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Abstract Background Although Tanzania adopted and has been implementing effective interventions to control and eventually eliminate malaria, the disease is still a leading public health problem, and the country experiences heterogeneous transmission. Recent studies reported the emergence of parasites with artemisinin partial resistance (ART-R) in Kagera region with high prevalence (> 10.0%) in two districts of Karagwe and Kyerwa. This study assessed the prevalence and predictors/risk of malaria infections among asymptomatic individuals living in a hyperendemic area where ART-R has emerged in Kyerwa District of Kagera region, north-western Tanzania. Methods This was a community-based cross-sectional survey which was conducted in July and August 2023 and involved individuals aged ≥ 6 months from five villages in Kyerwa district. Demographic, anthropometric, clinical, parasitological, type of house inhabited and socio-economic status (SES) data were collected using electronic capture tools run on Open Data Kit (ODK) software. Predictors/risks of malaria infections were determined by univariate and multivariate logistic regression, and the results were presented as crude (cORs) and adjusted odds ratios (aORs), with 95% confidence intervals (CIs). Results Overall, 4454 individuals were tested using rapid diagnostic tests (RDTs), and 1979 (44.4%) had positive results. The prevalence of malaria infections ranged from 14.4% to 68.5% and varied significantly among the villages (p

Abstract Background In 2021 and 2023, the World Health Organization approved RTS,S/AS01 and R21/Matrix M malaria vaccines, respectively, for routine immunization of children in African countries with moderate to high transmission. These vaccines are made of Plasmodium falciparum circumsporozoite protein (PfCSP), but polymorphisms in the gene raise concerns regarding strain-specific responses and the long-term efficacy of these vaccines. This study assessed the Pfcsp genetic diversity, population structure and signatures of selection among parasites from areas of different malaria transmission intensities in Mainland Tanzania, to generate baseline data before the introduction of the malaria vaccines in the country. Methods The analysis involved 589 whole genome sequences generated by and as part of the MalariaGEN Community Project. The samples were collected between 2013 and January 2015 from five regions of Mainland Tanzania: Morogoro and Tanga (Muheza) (moderate transmission areas), and Kagera (Muleba), Lindi (Nachingwea), and Kigoma (Ujiji) (high transmission areas). Wright’s inbreeding coefficient (Fws), Wright’s fixation index (FST), principal component analysis, nucleotide diversity, and Tajima’s D were used to assess within-host parasite diversity, population structure and natural selection. Results Based on Fws (

Abstract Background Artemisinin-based combination therapy (ACT) has been a major contributor to the substantial reductions in global malaria morbidity and mortality over the last decade. In Tanzania, artemether-lumefantrine (AL) was introduced as the first-line treatment for uncomplicated Plasmodium falciparum malaria in 2006. The World Health Organization (WHO) recommends regular assessment and monitoring of the efficacy of the first-line treatment, specifically considering that artemisinin resistance has been confirmed in the Greater Mekong sub-region. This study's main aim was to assess the efficacy and safety of AL for treating uncomplicated P. falciparum malaria in Tanzania. Methods This was a single-arm prospective antimalarial drug efficacy trial conducted in four of the eight National Malaria Control Programme (NMCP) sentinel sites in 2019. The trial was carried out in outpatient health facilities in Karume-Mwanza region, Ipinda-Mbeya region, Simbo-Tabora region, and Nagaga-Mtwara region. Children aged six months to 10 years with microscopy confirmed uncomplicated P. falciparum malaria who met the inclusion criteria were recruited based on the WHO protocol. The children received AL (a 6-dose regimen of AL twice daily for three days). Clinical and parasitological parameters were monitored during follow-up over 28 days to evaluate drug efficacy. Results A total of 628 children were screened for uncomplicated malaria, and 349 (55.6%) were enrolled between May and September 2019. Of the enrolled children, 343 (98.3%) completed the 28-day follow-up or attained the treatment outcomes. There were no early treatment failures; recurrent infections during follow-up were common at two sites (Karume 29.5%; Simbo 18.2%). PCR-corrected adequate clinical and parasitological response (ACPR) by survival analysis to AL on day 28 of follow-up varied from 97.7% at Karume to 100% at Ipinda and Nagaga sites. The commonly reported adverse events were cough, skin pallor, and abdominal pain. The drug was well tolerated, and no serious adverse event was reported. Conclusion This study showed that AL had adequate efficacy and safety for the treatment of uncomplicated falciparum malaria in Tanzania in 2019. The high recurrent infections were mainly due to new infections, highlighting the potential role of introducing alternative artemisinin-based combinations that offer improved post-treatment prophylaxis, such as artesunate-amodiaquine (ASAQ).

Abstract Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania.

