Urban and suburban malaria in Rondônia (Brazilian Western Amazon) II. Perennial transmissions with high anopheline densities are associated with human environmental changes

Longitudinal entomological surveys were performed in Vila Candelária and adjacent rural locality of Bate Estaca concomitantly with a clinical epidemiologic malaria survey. Vila Candelária is a riverside periurban neighborhood of Porto Velho, capital of the state of Rond?nia in the Brazilian Amazon. High anopheline densities were found accompanying the peak of rainfall, as reported in rural areas of the region. Moreover, several minor peaks of anophelines were recorded between the end of the dry season and the beginning of the next rainy season. These secondary peaks were related to permanent anopheline breeding sites resulting from human activities. Malaria transmission is, therefore, observed all over the year. In Vila Candelária, the risk of malaria infection both indoors and outdoors was calculated as being 2 and 10/infecting bites per year per inhabitant respectively. Urban malaria in riverside areas was associated with two factors: (1) high prevalence of asymptomatic carriers in a stable human population and (2) high anopheline densities related to human environmental changes. This association is probably found in other Amazonian urban and suburban communities. The implementation of control measures should include environmental sanitation and better characterization of the role of asymptomatic carriers in malaria transmission.     

Changing patterns of malaria epidemiology between 2002 and 2010 in Western Kenya: the fall and rise of malaria

Background

The impact of insecticide treated nets (ITNs) on reducing malaria incidence is shown mainly through data collection from health facilities. Routine evaluation of long-term epidemiological and entomological dynamics is currently unavailable. In Kenya, new policies supporting the provision of free ITNs were implemented nationwide in June 2006. To evaluate the impacts of ITNs on malaria transmission, we conducted monthly surveys in three sentinel sites with different transmission intensities in western Kenya from 2002 to 2010.

Methods and Findings

Longitudinal samplings of malaria parasite prevalence in asymptomatic school children and vector abundance in randomly selected houses were undertaken monthly from February 2002. ITN ownership and usage surveys were conducted annually from 2004 to 2010. Asymptomatic malaria parasite prevalence and vector abundances gradually decreased in all three sites from 2002 to 2006, and parasite prevalence reached its lowest level from late 2006 to early 2007. The abundance of the major malaria vectors, Anopheles funestus and An. gambiae, increased about 5–10 folds in all study sites after 2007. However, the resurgence of vectors was highly variable between sites and species. By 2010, asymptomatic parasite prevalence in Kombewa had resurged to levels recorded in 2004/2005, but the resurgence was smaller in magnitude in the other sites. Household ITN ownership was at 50–70% in 2009, but the functional and effective bed net coverage in the population was estimated at 40.3%, 49.4% and 28.2% in 2010 in Iguhu, Kombewa, and Marani, respectively.

Conclusion

The resurgence in parasite prevalence and malaria vectors has been observed in two out of three sentinel sites in western Kenya despite a high ownership of ITNs. The likely factors contributing to malaria resurgence include reduced efficacy of ITNs, insecticide resistance in mosquitoes and lack of proper use of ITNs. These factors should be targeted to avoid further resurgence of malaria transmission.     

Asymptomatic infection with Plasmodium falciparum and Plasmodium vivax in the Brazilian Amazon Basin: to treat or not to treat?

In this study, we determined whether the treatment of asymptomatic parasites carriers (APCs), which are frequently found in the riverside localities of the Brazilian Amazon that are highly endemic for malaria, would decrease the local malaria incidence by decreasing the overall pool of parasites available to infect mosquitoes. In one village, the treatment of the 19 Plasmodium falciparum-infected APCs identified among the 270 residents led to a clear reduction (Z = -2.39, p = 0.017) in the incidence of clinical cases, suggesting that treatment of APCs is useful for controlling falciparum malaria. For vivax malaria, 120 APCs were identified among the 716 residents living in five villages. Comparing the monthly incidence of vivax malaria in two villages where the APCs were treated with the incidence in two villages where APCs were not treated yielded contradictory results and no clear differences in the incidence were observed (Z = -0.09, p = 0.933). Interestingly, a follow-up study showed that the frequency of clinical relapse in both the treated and untreated APCs was similar to the frequency seen in patients treated for primary clinical infections, thus indicating that vivax clinical immunity in the population is not species specific but only strain specific.     

Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia

Recent studies have shown that Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named “Focused Screening and Treatment” (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as “high risk” and “low risk” based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow.     

Cross-sectional study defines difference in malaria morbidity in two Yanomami communities on Amazonian boundary between Brazil and Venezuela

It is well established that immunity to malaria is short-lived and is maintained by the continuous contact with the parasite. We now show that the stable transmission of malaria in Yanomami Amerindian communities maintains a degree of immunity in the exposed population capable to reduce prevalence and morbidity of malaria. We examined 508 Yanomami Amerindians living along Orinoco (407) and Mucajaí (101) rivers, on the Venezuelan and Brazilian Amazon region, respectively. At Orinoco villages, malaria was hyperendemic and presented stable transmission, while at Mucajaí villages it was mesoendemic and showed unstable transmission. The frequency of Plasmodium vivax and P. falciparum was roughly comparable in Venezuelan and Brazilian communities. Malaria presented different profiles at Orinoco and Mucajaí villages. In the former communities, malaria showed a lower prevalence (16% x 40.6%), particularly among those over 10 years old (5.2% x 34.8%), a higher frequency of asymptomatic cases (38.5% x 4.9%), and a lower frequency of cases of severe malaria (9.2% x 36.5%). Orinoco villagers also showed a higher reactivity of the immune system, measured by the frequency of splenomegaly (72.4% x 29.7%) and by the splenic index (71.4% over level 1 x 28.6), and higher prevalence (91.1% x 72.1%) and mean titer (1243 x 62) of antiplasmodial IgG antibodies, as well as a higher prevalence (77.4% x 24.7%) and mean titer (120 x 35) of antiplasmodial IgM antibodies. Our findings show that in isolated Yanomami communities the stability of malaria transmission, and the consequent continuous activation of the immune system of the exposed population, leads to the reduction of malaria prevalence and morbidity.     

A new challenge for malaria control in Brazil: asymptomatic Plasmodium infection--a review

The evolution of malaria in Brazil, its morbidity, the malaria control programs, and the new challenges for these programs in the light of the emergence of asymptomatic infection in the Amazon region of Brazil were reviewed. At least six Brazilian research groups have demonstrated that asymptomatic infection by Plasmodium is an important impediment to malaria control, among mineral prospectors in Mato Grosso and riverside communities in Rond?nia and, in our group, in the middle and upper reaches of the Negro river, in the state of Amazonas. Likewise, other researchers have studied the problem among indigenous communities in the Colombian, Peruvian, and Venezuelan parts of the Amazon basin, adjacent to Brazil. The frequency of positive results from the polymerase chain reaction (PCR) among asymptomatic individuals has ranged from 20.4 to 49.5%, and the presence of Plasmodium in the thick blood smears, from 4.2 to 38.5%. Infection with Anopheles darlingi has also been demonstrated by xenodiagnosis among asymptomatic patients with positive PCR results. If a mean of 25% is taken for the asymptomatic infection caused by Plasmodium sp. in the Amazon region of Brazil, malaria control will be difficult to achieve in that region with the measures currently utilized for such control.     

Routine delivery of artemisinin-based combination treatment at fixed health facilities reduces malaria prevalence in Tanzania: an observational study

