Population Pharmacokinetics of Antimalarial Naphthoquine in Combination with Artemisinin in Tanzanian Children and Adults: Dose Optimization.

The combination antimalarial therapy of artemisinin-naphthoquine (ART-NQ) was developed as a single-dose therapy, aiming to improve adherence relative to the multiday schedules of other artemisinin combination therapies. The pharmacokinetics of ART-NQ has not been well characterized, especially in children. A pharmacokinetic study was conducted in adults and children over 5 years of age (6 to 10, 11 to 17, and ≥18 years of age) with uncomplicated malaria in Tanzania. The median weights for the three age groups were 20, 37.5, and 55 kg, respectively. Twenty-nine patients received single doses of 20 mg/kg of body weight for artemisinin and 8 mg/kg for naphthoquine, and plasma drug concentrations were assessed at 13 time points over 42 days from treatment. We used nonlinear mixed-effects modeling to interpret the data, and allometric scaling was employed to adjust for the effect of body size. The pharmacokinetics of artemisinin was best described by one-compartment model and that of naphthoquine by a two-compartment disposition model. Clearance values for a typical patient (55-kg body weight and 44.3-kg fat-free mass) were estimated as 66.7 L/h (95% confidence interval [CI], 57.3 to 78.5 L/h) for artemisinin and 44.2 L/h (95% CI, 37.9 to 50.6 L/h) for naphthoquine. Nevertheless, we show via simulation that patients weighing ≥70 kg achieve on average a 30% lower day 7 concentration compared to a 48-kg reference patient at the doses tested, suggesting dose increases may be warranted to ensure adequate exposure. (This study has been registered at ClinicalTrials.gov under identifier NCT01930331.).

Optimal timing of primaquine to reduce Plasmodium falciparum gametocyte carriage when co-administered with artemether-lumefantrine.

BACKGROUND: Primaquine is an important gametocytocidal drug that is combined with conventional malaria treatment for prevention of Plasmodium falciparum malaria transmission. Primaquine has been administered together on the first or the last day of conventional treatment but the impact of primaquine timing has never been examined. This study aimed to assess safety, efficacy and optimal timing of single full-dose (0.75 mg/kg) primaquine when added to a standard 6-dose regimen of artemether-lumefantrine (AL). METHODS: In an individual-level randomized controlled trial, enrolled participants who were G6PD normal and had uncomplicated P. falciparum malaria were randomly assigned to receive: AL only; AL and a single 0.75 mg/kg primaquine dose on the first day of AL (day 1); or AL and single 0.75 mg//kg primaquine on the last day of AL (day 3). On days 2, 3, 4, 8, 11 and 15, gametocytes were assessed and quantified by microscope and quantitative nuclear acid sequence based quantification (QT-NASBA). RESULTS: Overall, 111 participants aged between 3 and 17 years were randomly allocated to receive AL only (36) or combined with primaquine on day 1 (38), or primaquine on day 3 (37). Day 4 gametocyte prevalence in AL + day 1 primaquine was half the level seen in either AL + day 3 primaquine or AL only arm (11% [4/35] vs 26% [8/31] and 27% [8/30], respectively) albeit not statistically significant. A similar trend of lower gametocyte in the AL + day 1 primaquine verses AL + day 3 primaquine or AL only arm was observed in mean gametocyte density. Mean (sd) haemoglobin level in AL + day 3 primaquine arm recovered from -0.42(1.2) g/dl on day 2 to 0.35 (1.5) g/dl on day 15 of follow up. This was not the case in AL only and AL + day 1 primaquine arms during the same follow-up period, although the difference was not statistically significant (p = 318). No serious adverse events reported in the study. Across arms, 23% (26/111) of participants reported a total of 31 mild adverse events and the difference was not statistically significant (p = 0.477). CONCLUSION: Primaquine administration on the first day of AL is well tolerated and as safe as later administration. Whilst the World Health Organization currently recommends a lower dose of primaquine (0.25 mg/kg), the findings are supportive of early primaquine administration when combined with artemisinin-combination therapy. ClinicalTrials.gov Registration NCT01906788.

The Candidate Blood-stage Malaria Vaccine P27A Induces a Robust Humoral Response in a Fast Track to the Field Phase 1 Trial in Exposed and Nonexposed Volunteers.

