 6-7 year-olds was the major surveillance tool, ICT positivity was also assessed in older children and adults (8-65 years old). CFA prevalence decreased in this group from 4.9% (39/800) to 3.5% (28/795) and 2.8% (50/1,812) in 2009, 2011 and 2012, respectively (trend χ 2 = 7.361, df =2, P = 0.0067). Some ICT-positive individuals were microfilaraemic in 2009 [2.6% (1/39)] and 2011 [8.3% (3/36)], but none were positive in 2012 or 2013. CONCLUSION: Although ICT rates in children increased over the 5-year surveillance period, the decrease in ICT prevalence in the older group suggests a reduction in transmission intensity. This was consistent with the failure to detect infective mosquitoes or microfilaraemia. The threshold of ICT positivity in children may need to be re-assessed and other adjunct surveillance tools considered.

Strategic emphases for tropical diseases research: a TDR perspective.

Setting priorities for health research is a difficult task, especially for the neglected diseases of the poor. A new approach to priority setting for tropical diseases research has been adopted by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (known as the TDR). Priorities are defined on the basis of a comprehensive analysis of research needs and research opportunities for each of the ten major tropical diseases in the TDR portfolio. The resulting strategic emphases matrix reflects the priorities for tropical diseases research from the perspective of the TDR. Its purpose is not to impose global research priorities, but we believe the results could be useful to other organizations.

The Impact of Six Annual Rounds of Mass Drug Administration on Wuchereria bancrofti Infections in Humans and in Mosquitoes in Mali.

Wuchereria bancrofti prevalence and transmission were assessed in six endemic villages in Sikasso, Mali prior to and yearly during mass drug administration (MDA) with albendazole and ivermectin from 2002 to 2007. Microfilaremia was determined by calibrated thick smear of night blood in adult volunteers and circulating filarial antigen was measured using immunochromatographic card test in children < 5 years of age. Mosquitoes were collected by human landing catch from July to December. None of the 686 subjects tested were microfilaremic 12 months after the sixth MDA round. More importantly, circulating antigen was not detected in any of the 120 children tested, as compared with 53% (103/194) before the institution of MDA. The number of infective bites/human/year decreased from 4.8 in 2002 to 0.04 in 2007, and only one mosquito containing a single infective larva was observed 12 months after the final MDA round. Whether this dramatic reduction in transmission will be sustained following cessation of MDA remains to be seen.

The Anopheles gambiae genome: next steps for malaria vector control.

Malaria remains a major public health problem that is made worse by poor implementation of control measures, and by the spread of drug- and insecticide-resistant parasites and vectors, respectively. Availability of the Anopheles gambiae genome sequence will accelerate identification and exploitation of new target genes in this insect vector. This provides unique opportunities to improve on existing vector control tools and to generate new tools within a global partnership. However, significant capacity needs to be built for investigators in disease-endemic countries to exploit the genome data. When integrated with existing strategies, the new tools will form an effective package for selective vector control in an effort to prevent mortality and morbidity due to malaria.

Strategic emphases for tropical diseases research: a TDR perspective.

Setting priorities for health research is a difficult task, especially for the neglected diseases of the poor. A new approach to priority setting for tropical diseases research has been adopted by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (known as the TDR). Priorities are defined on the basis of a comprehensive analysis of research needs and research opportunities for each of the ten major tropical diseases in the TDR portfolio. The resulting strategic emphases matrix reflects the priorities for tropical diseases research from the perspective of the TDR. Its purpose is not to impose global research priorities, but we believe the results could be useful to other organizations.

Frequency of multiple inseminations in field-collected Anopheles gambiae females revealed by DNA analysis of transferred sperm.

We investigated the frequencies of single and multiple matings in field-collected female Anopheles gambiae by conducting microsatellite DNA analyses on the sperm contained within their spermatheca. Amplifcation by a polymerase chain reaction (PCR) at four loci allowed the detection of sperm extracts exhibiting more than two alleles per locus, thereby revealing the occurrence of multiple inseminations. Polyandry was found in six of 239 females examined, or 2.5% of the samples. Previous analyses of the molecular form of the sperm and female extracts using a PCR-based diagnostic procedure showed that two of these multiple inseminations involved cross-mating between two chromosomal/molecular forms of An. gambiae s.s. Thus polyandry occurred within-form in 1.7% of examined females while other multiple inseminations may be linked to processes of reproductive isolation between forms of An. gambiae.

Malaria transmission in relation to rice cultivation in the irrigated Sahel of Mali.

Seven cross-sectional entomological surveys were carried out from September 1995 to February 1998 in three irrigated rice growing villages and three villages without irrigated agriculture in the area surrounding Niono, located 350km north-east of Bamako, Mali. The transmission pattern differed markedly between the two zones. In the irrigated zone, the transmission of malaria was fairly constant over the seasons at a low level. In the non-irrigated zone, transmission was mostly below detection level during the dry season, whereas it was high toward the end of the rainy season. In the irrigated zone, high densities of mosquitoes were correlated with low anthropophily, low sporozoite indices and probably low survival rates. In the non-irrigated zone, mosquito densities were lower and these relationships were less pronounced. Differential use of mosquito nets in the two zones may have been an important factor in the observed differences in transmission. The presence of cattle may also have played an important role. Two mosquito-catching methods (human landing catch and spray catch) were compared.