ABSTRACT: BACKGROUND: Artemisinin-based combination therapy (ACT) has been promoted as a means to reduce malaria transmission due to their ability to kill both asexual blood stages of malaria parasites, which sustain infections over long periods and the immature derived sexual stages responsible for infecting mosquitoes and onward transmission. Early studies reported a temporal association between ACT introduction and reduced malaria transmission in a number of ecological settings. However, these reports have come from areas with low to moderate malaria transmission, been confounded by the presence of other interventions or environmental changes that may have reduced malaria transmission, and have not included a comparison group without ACT. This report presents results from the first large-scale observational study to assess the impact of case management with ACT on population-level measures of malaria endemicity in an area with intense transmission where the benefits of effective infection clearance might be compromised by frequent and repeated re-infection. METHODS: A pre-post observational study with a non-randomized comparison group was conducted at two sites in Tanzania. Both sites used sulphadoxine-pyrimethamine (SP) monotherapy as a first-line anti-malarial from mid-2001 through 2002. In 2003, the ACT, artesunate (AS) coadministered with SP (AS + SP), was introduced in all fixed health facilities in the intervention site, including both public and registered non-governmental facilities. Population-level prevalence of Plasmodium falciparum asexual parasitaemia and gametocytaemia were assessed using light microscopy from samples collected during representative household surveys in 2001, 2002, 2004, 2005 and 2006. FINDINGS: Among 37,309 observations included in the analysis, annual asexual parasitaemia prevalence in persons of all ages ranged from 11% to 28% and gametocytaemia prevalence ranged from <1% to 2% between the two sites and across the five survey years. Amultivariable logistic regression model was fitted to adjust for age, socioeconomic status, bed net use and rainfall. In the presence of consistently high coverage and efficacy of SP monotherapy and AS + SP in the comparison and intervention areas, the introduction of ACT in the intervention site was associated with a modest reduction in the adjusted asexual parasitaemia prevalence of 5 percentage-points or 23% (p < 0.0001) relative to the comparison site. Gametocytaemia prevalence did not differ significantly (p = 0.30). Interpretation The introduction of ACT at fixed health facilities only modestly reduced asexual parasitaemia prevalence. ACT is effective for treatment of uncomplicated malaria and should have substantial public health impact on morbidity and mortality, but is unlikely to reduce malaria transmission substantially in much of sub-Saharan Africa where individuals are rapidly reinfected.     

A historical perspective on malaria control in Brazil

Malaria has always been an important public health problem in Brazil. The early history of Brazilian malaria and its control was powered by colonisation by Europeans and the forced relocation of Africans as slaves. Internal migration brought malaria to many regions in Brazil where, given suitableAnopheles mosquito vectors, it thrived. Almost from the start, officials recognised the problem malaria presented to economic development, but early control efforts were hampered by still developing public health control and ignorance of the underlying biology and ecology of malaria. Multiple regional and national malaria control efforts have been attempted with varying success. At present, the Amazon Basin accounts for 99% of Brazil’s reported malaria cases with regional increases in incidence often associated with large scale public works or migration. Here, we provide an exhaustive summary of primary literature in English, Spanish and Portuguese regarding Brazilian malaria control. Our goal was not to interpret the history of Brazilian malaria control from a particular political or theoretical perspective, but rather to provide a straightforward, chronological narrative of the events that have transpired in Brazil over the past 200 years and identify common themes.     

Asymptomatic Plasmodium vivax malaria in the Brazilian Amazon: Submicroscopic parasitemic blood infects Nyssorhynchus darlingi

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria.     

Identifying residual foci of Plasmodium falciparum infections for malaria elimination: the urban context of Khartoum, Sudan

Background

Identifying the location and size of residual foci of infections is critical where malaria elimination is the primary goal. Here the spatial heterogeneity of Plasmodium falciparum infections within the urban extent of Khartoum state in Sudan is investigated using data from cross-sectional surveys undertaken from 1999 to 2008 to inform the Khartoum Malaria Free Initiative (KMFI).

Methods

From 1999–2008 the KMFI undertook cross-sectional surveys of 256 clusters across 203 random samples of residential blocks in the urban Khartoum state in September of each year. Within sampled blocks, at least five persons, including at least one child under the age of five years, were selected from each household. Blood smears were collected from the sampled individuals to examine the presence of P. falciparum parasites. Residential blocks were mapped. Data were analysed for spatial clustering using the Bernoulli model and the significance of clusters were tested using the Kulldorff scan statistic.

Results

A total of 128,510 malaria slide examinations were undertaken during the study period. In 1999, overall prevalence was 2.5%, rising to 3.2% in 2000 and consistently staying below 1% in subsequent years. From 2006, over 90% of all surveyed clusters reported no infections. Spatial clustering of infections was present in each year but not statistically significant in the years 2001, 2002, 2004 and 2008. Spatial clusters of high infection were often located at the junction of the Blue and White Niles.