Background: P27A is an unstructured 104mer synthetic peptide from Plasmodium falciparum trophozoite exported protein 1 (TEX1), the target of human antibodies inhibiting parasite growth. The present project aimed at evaluating the safety and immunogenicity of P27A peptide vaccine in malaria-nonexposed European and malaria-exposed African adults. Methods: This study was designed as a staggered, fast-track, randomized, antigen and adjuvant dose-finding, multicenter phase 1a/1b trial, conducted in Switzerland and Tanzania. P27A antigen (10 or 50 µg), adjuvanted with Alhydrogel or glucopyranosil lipid adjuvant stable emulsion (GLA-SE; 2.5 or 5 µg), or control rabies vaccine (Verorab) were administered intramuscularly to 16 malaria-nonexposed and 40 malaria-exposed subjects on days 0, 28, and 56. Local and systemic adverse events (AEs) as well as humoral and cellular immune responses were assessed after each injection and during the 34-week follow-up. Results: Most AEs were mild to moderate and resolved completely within 48 hours. Systemic AEs were more frequent in the formulation with alum as compared to GLA-SE, whereas local AEs were more frequent after GLA-SE. No serious AEs occurred. Supported by a mixed Th1/Th2 cell-mediated immunity, P27A induced a marked specific antibody response able to recognize TEX1 in infected erythrocytes and to inhibit parasite growth through an antibody-dependent cellular inhibition mechanism. Incidence of AEs and antibody responses were significantly lower in malaria-exposed Tanzanian subjects than in nonexposed European subjects. Conclusions: The candidate vaccine P27A was safe and induced a particularly robust immunogenic response in combination with GLA-SE. This formulation should be considered for future efficacy trials. Clinical Trials Registration: NCT01949909, PACTR201310000683408.

Prevalence and assessment of malnutrition among children attending the Reproductive and Child Health clinic at Bagamoyo District Hospital, Tanzania.

BACKGROUND: Malnutrition has long been associated with poverty, poor diet and inadequate access to health care, and it remains a key global health issue that both stems from and contributes to ill-health, with 50 % of childhood deaths due to underlying undernutrition. The purpose of this study was to determine the prevalence of malnutrition among children under-five seen at Bagamoyo District Hospital (BDH) and three rural health facilities ranging between 25 and 55 km from Bagamoyo: Kiwangwa, Fukayosi, and Yombo. METHODS: A total of 63,237 children under-five presenting to Bagamoyo District Hospital and the three rural health facilities participated in the study. Anthropometric measures of age, height/length and weight and measurements of mid-upper arm circumference were obtained and compared with reference anthropometric indices to assess nutritional status for patients presenting to the hospital and health facilities. RESULTS: Overall proportion of stunting, underweight and wasting was 8.37, 5.74 and 1.41 % respectively. Boys were significantly more stunted, under weight and wasted than girls (p-value < 0.05). Children aged 24-59 months were more underweight than 6-23 months (p-value = <0.0001). But, there was no statistical significance difference between the age groups for stunting and wasting. Children from rural areas experienced increased rates of stunting, underweight and wasting than children in urban areas (p-value < 0.05). The results of this study concur with other studies that malnutrition remains a problem within Tanzania; however our data suggests that the population presenting to BDH and rural health facilities presented with decreased rates of malnutrition compared to the general population. CONCLUSIONS: Hospital and facility attending populations of under-five children in and around Bagamoyo suffer moderately high rates of malnutrition. Current nutrition programs focus on education for at risk children and referral to regional hospitals for malnourished children. Even though the general population has even greater malnutrition than the population presenting at the hospital, in areas of high malnutrition, hospital-based interventions should also be considered as centralized locations for reaching thousands of malnourished children under-five.

Impact of malaria preexposure on antiparasite cellular and humoral immune responses after controlled human malaria infection.

