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Background: Artemisinin-based combination therapies (ACTs) are the global mainstay treatment of uncomplicated Plasmodium falciparum infections. Pf MDR1 and Pf CRT are two transmembrane transporters, associated with sensitivity to several antimalarials, found in the parasite food vacuole. Herein, we explore if their relatedness extends to overlapping patterns of gene transcriptional activity before and during ACT administration. Methods: In a clinical trial performed in Tanzania, we explored the pfmdr1 and pfcrt transcription levels from 48 patients with uncomplicated P. falciparum malaria infections who underwent treatment with artemether-lumefantrine (AL). Samples analyzed were collected before treatment initiation and during the first 24 h of treatment. The frequency of Pf MDR1 N86Y and Pf CRT K76T was determined through PCR-RFLP or direct amplicon sequencing. Gene expression was analyzed by real-time quantitative PCR. Results: A wide range of pre-treatment expression levels was observed for both genes, approximately 10-fold for pfcrt and 50-fold for pfmdr1. In addition, a significant positive correlation demonstrates pfmdr1 and pfcrt co-expression. After AL treatment initiation, pfmdr1 and pfcrt maintained the positive co-expression correlation, with mild downregulation throughout the 24 h post-treatment. Additionally, a trend was observed for Pf MDR1 N86 alleles and higher expression before treatment initiation. Conclusion: pfmdr1 and pfcrt showed significant co-expression patterns in vivo , which were generally maintained during ACT treatment. This observation points to relevant related roles in the normal parasite physiology, which seem essential to be maintained when the parasite is exposed to drug stress. In addition, keeping the simultaneous expression of both transporters might be advantageous for responding to the drug action. [ABSTRACT FROM AUTHOR]

Background: Delayed parasite clearance and, consequently, reduced efficacy of artemisinin-based combination therapies have been linked with Plasmodium falciparum K13 gene SNPs in Southeast Asia. In Africa, significantly prolonged clearance has not yet been observed and the presently restricted variation in parasite clearance cannot be explained by K13 polymorphisms.Objectives: Our aim was to study the in vivo pfK13 transcriptional response in patients treated with artemether-lumefantrine and explore whether the pfk13 transcripts can explain the patients' parasite clearance outcomes.Patients and Methods: A total of 47 Tanzanian children with microscopically confirmed uncomplicated P. falciparum malaria were hospitalized and received artemether-lumefantrine treatment (clinical trial ID: NCT00336375). RNA was extracted from venous blood samples collected before treatment initiation and at five more timepoints after treatment. cDNA was synthesized and pfk13 transcripts measured by real-time PCR.Results: A wide range of pfk13 transcript variation was observed throughout all timepoints after artemether-lumefantrine treatment. Taking parasite clearance data together with the pfk13 transcripts profile, we observed a negative correlation inferring that pfk13 down-regulation is associated with longer parasite clearance time.Conclusions: The findings suggest that a reduced PfK13 transcriptional response may represent a first step towards artemisinin tolerance/resistance. [ABSTRACT FROM AUTHOR]