Conclusion

Persisting foci of malaria infection in Khartoum are likely to distort wide area assessments and disproportionately affect future transmission within the city limits. Improved investments in surveillance that combines both passive and active case detection linked to a geographic information system and a more detailed analysis of the location and stability of foci should be undertaken to facilitate and track malaria elimination in the state of Khartoum.     

Population and Land Use Effects on Malaria Prevalence in the Southern Brazilian Amazon

Malaria prevalence has been one of the most dramatic outcomes of the occupation of the Brazilian Amazon as exemplified by Northern Mato Grosso, one of the areas of highest malaria prevalence in the Americas in the early 1990s. This paper associates the dynamicsof high malaria prevalence in Northern Mato Grosso with three land uses—small-scalegold mining (garimpos), agricultural colonization/cattle ranching (rural), and urban activities—and their related population characteristics, which constitute riskprofiles. Furthermore, spatial proximity and population mobility between (a) garimpos and new rural settlements and (b) older rural settlements or urban areas are key factors explaining malaria diffusion throughout the region. The paper identifies and characterizes populations at high malaria risk and the effects of land use types on malaria diffusion, providing policymakers with information for regional and local policies to control malaria and minimize its effects on Amazonian populations.     

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.     

Effect of artesunate-mefloquine fixed-dose combination in malaria transmission in amazon basin communities

ABSTRACT: BACKGROUND: Studies in South-East Asia have suggested that early diagnosis and treatment with artesunate (AS) and mefloquine (MQ) combination therapy may reduce the transmission of Plasmodium falciparum malaria and the progression of MQ resistance. METHODS: The effectiveness of a fixed-dose combination of AS and MQ (ASMQ) in reducing malaria transmission was tested in isolated communities of the Jurua valley in the Amazon region. Priority municipalities within the Brazilian Legal Amazon area were selected according to pre-specified criteria. Routine national malaria control programmatic procedures were followed. Existing health structures were reinforced and health care workers were trained to treat with ASMQ all confirmed falciparum malaria cases that match inclusion criteria. A local pharmacovigilance structure was implemented. Incidence of malaria and hospitalizations were recorded two years before, during, and after the fixed-dose ASMQ intervention. In total, between July 2006 and December 2008, 23,845 patients received ASMQ. Two statistical modelling approaches were applied to monthly time series of P. falciparum malaria incidence rates, P. falciparum/Plasmodium vivax infection ratio, and malaria hospital admissions rates. All the time series ranged from January 2004 to December 2008, whilst the intervention period span from July 2006 to December 2008. RESULTS: The ASMQ intervention had a highly significant impact on the mean level of each time series, adjusted for trend and season, of 0.34 (95%CI 0.20 [EN DASH] 0.58) for the P. falciparum malaria incidence rates, 0.67 (95%CI 0.50 [EN DASH] 0.89) for the P. falciparum/P. vivax infection ratio, and 0.53 (95%CI 0.41 [EN DASH] 0.69) for the hospital admission rates. There was also a significant change in the seasonal (or monthly) pattern of the time series before and after intervention, with the elimination of the malaria seasonal peak in the rainy months of the years following the introduction of ASMQ. No serious adverse events relating to the use of fixed-dose ASMQ were reported. CONCLUSIONS: In the remote region of the Jurua valley, the early detection of malaria by health care workers and treatment with fixed-dose ASMQ was feasible and efficacious, and significantly reduced the incidence and morbidity of P. falciparum malaria.     

The search for new antimalarial drugs from plants used to treat fever and malaria or plants ramdomly selected: a review.

In this review we discuss the ongoing situation of human malaria in the Brazilian Amazon, where it is endemic causing over 610,000 new acute cases yearly, a number which is on the increase. This is partly a result of drug resistant parasites and new antimalarial drugs are urgently needed. The approaches we have used in the search of new drugs during decades are now reviewed and include ethnopharmocology, plants randomly selected, extracts or isolated substances from plants shown to be active against the blood stage parasites in our previous studies. Emphasis is given on the medicinal plant Bidens pilosa, proven to be active against the parasite blood stages in tests using freshly prepared plant extracts. The anti-sporozoite activity of one plant used in the Brazilian endemic area to prevent malaria is also described, the so called "Indian beer" (Ampelozizyphus amazonicus, Rhamnaceae). Freshly prepared extracts from the roots of this plant were totally inactive against blood stage parasites, but active against sporozoites of Plasmodium gallinaceum or the primary exoerythrocytic stages reducing tissue parasitism in inoculated chickens. This result will be of practical importance if confirmed in mammalian malaria. Problems and perspectives in the search for antimalarial drugs are discussed as well as the toxicological and clinical trials to validate some of the active plants for public health use in Brazil.     