To understand the effect of previous malaria exposure on antiparasite immune responses is important for developing successful immunization strategies. Controlled human malaria infections (CHMIs) using cryopreserved Plasmodium falciparum sporozoites provide a unique opportunity to study differences in acquisition or recall of antimalaria immune responses in individuals from different transmission settings and genetic backgrounds. In this study, we compared antiparasite humoral and cellular immune responses in two cohorts of malaria-naive Dutch volunteers and Tanzanians from an area of low malarial endemicity, who were subjected to the identical CHMI protocol by intradermal injection of P. falciparum sporozoites. Samples from both trials were analyzed in parallel in a single center to ensure direct comparability of immunological outcomes. Within the Tanzanian cohort, we distinguished one group with moderate levels of preexisting antibodies to asexual P. falciparum lysate and another that, based on P. falciparum serology, resembled the malaria-naive Dutch cohort. Positive P. falciparum serology at baseline was associated with a lower parasite density at first detection by quantitative PCR (qPCR) after CHMI than that for Tanzanian volunteers with negative serology. Post-CHMI, both Tanzanian groups showed a stronger increase in anti-P. falciparum antibody titers than Dutch volunteers, indicating similar levels of B-cell memory independent of serology. In contrast to the Dutch, Tanzanians failed to increase P. falciparum-specific in vitro recall gamma interferon (IFN-γ) production after CHMI, and innate IFN-γ responses were lower in P. falciparum lysate-seropositive individuals than in seronegative individuals. In conclusion, positive P. falciparum lysate serology can be used to identify individuals with better parasite control but weaker IFN-γ responses in circulating lymphocytes, which may help to stratify volunteers in future CHMI trials in areas where malaria is endemic.

Ultra-sensitive detection of Plasmodium falciparum by amplification of multi-copy subtelomeric targets.

BACKGROUND: Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data. METHODS AND FINDINGS: Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/µl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum-positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. CONCLUSIONS: Measured malaria prevalence in communities is largely determined by the sensitivity of the diagnostic tool used. Even when applying standard molecular diagnostics, prevalence in our study population was underestimated by 8% compared to the new assays. Our findings highlight the need for highly sensitive tools such as TARE-2 and varATS qPCR in community surveillance and for monitoring interventions to better describe malaria epidemiology and inform malaria elimination efforts.

Malaria transmission after artemether-lumefantrine and dihydroartemisinin-piperaquine: a randomized trial.

BACKGROUND: Artemisinin-based combination therapy (ACT) reduces the potential for malaria transmission, compared with non-ACTs. It is unclear whether this effect differs between ACTs. METHODS: A total of 298 children (age, 6 months to 10 years) with uncomplicated falciparum malaria were randomized to artemether-lumefantrine (AL; n = 153) or dihydroartemisinin-piperaquine (DP; n = 145) in Mbita, a community in western Kenya. Gametocyte carriage was determined by molecular methods on days 0, 1, 2, 3, 7, 14, 28, and 42 after treatment initiation. The gametocyte infectiousness to mosquitoes was determined by mosquito-feeding assays on day 7 after beginning therapy. RESULTS: The cumulative risk of recurrent parasitemia on day 42 after initiation of treatment, unadjusted by polymerase chain reaction findings, was 20.7% (95% confidence interval [CI], 14.4-28.2) for AL, compared with 3.7% (95% CI, 1.2-8.5) for DP (P < .001). The mean duration of gametocyte carriage was 5.5 days (95% CI, 3.6-8.5) for AL and 15.3 days (95% CI, 9.7-24.2) for DP (P = .001). The proportion of mosquitoes that became infected after feeding on blood from AL-treated children was 1.88% (43 of 2293), compared with 3.50% (83 of 2371) for those that fed on blood from DP-treated children (P = .06); the oocyst burden among mosquitoes was lower among those that fed on blood from AL-treated children (P = .005) CONCLUSIONS: While DP was associated with a longer prophylactic time after treatment, gametocyte carriage and malaria transmission to mosquitoes was lower after AL treatment. CLINICAL TRIALS REGISTRATION: NCT00868465.

Residual Plasmodium falciparum parasitemia in Kenyan children after artemisinin-combination therapy is associated with increased transmission to mosquitoes and parasite recurrence.

