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OBJECTIVE To measure pyrethroid susceptibility in populations of malaria vectors and nuisance-biting mosquitoes in Tanzania and to test the biological efficacy of current insecticide formulations used for net treatment. METHODS Anopheles gambiae Giles s.l., An. funestus Giles s.l. and Culex quinquefasciatus Say were collected during three national surveys and two insecticide-treated net (ITN) studies in Tanzania. Knockdown effect and mortality were measured in standard WHO susceptibility tests and ball-frame bio-efficacy tests. Test results from 1999 to 2004 were compared to determine trends in resistance development. RESULTS Anopheles gambiae s.l. and An. funestus s.l. were highly susceptible to permethrin (range 87-100%) and deltamethrin (consistently 100%) in WHO tests in 1999 and 2004, while Culex quinquefasciatus susceptibility to these pyrethroids was much lower (range 7-100% and 0-84% respectively). Efficacy of pyrethroid-treated nets was similarly high against An. gambiae s.l. and An. funestus s.l. (range 82-100%) while efficacy against Cx. quinquefasciatus was considerably lower (range 2-100%). There was no indication of development of resistance in populations of An. gambiae s.l. or An. funestus s.l. where ITNs have been extensively used; however, susceptibility of nuisance-biting Cx. quinquefasciatus mosquitoes declined in some areas between 1999 and 2004. CONCLUSION The sustained pyrethroid susceptibility of malaria vectors in Tanzania is encouraging for successful malaria control with ITNs. Continued monitoring is essential to ensure early resistance detection, particularly in areas with heavy agricultural or public health use of insecticides where resistance is likely to develop. Widespread low susceptibility of nuisance-biting Culex mosquitoes to ITNs raises concern for user acceptance of nets.

OBJECTIVE: Recently developed molecular gametocyte detection techniques have shown that submicroscopic Plasmodium falciparum gametocytes are common in symptomatic patients and can infect mosquitoes. The relevance for the infectious reservoir of malaria in the general population remains unknown. In this study, we investigated submicroscopic asexual parasitaemia and gametocytaemia in inhabitants of an area of hypoendemic and seasonal malaria in Tanzania. METHODS: Two cross-sectional malariometric surveys were conducted in the dry and wet seasons of 2005 in villages in lower Moshi, Tanzania. Finger prick blood samples were taken to determine the prevalence of P. falciparum parasites by microscopy, rapid diagnostic test and real-time nucleic acid sequence-based amplification (QT-NASBA). RESULTS: 2752 individuals participated in the surveys, of whom 1.9% (51/2721) had microscopically confirmed asexual parasites and 0.4% (10/2721) had gametocytes. In contrast, QT-NASBA revealed that 32.5% (147/453) of the individuals harboured asexual parasites and 15.0% (68/453) had gametocytes. No age dependency or seasonality was observed in submicroscopic parasite carriage. DISCUSSION: Molecular detection techniques reveal that carriage of submicroscopic asexual parasite and gametocyte densities is relatively common in this low transmission area. Submicroscopic gametocytaemia is likely to be responsible for maintaining malarial transmission in the study area.

BACKGROUND: Insecticide-treated nets are the primary method of preventing malaria. To remain effective, the pyrethroid insecticide must withstand multiple washes over the lifetime of the net. ICON(®) Maxx is a 'dip-it-yourself' kit for long-lasting treatment of polyester nets. The twin-sachet kit contains a slow-release capsule suspension of lambda-cyhalothrin plus binding agent. To determine whether ICON Maxx meets the standards required by the World Health Organization Pesticide Evaluation Scheme (WHOPES), the efficacy and wash fastness of ICON Maxx was evaluated against wild, free-flying anopheline mosquitoes. METHODS: ICON Maxx was subjected to bioassay evaluation and experimental hut trial against pyrethroid-susceptible Anopheles gambiae, Anopheles arabiensis and Anopheles funestus. Mosquito mortality, blood feeding inhibition and personal protection were compared between untreated nets, conventional lambda-cyhalothrin treated nets (CTN) washed either four times (cut-off threshold) or 20 times, and ICON Maxx-treated nets either unwashed or washed 20 times. RESULTS: In bioassay, ICON Maxx demonstrated superior wash resistance to the CTN. In the experimental hut trial, ICON Maxx killed 75 % of An. funestus, 71 % of An. gambiae and 47 % of An. arabiensis when unwashed and 58, 66 and 42 %, respectively, when 20 times washed. The CTN killed 52 % of An. funestus, 33 % of An. gambiae and 30 % of An. arabiensis when washed to the cut-off threshold of four washes and 40, 40 and 36 %, respectively, when 20 times washed. Percentage mortality with ICON Maxx 20 times washed was similar (An. funestus) or significantly higher (An. gambiae, An. arabiensis) than with CTN washed to the WHOPES cut-off threshold. Blood-feeding inhibition with ICON Maxx 20 times washed was similar to the CTN washed to cut-off for all three species. Personal protection was significantly higher with ICON Maxx 20 times washed (66-79 %) than with CTN washed to cut-off (48-60 %). CONCLUSIONS: Nets treated with ICON Maxx and washed 20 times met the approval criteria set by WHOPES for Phase II trials in terms of mortality and blood-feeding inhibition. This finding raises the prospect of conventional polyester nets and other materials being made long-lastingly insecticidal through simple dipping in community or home, and thus represents a major advance over conventional pyrethroid treatments.