We assessed the influence that consecutive-day blood sampling, compared with single-day blood sampling, had on polymerase chain reaction (PCR)-adjusted parasitological cure after stepwise genotyping of merozoite surface proteins 2 (msp2) and 1 (msp1) in 106 children in Tanzania who had uncomplicated falciparum malaria treated with either sulfadoxine-pyrimethamine or artemether-lumefantrine; 78 of these children developed recurrent parasitemia during the 42-day follow-up period. Initial msp2 genotyping identified 27 and 33 recrudescences by use of single- and consecutive-day sampling, respectively; in subsequent msp1 genotyping, 17 and 21 of these episodes, respectively, were still classified as recrudescences; these results indicate a similar sensitivity of the standard single-day PCR protocol--that is, 82% (27/33) and 81% (17/21), in both genotyping steps. Interpretation of PCR-adjusted results will significantly depend on methodology. Copyright © 2007 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Abstract Background This study aimed to explore Plasmodium falciparum population dynamics during the early phase of anti-malarial drug treatment with artemisinin-based combination therapy in children with clinical malaria in a high transmission area in Africa. Methods A total of 50 children aged 1-10 years with acute uncomplicated P. falciparum malaria in Bagamoyo District, Tanzania, were enrolled. Participants were hospitalized and received supervised standard treatment with artemether-lumefantrine according to body weight in six doses over 3 days. Blood samples were collected 11 times, i.e. at time of diagnosis (-2 h) and 0, 2, 4, 8, 16, 24, 36, 48, 60 and 72 h after initiation of treatment. Parasite population dynamics were assessed using nested polymerase chain reaction (PCR)-genotyping of merozoite surface protein (msp) 1 and 2. Results PCR-analyses from nine sequential blood samples collected after initiation of treatment identified 20 and 21 additional genotypes in 15/50 (30%) and 14/50 (28%) children with msp1 and msp2, respectively, non-detectable in the pre-treatment samples (-2 and 0 h combined). Some 15/20 (75%) and 14/21 (67%) of these genotypes were identified within 24 h, whereas 17/20 (85%) and 19/21 (90%) within 48 h for msp1 and msp2, respectively. The genotype profile was diverse, and varied considerably over time both within and between patients, molecular markers and their respective families. Conclusion PCR analyses from multiple blood samples collected during the early treatment phase revealed a complex picture of parasite sub-populations. This underlines the importance of interpreting PCR-outcomes with caution and suggests that the present use of PCR-adjustment from paired blood samples in anti-malarial drug trials may overestimate assessment of drug efficacy in high transmission areas in Africa. The study is registered at http://www.clinicaltrials.gov with identifier NCT00336375.

Abstract Background Home-management of malaria (HMM) strategy improves early access of anti-malarial medicines to high-risk groups in remote areas of sub-Saharan Africa. However, limited data are available on the effectiveness of using artemisinin-based combination therapy (ACT) within the HMM strategy. The aim of this study was to assess the effectiveness of artemether-lumefantrine (AL), presently the most favoured ACT in Africa, in under-five children with uncomplicated Plasmodium falciparum malaria in Tanzania, when provided by community health workers (CHWs) and administered unsupervised by parents or guardians at home. Methods An open label, single arm prospective study was conducted in two rural villages with high malaria transmission in Kibaha District, Tanzania. Children presenting to CHWs with uncomplicated fever and a positive rapid malaria diagnostic test (RDT) were provisionally enrolled and provided AL for unsupervised treatment at home. Patients with microscopy confirmed P. falciparum parasitaemia were definitely enrolled and reviewed weekly by the CHWs during 42 days. Primary outcome measure was PCR corrected parasitological cure rate by day 42, as estimated by Kaplan-Meier survival analysis. This trial is registered with ClinicalTrials.gov, number NCT00454961. Results A total of 244 febrile children were enrolled between March-August 2007. Two patients were lost to follow up on day 14, and one patient withdrew consent on day 21. Some 141/241 (58.5%) patients had recurrent infection during follow-up, of whom 14 had recrudescence. The PCR corrected cure rate by day 42 was 93.0% (95% CI 88.3%-95.9%). The median lumefantrine concentration was statistically significantly lower in patients with recrudescence (97 ng/mL [IQR 0-234]; n = 10) compared with reinfections (205 ng/mL [114-390]; n = 92), or no parasite reappearance (217 [121-374] ng/mL; n = 70; p ≤ 0.046). Conclusions Provision of AL by CHWs for unsupervised malaria treatment at home was highly effective, which provides evidence base for scaling-up implementation of HMM with AL in Tanzania.