BACKGROUND: Parasite clearance time after artemisinin-based combination therapy (ACT) may be increasing in Asian and African settings. The association between parasite clearance following ACT and transmissibility is currently unknown. METHODS: We determined parasite clearance dynamics by duplex quantitative polymerase chain reaction (qPCR) in samples collected in the first 3 days after treatment of uncomplicated malaria with ACT. Gametocyte carriage was determined by Pfs25 quantitative nucleic acid sequence-based amplification assays; infectiousness to mosquitoes by membrane-feeding assays on day 7 after treatment. RESULTS: Residual parasitemia was detected by qPCR in 31.8% (95% confidence interval [CI], 24.6-39.8) of the children on day 3 after initiation of treatment. Residual parasitemia was associated with a 2-fold longer duration of gametocyte carriage (P = .0007), a higher likelihood of infecting mosquitoes (relative risk, 1.95; 95% CI, 1.17-3.24; P = .015), and a higher parasite burden in mosquitoes (incidence rate ratio, 2.92; 95% CI, 1.61-5.31; P < .001). Children with residual parasitemia were also significantly more likely to experience microscopically detectable parasitemia during follow-up (relative risk, 11.25; 95% CI, 4.08-31.01; P < .001). CONCLUSIONS: Residual submicroscopic parasitemia is common after ACT and is associated with a higher transmission potential. Residual parasitemia may also have consequences for individual patients because of its higher risk of recurrent parasitemia.

Clinical performance of an automated reader in interpreting malaria rapid diagnostic tests in Tanzania.

BACKGROUND: Parasitological confirmation of malaria is now recommended in all febrile patients by the World Health Organization (WHO) to reduce inappropriate use of anti-malarial drugs. Widespread implementation of rapid diagnostic tests (RDTs) is regarded as an effective strategy to achieve this goal. However, the quality of diagnosis provided by RDTs in remote rural dispensaries and health centres is not ideal. Feasible RDT quality control programmes in these settings are challenging. Collection of information regarding diagnostic events is also very deficient in low-resource countries. METHODS: A prospective cohort of consecutive patients aged more than one year from both genders, seeking routine care for febrile episodes at dispensaries located in the Bagamoyo district of Tanzania, were enrolled into the study after signing an informed consent form. Blood samples were taken for thick blood smear (TBS) microscopic examination and malaria RDT (SD Bioline Malaria Antigen Pf/Pan™ (SD RDT)). RDT results were interpreted by both visual interpretation and Deki Reader™ device. Results of visual interpretation were used for case management purposes. Microscopy was considered the "gold standard test" to assess the sensitivity and specificity of the Deki Reader interpretation and to compare it to visual interpretation. RESULTS: In total, 1,346 febrile subjects were included in the final analysis. The SD RDT, when used in conjunction with the Deki Reader and upon visual interpretation, had sensitivities of 95.3% (95% CI, 90.6-97.7) and 94.7% (95% CI, 89.8-97.3) respectively, and specificities of 94.6% (95% CI, 93.5-96.1) and 95.6% (95% CI, 94.2-96.6), respectively to gold standard. There was a high percentage of overall agreement between the two methods of interpretation. CONCLUSION: The sensitivity and specificity of the Deki Reader in interpretation of SD RDTs were comparable to previous reports and showed high agreement to visual interpretation (>98%). The results of the study reflect the situation in real practice and show good performance characteristics of Deki Reader on interpreting malaria RDTs in the hands of local laboratory technicians. They also suggest that a system like this could provide great benefits to the health care system. Further studies to look at ease of use by community health workers, and cost benefit of the system are warranted.

Experience and challenges from clinical trials with malaria vaccines in Africa.

Malaria vaccines are considered amongst the most important modalities for potential elimination of malaria disease and transmission. Research and development in this field has been an area of intense effort by many groups over the last few decades. Despite this, there is currently no licensed malaria vaccine. Researchers, clinical trialists and vaccine developers have been working on many approached to make malaria vaccine available.African research institutions have developed and demonstrated a great capacity to undertake clinical trials in accordance to the International Conference on Harmonization-Good Clinical Practice (ICH-GCP) standards in the last decade; particularly in the field of malaria vaccines and anti-malarial drugs. This capacity is a result of networking among African scientists in collaboration with other partners; this has traversed both clinical trials and malaria control programmes as part of the Global Malaria Action Plan (GMAP). GMAP outlined and support global strategies toward the elimination and eradication of malaria in many areas, translating in reduction in public health burden, especially for African children. In the sub-Saharan region the capacity to undertake more clinical trials remains small in comparison to the actual need.However, sustainability of the already developed capacity is essential and crucial for the evaluation of different interventions and diagnostic tools/strategies for other diseases like TB, HIV, neglected tropical diseases and non-communicable diseases. There is urgent need for innovative mechanisms for the sustainability and expansion of the capacity in clinical trials in sub-Saharan Africa as the catalyst for health improvement and maintained.