Survivorship and distribution of immature Anopheles gambiae s.l. (Diptera: Culicidae) in Banambani village, Mali.

We observed the survivorship and distribution of larvae and pupae of Anopheles gambiae s.l. Giles immature stages in three habitats (rock pools, swamp, and puddles) in Banambani village. Mali, West Africa, during the mid-rainy season of 2000. Horizontal life tables were constructed for immatures in the laboratory. Times spent in the various immature stages were determined, and laboratory survival was measured. Vertical life tables were obtained from each habitat. We found large day-to-day variation for age class composition within habitats across days. The swamp samples had small but statistically significant different distributions in some instar stages compared with rock pools and puddles as affected by precipitation history. There were obviously unstable age distributions in the swamp and puddles and to some extent in rock pools. There were more individuals in some later age classes than in earlier ones. The daily survival estimates using an exponential decay model were 0.807 in rock pools, 0.899 in the swamp, 0.818 in puddles, and 0.863 in the overall village. Possible reasons for the departure from stable age distribution were cannibalism, predation and other complex interactions, rainfall effects, sampling bias, and differences in physicochemical properties of the water in the habitats.

Spatial and habitat distribution of Anopheles gambiae and Anopheles arabiensis (Diptera: Culicidae) in Banambani village, Mali.

We studied the larval distribution and composition of Anopheles arabiensis Patton, An. gambiae s.s. Giles, and its forms, among local habitats; and their association with the adults between these habitats in Banambani village, Mali during the mid-rainy seasons of 1997-1999. For species and form identification we used polymerase chain reaction (PCR) and PCR-restriction fragment-length polymorphism (RFLP). Differences among species in the distribution of larvae were observed in 1998, but not in 1997 or 1999, although they were on the borderline of statistical significance. Differences among the M and S molecular forms were statistically significant in 1999 when rainfall was high, but not in the two prior, drier sampling periods. Combining all information into the Fisher multiple comparisons test, there were statistically significant differences between species and molecular forms during the 3-yr study period. Hybrid larvae between the M and S forms were observed (0.57%), the first such observation to our knowledge. In spite of differences among larval distribution, no differences of adult species composition were observed among habitats. Factors that influence the distributions of An. gambiae larval populations are discussed.

Informed consent in international research: the rationale for different approaches.

In developed countries, informed consent is based on the autonomy of the individual, a written description of the studies proposed, and previous experience of the participant with Western medicine. Consent is documented by the signature of the participant and supervised by institutional review boards (IRBs), which have conflicts of interest because they are also responsible for limiting institutional liability. In developing countries, the initial decision-making for informed consent is typically vested in the community rather than the individual, and illiteracy is common-limiting the value of written documents and signatures. The challenges in developing countries are exacerbated by the fact that persons at greatest risk of disease are often illiterate, have limited experience with Western medicine, and have limited understanding of the scientific rationale for the studies proposed. Given these differences, it is unrealistic to expect that consent strategies used in developed countries would be effective in such diverse settings.

Mosaic genome architecture of the Anopheles gambiae species complex.

BACKGROUND: Attempts over the last three decades to reconstruct the phylogenetic history of the Anopheles gambiae species complex have been important for developing better strategies to control malaria transmission. METHODOLOGY: We used fingerprint genotyping data from 414 field-collected female mosquitoes at 42 microsatellite loci to infer the evolutionary relationships of four species in the A. gambiae complex, the two major malaria vectors A. gambiae sensu stricto (A. gambiae s.s.) and A. arabiensis, as well as two minor vectors, A. merus and A. melas. PRINCIPAL FINDINGS: We identify six taxonomic units, including a clear separation of West and East Africa A. gambiae s.s. S molecular forms. We show that the phylogenetic relationships vary widely between different genomic regions, thus demonstrating the mosaic nature of the genome of these species. The two major malaria vectors are closely related and closer to A. merus than to A. melas at the genome-wide level, which is also true if only autosomes are considered. However, within the Xag inversion region of the X chromosome, the M and two S molecular forms are most similar to A. merus. Near the X centromere, outside the Xag region, the two S forms are highly dissimilar to the other taxa. Furthermore, our data suggest that the centromeric region of chromosome 3 is a strong discriminator between the major and minor malaria vectors. CONCLUSIONS: Although further studies are needed to elucidate the basis of the phylogenetic variation among the different regions of the genome, the preponderance of sympatric admixtures among taxa strongly favor introgression of different genomic regions between species, rather than lineage sorting of ancestral polymorphism, as a possible mechanism.

The mosquito genome--a breakthrough for public health.

The Anopheles gambiae genome sequence will accelerate identification of new insect vector target genes leading to improved strategies for malaria control.