Enhanced understanding of infectious diseases by fusing multiple datasets: a case study on malaria in the Western Brazilian Amazon region

Background

A common challenge to the study of several infectious diseases consists in combining limited cross-sectional survey data, collected with a more sensitive detection method, with a more extensive (but biased) syndromic sentinel surveillance data, collected with a less sensitive method. Our article describes a novel modeling framework that overcomes this challenge, resulting in enhanced understanding of malaria in the Western Brazilian Amazon.

Methodology/Principal Findings

A cohort of 486 individuals was monitored using four cross-sectional surveys, where all participants were sampled regardless of symptoms (aggressive-active case detection), resulting in 1,383 microscopy and 1,400 polymerase chain reaction tests. Data on the same individuals were also obtained from the local surveillance facility (i.e., passive and active case detection), totaling 1,694 microscopy tests. Our model accommodates these multiple pathogen and case detection methods. This model is shown to outperform logistic regression in terms of interpretability of its parameters, ability to recover the true parameter values, and predictive performance. We reveal that the main infection determinant was the extent of forest, particularly during the rainy season and in close proximity to water bodies, and participation on forest activities. We find that time residing in Acrelandia (as a proxy for past malaria exposure) decreases infection risk but surprisingly increases the likelihood of reporting symptoms once infected, possibly because non-naïve settlers are only susceptible to more virulent Plasmodium strains. We suggest that the search for asymptomatic carriers should focus on those at greater risk of being infected but lower risk of reporting symptoms once infected.

Conclusions/Significance

The modeling framework presented here combines cross-sectional survey data and syndromic sentinel surveillance data to shed light on several aspects of malaria that are critical for public health policy. This framework can be adapted to enhance inference on infectious diseases whenever asymptomatic carriers are important and multiple datasets are available.     

Survivorship of Anopheles darlingi (Diptera: Culicidae) in relation with malaria incidence in the Brazilian Amazon

We performed a longitudinal study of adult survival of Anopheles darlingi, the most important vector in the Amazon, in a malarigenous frontier zone of Brazil. Survival rates were determined from both parous rates and multiparous dissections. Anopheles darlingi human biting rates, daily survival rates and expectation of life where higher in the dry season, as compared to the rainy season, and were correlated with malaria incidence. The biting density of mosquitoes that had survived long enough for completing at least one sporogonic cycle was related with the number of malaria cases by linear regression. Survival rates were the limiting factor explaining longitudinal variations in Plasmodium vivax malaria incidence and the association between adult mosquito survival and malaria was statistically significant by logistic regression (P<0.05). Survival rates were better correlated with malaria incidence than adult mosquito biting density. Mathematical modeling showed that P. falciparum and P. malariae were more vulnerable to changes in mosquito survival rates because of longer sporogonic cycle duration, as compared to P. vivax, which could account for the low prevalence of the former parasites observed in the study area. Population modeling also showed that the observed decreases in human biting rates in the wet season could be entirely explained by decreases in survival rates, suggesting that decreased breeding did not occur in the wet season, at the sites where adult mosquitoes were collected. For the first time in the literature, multivariate methods detected a statistically significant inverse relation (P<0.05) between the number of rainy days per month and daily survival rates, suggesting that rainfall may cause adult mortality.     