Comparison of bioavailability between the most available generic tablet formulation containing artemether and lumefantrine on the Tanzanian market and the innovator's product.

BACKGROUND: Existence of anti-malarial generic drugs with low bioavailability marketed on sub-Saharan Africa raises a concern on patients achieving therapeutic concentrations after intake of such products. This work compared bioavailability of one generic tablet formulation with innovator's product. Both were fixed dose combination tablet formulations containing artemether and lumefantrine. METHODOLOGY: The study was conducted in Dar Es Salaam, Tanzania, in which a survey of the most abundant generic containing artemether-lumefantrine tablet formulation was carried out in retail pharmacies. The most widely available generic (Artefan®, Ajanta Pharma Ltd, Maharashtra, India) was sampled for bioavailability comparison with Coartem® (Novartis Pharma, Basel, Switzerland)--the innovator's product. A randomized, two-treatment cross-over study was conducted in 18 healthy Tanzanian black male volunteers. Each volunteer received Artefan® (test) and Coartem® (as reference) formulation separated by 42 days of drug-free washout period. Serial blood samples were collected up to 168 hours after oral administration of a single dose of each treatment. Quantitation of lumefantrine plasma levels was done using HPLC with UV detection. Bioequivalence of the two products was assessed in accordance with the US Food and Drug Authority (FDA) guidelines. RESULTS: The most widely available generic in pharmacies was Artefan® from India. All eighteen enrolled volunteers completed the study and both test and reference tablet formulations were well tolerated. It was possible to quantify lumefantrine alone, therefore, the pharmacokinetic parameters reported herein are for lumefantrine. The geometric mean ratios for Cmax, AUC0-t and AUC0-∞ were 84% in all cases and within FDA recommended bioequivalence limits of 80%-125%, but the 90% confidence intervals were outside FDA recommended limits (CI 49-143%, 53-137%, 52-135% respectively). There were no statistical significant differences between the two formulations with regard to PK parameters (P > 0.05). CONCLUSIONS: Although the ratios of AUCs and Cmax were within the acceptable FDA range, bioequivalence between Artefan® and Coartem® tablet formulations was not demonstrated due to failure to comply with the FDA 90% confidence interval criteria. Based on the observed total drug exposure (AUCs), Artefan® is likely to produce a similar therapeutic response as Coartem®.

Malaria diagnostic testing and treatment practices in three different Plasmodium falciparum transmission settings in Tanzania: before and after a government policy change.

BACKGROUND: Patterns of decreasing malaria transmission intensity make presumptive treatment of malaria an unjustifiable approach in many African settings. The controlled use of anti-malarials after laboratory confirmed diagnosis is preferable in low endemic areas. Diagnosis may be facilitated by malaria rapid diagnostic tests (RDTs). In this study, the impact of a government policy change, comprising the provision of RDTs and advice to restrict anti-malarial treatment to RDT-positive individuals, was assessed by describing diagnostic behaviour and treatment decision-making in febrile outpatients <10 years of age in three hospitals in the Kagera and Mwanza Region in northern Tanzania. METHODS: Prospective data from Biharamulo and Rubya Designated District Hospital (DDH) were collected before and after policy change, in Sumve DDH no new policy was implemented. Diagnosis of malaria was confirmed by RDT; transmission intensity was evaluated by a serological marker of malaria exposure in hospital attendees. RESULTS: Prior to policy change, there was no evident association between the actual level of transmission intensity and drug-prescribing behaviour. After policy change, there was a substantial decrease in anti-malarial prescription and an increase in prescription of antibiotics. The proportion of parasite-negative individuals who received anti-malarials decreased from 89.1% (244/274) to 38.7% (46/119) in Biharamulo and from 76.9% (190/247) to 10.0% (48/479) in Rubya after policy change. CONCLUSION: This study shows that an official policy change, where RDTs were provided and healthcare providers were advised to adhere to RDT results in prescribing drugs can be followed by more rational drug-prescribing behaviour. The current findings are promising for improving treatment policy in Tanzanian hospitals.