Contains fulltext : 81523.pdf (Publisher’s version ) (Open Access) Malaria infection induces oxidative stress in the host cells. Antioxidant enzymes such as glutathione S-transferases (GSTs) are responsible for fighting reactive oxygen species and reduction of oxidative stress. Common GST polymorphisms have been associated with susceptibility to different diseases whose pathologies involve oxidative stress. In this study, we tested the hypothesis that GST polymorphisms that lead to reduced or lack of enzyme activity are associated with severe Plasmodium falciparum malarial anemia. We studied the genotypic distribution of GSTM1, GSTT1, and GSTP1 polymorphisms between mild malaria (N = 107) and severe malarial anemia (N = 50) in Tanzanian children. We did not find a significant relationship with the GSTT1 polymorphism. GSTM1-null was higher in the severe malaria anemia group but the difference was not significant (P = 0.08). However, a significant association of GSTP1 I105V genotype with severe malarial anemia was discovered (26.0% against 10.3% mild malaria, P = 0.004). We concluded that GSTP1 and possibly GSTM1 may protect against severe falciparum malaria in children.

Background Plasmodium falciparum resistance to anti-malarial drugs remains a major obstacle to the control of malaria. In 2001 Tanzania replaced chloroquine (CQ) with sulphadoxine-pyrimethamine (SP) as first-line drug, which in turn was replaced by artemisinin combination therapy in 2006. SP has however, continued to be used in intermittent preventive treatment of malaria in pregnancy (IPTp) despite reports of high levels of resistance to SP due to the lack of alternatives to SP for IPTp. Recent reports have indicated recovery of CQ-susceptibility in Malawi, Kenya, Mozambique, and Tanzania based on the prevalence of wild types at codon 76 of the Pfcrt gene in indigenous P. falciparum populations. The current prevalence of this Pfcrt- 76 CQ resistance marker from six regions of Tanzania mainland is hereby reported. Methods DNA extracted from filter-paper dried blood spots and rapid diagnostics kit strips collected from finger-prick blood were used to genotype the Pfcrt-76 resistance marker using PCR-RFLP. Data from previously published studies were used to generate CQ susceptibility recovery trends using logistic regression model. Results Seven hundred and forty one (741) samples were genotyped. The current frequency of the CQ-susceptible Pfcrt-K76 was above 92% and did not differ between regions in Tanzania (χ 2 = 2.37; p = 0.795). The K76 allelic prevalence was between 85.7 and 93% in regions (χ 2 = 7.88, p = 0.163). The CQ resistance recovery trends showed regional variability that may be caused by differences in malaria transmission intensity, but overall the trends converge as the susceptibility levels in all regions approach >90%. Conclusions CQ withdrawal in Tanzania has resulted into >90% recovery of susceptibility in ten years of withdrawal. These findings are in support of the search for CQ-based combination drugs as a possible future alternative to SP for IPTp in places where full recovery of CQ-susceptibility will be evident.