Mosquito anthropophily: implications on malaria transmission in the Northern Brazilian Amazon

Seasonal variation of adults and larvae of anophelines was studied during 2003 and 2004 in Roraima State, located in the Northern region of Brazilian Amazon. Species diversity increased with distance of capture to human dwellings. Greater diversity was found in extradomiciliary collections than in peridomiciliary or intradomiciliary. A significant association between Anopheles darlingi Root and An. albitarsis (s.l.) Arribálzaga (Diptera: Culicidae) breeding sites and the proximity to human dwellings was observed. Malaria Sporozoite Antigen Panel Assay (VectestTM Malaria) indicated An. albitarsis s.l. as one of the local vectors in the studied area. In this study, an index to describe the anthropophilic behavior of each anopheline species is proposed.     

Surveillance of vivax malaria vectors and civilian patients for malaria high-risk areas in northern Gyeonggi and Gangwon Provinces near the demilitarized zone,Republic of Korea, 2003–2006

After re-emergence of malaria in 1993, a continued increase in Plasmodium vivax cases was observed from 1993 to 2006 in northern Gyeonggi and Gangwon Provinces adjacent to the demilitarized zone separating North from South Korea. Annual parasite incidence per 1000 people ranged from 0.33 in 2004 to 0.89 in 2006. While malaria case rates declined (22.6%) in 2004, they increased 75.1% in 2005 and 51.7% in 2006 from the previous years. An initial incorrect diagnosis of 46.8% of malaria cases as common cold resulted in a mean delay of 1.3 days for the detection malarial parasites. Of the total cases, 10.2% from December to May were due to latent intrinsic incubation infections acquired the previous malaria season and the rest of the cases from June to November were either latent or short incubation infections. Overall, the peak anopheline population occurred from July to September, resulting in a similar peak in malaria cases. While malaria cases increased during 2005–2006, anopheline populations, based on trap indices, were not significantly different during 4 years of surveillance. To decrease the malaria patient infective period to mosquitoes, public health centers in Paju and Cheorwon in 2006 prescribed chloroquine + primaquine at days 0–3 after initial malaria diagnosis followed by an additional 11 days of primaquine (early primaquine treatment), rather than chloroquine on days 0–3 and primaquine on days 4–17 (delayed primaquine treatment). A reduction in the malaria parasite incidence during 2007 was recorded for the two locations offering the early primaquine treatment relative to other locations using the delayed primaquine treatment.     

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rond?nia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.     

Epidemiology of Disappearing Plasmodium vivax Malaria: A Case Study in Rural Amazonia

Background

New frontier settlements across the Amazon Basin pose a major challenge for malaria elimination in Brazil. Here we describe the epidemiology of malaria during the early phases of occupation of farming settlements in Remansinho area, Brazilian Amazonia. We examine the relative contribution of low-density and asymptomatic parasitemias to the overall Plasmodium vivax burden over a period of declining transmission and discuss potential hurdles for malaria elimination in Remansinho and similar settings.

Methods

Eight community-wide cross-sectional surveys, involving 584 subjects, were carried out in Remansinho over 3 years and complemented by active and passive surveillance of febrile illnesses between the surveys. We used quantitative PCR to detect low-density asexual parasitemias and gametocytemias missed by conventional microscopy. Mixed-effects multiple logistic regression models were used to characterize independent risk factors for P. vivax infection and disease.

Principal Findings/Conclusions

P. vivax prevalence decreased from 23.8% (March–April 2010) to 3.0% (April–May 2013), with no P. falciparum infections diagnosed after March–April 2011. Although migrants from malaria-free areas were at increased risk of malaria, their odds of having P. vivax infection and disease decreased by 2–3% with each year of residence in Amazonia. Several findings indicate that low-density and asymptomatic P. vivax parasitemias may complicate residual malaria elimination in Remansinho: (a) the proportion of subpatent infections (i.e. missed by microscopy) increased from 43.8% to 73.1% as P. vivax transmission declined; (b) most (56.6%) P. vivax infections were asymptomatic and 32.8% of them were both subpatent and asymptomatic; (c) asymptomatic parasite carriers accounted for 54.4% of the total P. vivax biomass in the host population; (d) over 90% subpatent and asymptomatic P. vivax had PCR-detectable gametocytemias; and (e) few (17.0%) asymptomatic and subpatent P. vivax infections that were left untreated progressed to clinical disease over 6 weeks of follow-up and became detectable by routine malaria surveillance.