A cluster-randomized trial of mass drug administration with a gametocytocidal drug combination to interrupt malaria transmission in a low endemic area in Tanzania.

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 1 km 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.

In Tanzania, hemolysis after a single dose of primaquine coadministered with an artemisinin is not restricted to glucose-6-phosphate dehydrogenase-deficient (G6PD A-) individuals.

The current interest in malaria elimination has led to a renewed interest in drugs that can be used for mass administration to minimize malaria transmission. Primaquine (PQ) is the only generally available drug with a strong activity against mature Plasmodium falciparum gametocytes, the parasite stage responsible for transmission. Despite concerns about PQ-induced hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals, a single dose of PQ may be safe and efficacious in clearing gametocytes that persist after conventional treatment. As part of a mass drug intervention, we determined the hemolytic effect of sulfadoxine-pyrimethamine (SP) plus artesunate (AS) plus a single dose of primaquine (PQ; 0.75 mg/kg of body weight) in children aged 1 to 12 years. Children were randomized to receive SP+AS+PQ or placebo; those with a hemoglobin (Hb) level below 8 g/dl were excluded from receiving PQ and received SP+AS. The Hb concentration was significantly reduced 7 days after SP+AS+PQ treatment but not after placebo or SP+AS treatment. This reduction in Hb was most pronounced in G6PD-deficient (G6PD A-) individuals (-2.5 g/dl; 95% confidence interval [95% CI], -1.2 to -3.8 g/dl) but was also observed in heterozygotes (G6PD A) (-1.6 g/dl; 95% CI, -0.9 to -2.2 g/dl) and individuals with the wild-type genotype (G6PD B) (-0.5 g/dl; 95% CI, -0.4 to -0.6 g/dl). Moderate anemia (Hb level of <8 g/dl) was observed in 40% (6/15 individuals) of the G6PD A-, 11.1% (3/27 individuals) of the G6PD A, and 4.5% (18/399 individuals) of the G6PD B individuals; one case of severe anemia (Hb level of <5 g/dl) was observed. PQ may cause moderate anemia when coadministered with artemisinins, and excluding individuals based on G6PD status alone may not be sufficient to prevent PQ-induced hemolysis.

Revisiting the circulation time of Plasmodium falciparum gametocytes: molecular detection methods to estimate the duration of gametocyte carriage and the effect of gametocytocidal drugs.

BACKGROUND: There is renewed acknowledgement that targeting gametocytes is essential for malaria control and elimination efforts. Simple mathematical models were fitted to data from clinical trials in order to determine the mean gametocyte circulation time and duration of gametocyte carriage in treated malaria patients. METHODS: Data were used from clinical trials from East Africa. The first trial compared non-artemisinin combination therapy (non-ACT: sulphadoxine-pyrimethamine (SP) plus amodiaquine) and artemisinin-based combination therapy (ACT: SP plus artesunate (AS) or artemether-lumefantrine). The second trial compared ACT (SP+AS) with ACT in combination with a single dose of primaquine (ACT-PQ: SP+AS+PQ). Mature gametocytes were quantified in peripheral blood samples by nucleic acid sequence based amplification. A simple deterministic compartmental model was fitted to gametocyte densities to estimate the circulation time per gametocyte; a similar model was fitted to gametocyte prevalences to estimate the duration of gametocyte carriage after efficacious treatment. RESULTS: The mean circulation time of gametocytes was 4.6-6.5 days. After non-ACT treatment, patients were estimated to carry gametocytes for an average of 55 days (95% CI 28.7 - 107.7). ACT reduced the duration of gametocyte carriage fourfold to 13.4 days (95% CI 10.2-17.5). Addition of PQ to ACT resulted in a further fourfold reduction of the duration of gametocyte carriage. CONCLUSIONS: These findings confirm previous estimates of the circulation time of gametocytes, but indicate a much longer duration of (low density) gametocyte carriage after apparently successful clearance of asexual parasites. ACT shortened the period of gametocyte carriage considerably, and had the most pronounced effect on mature gametocytes when combined with PQ.