Background Effective mass drug administration (MDA) with anti-malarial drugs can clear the human infectious reservoir for malaria and thereby interrupt malaria transmission. The likelihood of success of MDA depends on the intensity and seasonality of malaria transmission, the efficacy of the intervention in rapidly clearing all malaria parasite stages and the degree to which symptomatic and asymptomatic parasite carriers participate in the intervention. The impact of MDA with the gametocytocidal drug combination sulphadoxine-pyrimethamine (SP) plus artesunate (AS) plus primaquine (PQ, single dose 0.75 mg/kg) on malaria transmission was determined in an area of very low and seasonal malaria transmission in northern Tanzania. Methods In a cluster-randomized trial in four villages in Lower Moshi, Tanzania, eight clusters (1,110 individuals; cluster size 47- 209) were randomized to observed treatment with SP+AS+PQ and eight clusters (2,347 individuals, cluster size 55- 737) to treatment with placebo over three days. Intervention and control clusters were 1km apart; households that were located between clusters were treated as buffer zones where all individuals received SP+AS+PQ but were not selected for the evaluation. Passive case detection was done for the entire cohort and active case detection in 149 children aged 1-10 year from the intervention arm and 143 from the control arm. Four cross-sectional surveys assessed parasite carriage by microscopy and molecular methods during a five-month follow-up period. Results The coverage rate in the intervention arm was 93.0% (1,117/1,201). Parasite prevalence by molecular detection methods was 2.2-2.7% prior to the intervention and undetectable during follow-up in both the control and intervention clusters. None of the slides collected during cross-sectional surveys had microscopically detectable parasite densities. Three clinical malaria episodes occurred in the intervention (n = 1) and control clusters (n = 2). Conclusions This study illustrates the possibility to achieve high coverage with a three-day intervention but also the difficulty in defining suitable outcome measures to evaluate interventions in areas of very low malaria transmission intensity. The decline in transmission intensity prior to the intervention made it impossible to assess the impact of MDA in the chosen study setting. Trial Registration ClinicalTrials.gov: NCT00509015

BACKGROUND: Intermittent preventive treatment in infants (IPTi) is a new malaria control tool. However, it is uncertain whether IPTi works mainly through chemoprophylaxis or treatment of existing infections. Understanding the mechanism is essential for development of replacements for sulfadoxine-pyrimethamine (SP) where it is no longer effective. This study investigated how protection against malaria given by SP, chlorproguanil-dapsone (CD) and mefloquine (MQ), varied with time since administration of IPTi. METHODS AND FINDINGS: A secondary analysis of data from a randomised, placebo-controlled trial in an area of high antifolate resistance in Tanzania was conducted. IPTi using SP, CD, MQ or placebo was given to 1280 infants at 2, 3 and 9 months of age. Poisson regression with random effects to adjust for potential clustering of malaria episodes within children was used to calculate incidence rate ratios for clinical malaria in defined time strata following IPTi. The short-acting antimalarial CD gave no protection against clinical malaria, whereas long-acting MQ gave two months of substantial protection (protective efficacy (PE) 73.1% (95% CI: 23.9, 90.5) and 73.3% (95% CI: 0, 92.9) in the first and second month respectively). SP gave some protection in the first month after treatment (PE 64.5% (95% CI: 10.6, 85.9)) although it did not reduce the incidence of malaria up to 12 months of age. There was no evidence of either long-term protection or increased risk of malaria for any of the regimens. CONCLUSION: Post-treatment chemoprophylaxis appears to be the main mechanism by which IPTi protects children against malaria. Long-acting antimalarials are therefore likely to be the most effective drugs for IPTi, but as monotherapies could be vulnerable to development of drug resistance. Due to concerns about tolerability, the mefloquine formulation used in this study is not suitable for IPTi. Further investigation of combinations of long-acting antimalarials for IPTi is needed. TRIAL REGISTRATION: Clinicaltrials.gov NCT00158574.

Item does not contain fulltext BACKGROUND: Variation in the risk of malaria within populations is a frequently described but poorly understood phenomenon. This heterogeneity creates opportunities for targeted interventions but only if hot spots of malaria transmission can be easily identified. METHODS: We determined spatial patterns in malaria transmission in a district in northeastern Tanzania, using malaria incidence data from a cohort study involving infants and household-level mosquito sampling data. The parasite prevalence rates and age-specific seroconversion rates (SCRs) of antibodies against Plasmodium falciparum antigens were determined in samples obtained from people attending health care facilities. RESULTS: Five clusters of higher malaria incidence were detected and interpreted as hot spots of transmission. These hot spots partially overlapped with clusters of higher mosquito exposure but could not be satisfactorily predicted by a probability model based on environmental factors. Small-scale local variation in malaria exposure was detected by parasite prevalence rates and SCR estimates for samples of health care facility attendees. SCR estimates were strongly associated with local malaria incidence rates and predicted hot spots of malaria transmission with 95% sensitivity and 85% specificity. CONCLUSIONS: Serological markers were able to detect spatial variation in malaria transmission at the microepidemiological level, and they have the potential to form an effective method for spatial targeting of malaria control efforts.