Abstract Background Several criteria have been used to assess agreement between replicate slide readings of malaria parasite density. Such criteria may be based on percent difference, or absolute difference, or a combination. Neither the rationale for choosing between these types of criteria, nor that for choosing the magnitude of difference which defines acceptable agreement, are clear. The current paper seeks a procedure which avoids the disadvantages of these current options and whose parameter values are more clearly justified. Methods and Results Variation of parasite density within a slide is expected, even when it has been prepared from a homogeneous sample. This places lower limits on sensitivity and observer agreement, quantified by the Poisson distribution. This means that, if a criterion of fixed percent difference criterion is used for satisfactory agreement, the number of discrepant readings is over-estimated at low parasite densities. With a criterion of fixed absolute difference, the same happens at high parasite densities. For an ideal slide, following the Poisson distribution, a criterion based on a constant difference in square root counts would apply for all densities. This can be back-transformed to a difference in absolute counts, which, as expected, gives a wider range of acceptable agreement at higher average densities. In an example dataset from Tanzania, observed differences in square root counts correspond to a 95% limits of agreement of -2,800 and +2,500 parasites/μl at average density of 2,000 parasites/μl, and -6,200 and +5,700 parasites/μl at 10,000 parasites/μl. However, there were more outliers beyond those ranges at higher densities, meaning that actual coverage of these ranges was not a constant 95%, but decreased with density. In a second study, a trial of microscopist training, the corresponding ranges of agreement are wider and asymmetrical: -8,600 to +5,200/μl, and -19,200 to +11,700/μl, respectively. By comparison, the optimal limits of agreement, corresponding to Poisson variation, are ± 780 and ± 1,800 parasites/μl, respectively. The focus of this approach on the volume of blood read leads to other conclusions. For example, no matter how large a volume of blood is read, some densities are too low to be reliably detected, which in turn means that disagreements on slide positivity may simply result from within-slide variation, rather than reading errors. Conclusions The proposed method defines limits of acceptable agreement in a way which allows for the natural increase in variability with parasite density. This includes defining the levels of between-reader variability, which are consistent with random variation: disagreements within these limits should not trigger additional readings. This approach merits investigation in other settings, in order to determine both the extent of its applicability, and appropriate numerical values for limits of agreement.

Abstract Background Prescribing antimalarial medicines based on parasite confirmed diagnosis of malaria is critical to rational drug use and optimal outcome of febrile illness. The impact of microscopy-based versus clinical-based diagnosis of childhood malaria was assessed at primary health care (PHC) facilities using a cluster randomized controlled training intervention trial. Methods Sixteen PHC facilities in rural Tanzania were randomly allocated to training of health staff in clinical algorithm plus microscopy (Arm-I, n = 5) or clinical algorithm only (Arm-II, n = 5) or no training (Arm-III, n = 6). Febrile under-five children presenting at these facilities were assessed, treated and scheduled for follow up visit after 7 days. Blood smears on day 0 were only done in Arm-I but on Day 7 in all arms. Primary outcome was antimalarial drug prescription. Other outcomes included antibiotic prescription and health outcome. Multilevel regression models were applied with PHC as level of clustering to compare outcomes in the three study arms. Results A total of 973, 1,058 and 1,100 children were enrolled in arms I, II and III, respectively, during the study period. Antimalarial prescriptions were significantly reduced in Arm-I (61.3%) compared to Arms-II (95.3%) and III (99.5%) (both P < 0.001), whereas antibiotic prescriptions did not vary significantly between the arms (49.9%, 54.8% and 34.2%, respectively). In Arm-I, 99.1% of children with positive blood smear readings received antimalarial prescriptions and so did 11.3% of children with negative readings. Those with positive readings were less likely to be prescribed antibiotics than those with negative (relative risk = 0.66, 95% confidence interval: 0.55, 0.72). On day 7 follow-up, more children reported symptoms in Arm-I compared to Arm-III, but fewer children had malaria parasitaemia (p = 0.049). The overall sensitivity of microscopy reading at PHC compared to reference level was 74.5% and the specificity was 59.0% but both varied widely between PHCs. Conclusion Microscopy based diagnosis of malaria at PHC facilities reduces prescription of antimalarial drugs, and appears to improve appropriate management of non-malaria fevers, but major variation in accuracy of the microscopy readings was found. Lack of qualified laboratory technicians at PHC facilities and the relatively short training period may have contributed to the shortcomings. Trial registration This study is registered at Clinicaltrials.gov with the identifier NCT00687895.