Low density parasitaemia, red blood cell polymorphisms and Plasmodium falciparum specific immune responses in a low endemic area in northern Tanzania.

BACKGROUND: Low density Plasmodium falciparum infections, below the microscopic detection limit, may play an important role in maintaining malaria transmission in low endemic areas as well as contribute to the maintenance of acquired immunity. Little is known about factors influencing the occurrence of sub-microscopic parasitaemia or the relation with immune responses.We investigated possible associations between the occurrence of sub-microscopic P. falciparum parasite carriage and antibody responses to the asexual stage antigens, G6PD deficiency and alpha+-thalassaemia in 464 subjects from a low endemic area in northern Tanzania. METHODS: We used samples collected from two cross sectional surveys conducted during dry and wet season in 2005. Submicroscopic parasitaemia was detected by using quantitative nucleic acid sequence based amplification (QT-NASBA). Genotyping for G6PD and alpha+-thalassaemia were performed by high throughput PCR; the prevalence and level of total IgG antibodies against MSP-1, MSP-2 and AMA-1 were determined by ELISA. RESULTS: Compared to parasite free individuals, individuals carrying sub-microscopic densities of P. falciparum parasites had significantly higher median antibody levels to MSP-1 (p = 0.042) and MSP-2 (p = 0.034) but not to AMA-1 (p = 0.14) while no clear relation between sub-microscopic parasite carriage and G6PD deficiency or alpha+-thalassaemia was observed. CONCLUSION: Our data suggest a role for sub-microscopic parasite densities in eliciting or maintaining humoral immune responses without evidence for a modulating effect of G6PD deficiency or alpha+-thalassaemia.

Evaluation of uptake and attitude to voluntary counseling and testing among health care professional students in Kilimanjaro region, Tanzania.

BACKGROUND: Voluntary counseling and testing (VCT) is a corner stone for successful implementation of prevention, care and support services among HIV negative and positive individuals. VCT is also perceived to be an effective strategy in risk reduction among sexually active young people.. This study aimed to assess the acceptability of VCT and its actual uptake among young health care professional students at KCM College of Tumaini University and Allied health schools. METHODS: This was a cross-sectional study. A structured questionnaire was used among health care professional students aged 18-25 years who were enrolled in degrees, diplomas and certificates courses at Kilimanjaro Christian Medical College and all other Allied health schools RESULTS: A total of 309 students were recruited, among these 197 (63.8%) were females. All respondents were aware of the benefits of VCT. Only 107 (34.6%) of students have had VCT done previously. About 59 (19.1%) of the students had negative for health care professional to attend VCT. Risk perception among the students was low (37.2%) even though they were found to have higher risk behaviors that predispose them to get HIV infection. CONCLUSION: Awareness of VCT services and willingness to test is high among students; however its uptake is low. In order to promote these services, a comprehensive training module on VCT needs to be included in their training curricula. In particular, more emphasis should focus on the benefits of VCT and to help the students to internalize the risk of HIV so that they can take preventive measures.

Overuse of artemisinin-combination therapy in Mto wa Mbu (river of mosquitoes), an area misinterpreted as high endemic for malaria.

BACKGROUND: Adequate malaria diagnosis and treatment remain major difficulties in rural sub-Saharan Africa. These issues deserve renewed attention in the light of first-line treatment with expensive artemisinin-combination therapy (ACT) and changing patterns of transmission intensity. This study describes diagnostic and treatment practices in Mto wa Mbu, an area that used to be hyperendemic for malaria, but where no recent assessments of transmission intensity have been conducted. METHODS: Retrospective and prospective data were collected from the two major village health clinics. The diagnosis in prospectively collected data was confirmed by microscopy. The level of transmission intensity was determined by entomological assessment and by estimating sero-conversion rates using anti-malarial antibody responses. RESULTS: Malaria transmission intensity by serological assessment was equivalent to < 1 infectious bites per person per year. Despite low transmission intensity, > 40% of outpatients attending the clinics in 2006-2007 were diagnosed with malaria. Prospective data demonstrated a very high overdiagnosis of malaria. Microscopy was unreliable with < 1% of slides regarded as malaria parasite-positive by clinic microscopists being confirmed by trained research microscopists. In addition, many 'slide negatives' received anti-malarial treatment. As a result, 99.6% (248/249) of the individuals who were treated with ACT were in fact free of malaria parasites. CONCLUSION: Transmission intensity has dropped considerably in the area of Mto wa Mbu. Despite this, most fevers are still regarded and treated as malaria, thereby ignoring true causes of febrile illness and over-prescribing ACT. The discrepancy between the perceived and actual level of transmission intensity may be present in many areas in sub-Saharan Africa and calls for greater efforts in defining levels of transmission on a local scale to help rational drug-prescribing behaviour.