Summary Background Administration of sulfadoxine-pyrimethamine at times of vaccination—intermittent preventive treatment in infants (IPTi)—is a promising strategy to prevent malaria. However, rising resistance to this combination is a concern. We investigated a shortacting and longacting antimalarial drug as alternative regimens for IPTi. Methods We undertook a double-blind, placebo-controlled trial of IPTi in an area of high resistance to sulfadoxine-pyrimethamine at sites of moderate (n=1280 infants enrolled) and low (n=1139) intensity of malaria transmission in Tanzania. Infants aged 8–16 weeks were randomly assigned in blocks of 16 to sulfadoxine (250 mg) plus pyrimethamine (12·5 mg; n=319 in moderate-transmission and 283 in low-transmission sites), chlorproguanil (15 mg) plus dapsone (18·75 mg; n=317 and 285), mefloquine (125 mg; n=320 and 284), or placebo (n=320 and 284), given at the second and third immunisations for diphtheria, pertussis, and tetanus, and for measles. Research team and child were masked to treatment. Recruitment was stopped early at the low-transmission site because of low malaria incidence. The primary endpoint was protective efficacy against all episodes of clinical malaria at 2–11 months of age. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00158574. Findings All randomly assigned infants were analysed. At the moderate-transmission site, mefloquine had a protective efficacy of 38·1% (95% CI 11·8–56·5, p=0·008) against clinical malaria in infants aged 2–11 months, but neither sulfadoxine-pyrimethamine (−6·7%, −45·9 to 22·0) nor chlorproguanil-dapsone (10·8%, −24·6 to 36·1) had a protective effect. No regimen had any protective efficacy against anaemia or hospital admission. Mefloquine caused vomiting in 141 of 1731 (8%) doses given on day 1 (odds ratio vs placebo 5·50, 95% CI 3·56–8·46). More infants died in the chlorproguanil-dapsone and mefloquine groups (18 and 15, respectively) than in the sulfadoxine-pyrimethamine or placebo groups (eight deaths per group; p=0·05 for difference between chlorproguanil-dapsone and placebo). Interpretation IPTi with a longacting, efficacious drug such as mefloquine can reduce episodes of malaria in infants in a moderate-transmission setting. IPTi with sulfadoxine-pyrimethamine has no benefit in areas of very high resistance to this combination. The appropriateness of IPTi should be measured by the expected incidence of malaria and the efficacy, tolerability, and safety of the drug. Funding IPTi Consortium and the Gates Malaria Partnership.

Malaria infection induces oxidative stress in the host cells. Antioxidant enzymes such as glutathione S-transferases (GSTs) are responsible for fighting reactive oxygen species and reduction of oxidative stress. Common GST polymorphisms have been associated with susceptibility to different diseases whose pathologies involve oxidative stress. In this study, we tested the hypothesis that GST polymorphisms that lead to reduced or lack of enzyme activity are associated with severe Plasmodium falciparum malarial anemia. We studied the genotypic distribution of GSTM1, GSTT1, and GSTP1 polymorphisms between mild malaria (N = 107) and severe malarial anemia (N = 50) in Tanzanian children. We did not find a significant relationship with the GSTT1 polymorphism. GSTM1-null was higher in the severe malaria anemia group but the difference was not significant (P = 0.08). However, a significant association of GSTP1 I105V genotype with severe malarial anemia was discovered (26.0% against 10.3% mild malaria, P = 0.004). We concluded that GSTP1 and possibly GSTM1 may protect against severe falciparum malaria in children.

Background Sulphadoxine-pyrimethamine (SP) a widely used treatment for uncomplicated malaria and recommended for intermittent preventive treatment of malaria in pregnancy, is being investigated for intermittent preventive treatment of malaria in infants (IPTi). High levels of drug resistance to SP have been reported from north-eastern Tanzania associated with mutations in parasite genes. This study compared the in vivo efficacy of SP in symptomatic 6–59 month children with uncomplicated malaria and in asymptomatic 2–10 month old infants. Methodology and Principal Findings An open label single arm (SP) standard 28 day in vivo WHO antimalarial efficacy protocol was used in 6 to 59 months old symptomatic children and a modified protocol used in 2 to 10 months old asymptomatic infants. Enrolment was stopped early (87 in the symptomatic and 25 in the asymptomatic studies) due to the high failure rate. Molecular markers were examined for recrudescence, re-infection and markers of drug resistance and a review of literature of studies looking for the 581G dhps mutation was carried out. In symptomatic children PCR-corrected early treatment failure was 38.8% (95% CI 26.8–50.8) and total failures by day 28 were 82.2% (95% CI 72.5–92.0). There was no significant difference in treatment failures between asymptomatic and symptomatic children. 96% of samples carried parasites with mutations at codons 51, 59 and 108 in the dhfr gene and 63% carried a double mutation at codons 437 and 540. 55% carried a third mutation with the addition of a mutation at codon 581 in the dhps gene. This triple: triple haplotype maybe associated with earlier treatment failure. Conclusion In northern Tanzania SP is a failed drug for treatment and its utility for prophylaxis is doubtful. The study found a new combination of parasite mutations that maybe associated with increased and earlier failure. Trial Registration ClinicalTrials.gov NCT00361114