Safety, Immunogenicity, and Protective Efficacy against Controlled Human Malaria Infection of Plasmodium falciparum Sporozoite Vaccine in Tanzanian Adults.

We are using controlled human malaria infection (CHMI) by direct venous inoculation (DVI) of cryopreserved, infectious Plasmodium falciparum (Pf) sporozoites (SPZ) (PfSPZ Challenge) to try to reduce time and costs of developing PfSPZ Vaccine to prevent malaria in Africa. Immunization with five doses at 0, 4, 8, 12, and 20 weeks of 2.7 × 105 PfSPZ of PfSPZ Vaccine gave 65% vaccine efficacy (VE) at 24 weeks against mosquito bite CHMI in U.S. adults and 52% (time to event) or 29% (proportional) VE over 24 weeks against naturally transmitted Pf in Malian adults. We assessed the identical regimen in Tanzanians for VE against PfSPZ Challenge. Twenty- to thirty-year-old men were randomized to receive five doses normal saline or PfSPZ Vaccine in a double-blind trial. Vaccine efficacy was assessed 3 and 24 weeks later. Adverse events were similar in vaccinees and controls. Antibody responses to Pf circumsporozoite protein were significantly lower than in malaria-naïve Americans, but significantly higher than in Malians. All 18 controls developed Pf parasitemia after CHMI. Four of 20 (20%) vaccinees remained uninfected after 3 week CHMI (P = 0.015 by time to event, P = 0.543 by proportional analysis) and all four (100%) were uninfected after repeat 24 week CHMI (P = 0.005 by proportional, P = 0.004 by time to event analysis). Plasmodium falciparum SPZ Vaccine was safe, well tolerated, and induced durable VE in four subjects. Controlled human malaria infection by DVI of PfSPZ Challenge appeared more stringent over 24 weeks than mosquito bite CHMI in United States or natural exposure in Malian adults, thereby providing a rigorous test of VE in Africa.

Prevalence of submicroscopic Plasmodium falciparum infections in asymptomatic children in low transmission settings in Bagamoyo, Tanzania.

Background: Falciparum malaria in endemic areas continues to occur in asymptomatic cases, which contribute to the persistence of transmission as well as the size of the parasite reservoirs. Recent successes in malaria control have resulted in renewed interest in malaria eradication and identification of the human infectious reservoir is essential for this. In this study, we evaluated prevalence of microscopic and submicroscopic gametocytes that were obtained from asymptomatic primary school children from Bagamoyo rural in Tanzania. Materials and methods: Samples were collected from 501 asymptomatic primary school children (6-14 years of age) from 7 villages in Bagamoyo district. Participants were screened for malaria in the field using RDT, and samples were brought to the laboratory for microscopy and molecular analysis. Parasite density was determined by microscopy, and gametocyte carriage identification was performed by RT-qPCR targeting gametocyte-specific genes. Results: Asymptomatic infection was found to be 45.1% (95% : CI=40.7-49.6) by RT-qPCR, followed by RDT, 14.2% (95%: CI=11.2-17.5) and microscopy 6.8% (95%: CI=4.7-9.4). Parasite prevalence by microscopy was 12% (23/191) in boys compared to 3.6% (11/310) in girls (p<0.001). Gametocytes were detected in 12.6% (226/501) of the asymptomatic school children by RT-qPCR compared to only 0.8% (4/501) of the children by microscopy (P=0.008). Conclusions: Asymptomatic infection and submicroscopic gametocyte carriage were high in the study area. The detection of asymptomatic cases with circulating submicroscopic P. falciparum gametocytes in school children indicates that these form a substantive gametocyte reservoir that sustains malaria transmission. Asymptomatic carriers and submicroscopic infections should therefore be considered when implementing elimination strategies of the disease.