To measure pyrethroid susceptibility in populations of malaria vectors and nuisance-biting mosquitoes in Tanzania and to test the biological efficacy of current insecticide formulations used for net treatment.Anopheles gambiae Giles s.l., An. funestus Giles s.l. and Culex quinquefasciatus Say were collected during three national surveys and two insecticide-treated net (ITN) studies in Tanzania. Knockdown effect and mortality were measured in standard WHO susceptibility tests and ball-frame bio-efficacy tests. Test results from 1999 to 2004 were compared to determine trends in resistance development.Anopheles gambiae s.l. and An. funestus s.l. were highly susceptible to permethrin (range 87-100%) and deltamethrin (consistently 100%) in WHO tests in 1999 and 2004, while Culex quinquefasciatus susceptibility to these pyrethroids was much lower (range 7-100% and 0-84% respectively). Efficacy of pyrethroid-treated nets was similarly high against An. gambiae s.l. and An. funestus s.l. (range 82-100%) while efficacy against Cx. quinquefasciatus was considerably lower (range 2-100%). There was no indication of development of resistance in populations of An. gambiae s.l. or An. funestus s.l. where ITNs have been extensively used; however, susceptibility of nuisance-biting Cx. quinquefasciatus mosquitoes declined in some areas between 1999 and 2004.The sustained pyrethroid susceptibility of malaria vectors in Tanzania is encouraging for successful malaria control with ITNs. Continued monitoring is essential to ensure early resistance detection, particularly in areas with heavy agricultural or public health use of insecticides where resistance is likely to develop. Widespread low susceptibility of nuisance-biting Culex mosquitoes to ITNs raises concern for user acceptance of nets.

Contains fulltext : 53378.pdf (Publisher’s version ) (Open Access) BACKGROUND: P. falciparum gametocytes may persist after treatment with sulphadoxine-pyrimethamine (SP) plus artesunate (AS) and contribute considerably to malaria transmission. We determined the efficacy of SP+AS plus a single dose of primaquine (PQ, 0.75 mg/kg) on clearing gametocytaemia measured by molecular methods. METHODOLOGY: The study was conducted in Mnyuzi, an area of hyperendemic malaria in north-eastern Tanzania. Children aged 3-15 years with uncomplicated P. falciparum malaria with an asexual parasite density between 500-100,000 parasites/microL were randomized to receive treatment with either SP+AS or SP+AS+PQ. P. falciparum gametocyte prevalence and density during the 42-day follow-up period were determined by real-time nucleic acid sequence-based amplification (QT-NASBA). Haemoglobin levels (Hb) were determined to address concerns about haemolysis in G6PD-deficient individuals. RESULTS: 108 individuals were randomized. Pfs25 QT-NASBA gametocyte prevalence was 88-91% at enrolment and decreased afterwards for both treatment arms. Gametocyte prevalence and density were significantly lower in children treated with SP+AS+PQ. On day 14 after treatment 3.9% (2/51) of the SP+AS+PQ treated children harboured gametocytes compared to 62.7% (32/51) of those treated with SP+AS (p

Background Molecular markers of insecticide resistance can provide sensitive indicators of resistance development in malaria vector populations. Monitoring of insecticide resistance in vector populations is an important component of current malaria control programmes. Knockdown resistance (kdr) confers resistance to the pyrethroid class of insecticides with cross-resistance to DDT through single nucleotide polymorphisms (SNPs) in the voltage-gated sodium channel gene. Methods To enable detection of kdr mutations at low frequency a method was developed that uses polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA)-based technology, allowing rapid, reliable and cost-effective testing of large numbers of individual mosquitoes. This was used to assay mosquitoes from sites in lower Moshi, Tanzania. Results Sequence-specific oligonucleotide probes (SSOP) were used for simultaneous detection of both East and West African kdr mutations with high specificity and sensitivity. Application of the SSOP-ELISA method to 1,620 field-collected Anopheles arabiensis from Tanzania identified the West African leucine-phenylalanine kdr mutation in two heterozygous individuals, indicating the potential for resistance development that requires close monitoring. Conclusion The presence of the West African kdr mutation at low frequency in this East African population of An. arabiensis has implications for the spread of the kdr gene across the African continent.