Abstract Background Recent studies point to the need to incorporate the detection of non-falciparum species into malaria surveillance activities in sub-Saharan Africa, where 95% of the world’s malaria cases occur. Although malaria caused by infection with Plasmodium falciparum is typically more severe than malaria caused by the non-falciparum Plasmodium species P. malariae, P. ovale spp. and P. vivax, the latter may be more challenging to diagnose, treat, control and ultimately eliminate. The prevalence of non-falciparum species throughout sub-Saharan Africa is poorly defined. Tanzania has geographical heterogeneity in transmission levels but an overall high malaria burden. Methods To estimate the prevalence of malaria species in Mainland Tanzania, we randomly selected 1428 samples from 6005 asymptomatic isolates collected in previous cross-sectional community surveys across four regions and analyzed these by quantitative PCR to detect and identify the Plasmodium species. Results Plasmodium falciparum was the most prevalent species in all samples, with P. malariae and P. ovale spp. detected at a lower prevalence (

Abstract Background Tanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of anti-malarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes, merozoite surface proteins 1 and 2 (msp1 and msp2), and replacing the glutamate-rich protein (glurp) gene with one of the highly polymorphic microsatellites in Plasmodium falciparum to adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which is routinely performed in Tanzania. Methods Plasmodium falciparum samples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro) and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was done using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and M2490) by capillary method, and the data were analysed to determine the extent of their polymorphisms and genetic diversity at the four sites. Results Overall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥ 50.0% of the markers), and > 50.0% of the samples (range = 47.6–59.1%) were polyclonal, with a mean multiplicity of infection (MOI) ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (RS = 7.48, range = 7.27–8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (H e = 0.83, range = 0.80–0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (F ST ) did not reveal population structure or clustering of parasites according to geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while M2490 was the least polymorphic. Conclusion Microsatellite genotyping revealed high polyclonality and genetic diversity but no significant population structure. Poly-α, C2M34, TA1 and C3M69 were the most polymorphic markers, and Poly-α alone or with any of the other three markers could be adopted for use in TES in Tanzania.

Abstract Background Therapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021. Methods A total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1). Results Sequencing success was ≥ 87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by the WHO as molecular markers of resistance. One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%. Conclusion This is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of artemisinin partial resistance in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACT.

Abstract Background Despite significant decline in the past two decades, malaria is still a major public health concern in Tanzania; with over 93% of the population still at risk. Community knowledge, attitudes and practices (KAP), and beliefs are key in enhancing uptake and utilization of malaria control interventions, but there is a lack of information on their contribution to effective control of the disease. This study was undertaken to determine KAP and beliefs of community members and service providers on malaria, and how they might be associated with increased risk and persistence of the disease burden in North-western and Southern regions of Tanzania. Methods This was an exploratory study that used qualitative methods including 16 in-depth interviews (IDI) and 32 focus group discussions (FGDs) to collect data from health service providers and community members, respectively. The study was conducted from September to October 2017 and covered 16 villages within eight districts from four regions of mainland Tanzania (Geita, Kigoma, Mtwara and Ruvuma) with persistently high malaria transmission for more than two decades. Results Most of the participants had good knowledge of malaria and how it is transmitted but some FGD participants did not know the actual cause of malaria, and thought that it is caused by bathing and drinking un-boiled water, or consuming contaminated food that has malaria parasites without warming it. Reported barriers to malaria prevention and control (by FGD and IDI participants) included shortage of qualified health workers, inefficient health financing, low care-seeking behaviour, consulting traditional healers, use of local herbs to treat malaria, poverty, increased breeding sites by socio-economic activities and misconceptions related to the use of bed nets and indoor residual spraying (IRS). Among the misconceptions, some participants believed that bed nets provided for free by the government came with bedbugs while others reported that free bed nets caused impotence among men. Conclusion Despite good knowledge of malaria, several risk factors, such as socio-economic and behavioural issues, and misconceptions related to the use of bed nets and IRS were reported. Other key factors included unavailability or limited access to health services, poor health financing and economic activities that potentially contributed to persistence of malaria burden in these regions. Relevant policies and targeted malaria interventions, focusing on understanding socio-cultural factors, should be implemented to reduce and finally eliminate the disease in the study regions and others with persistent transmission.

Abstract Plasmodium malariae, a neglected human malaria parasite, contributes up to 10% of malaria infections in sub-Saharan Africa (sSA). Though P. malariae infection is considered clinically benign, it presents mostly as coinfections with the dominant P. falciparum. Completion of its reference genome has paved the way to further understand its biology and interactions with the human host, including responses to antimalarial interventions. We characterized 75 P. malariae isolates from seven endemic countries in sSA using highly divergent microsatellites. The P. malariae infections were highly diverse and five subpopulations from three ancestries (independent of origin of isolates) were determined. Sequences of 11 orthologous antimalarial resistance genes, identified low frequency single nucleotide polymorphisms (SNPs), strong linkage disequilibrium between loci that may be due to antimalarial drug selection. At least three sub-populations were detectable from a subset of denoised SNP data from mostly the mitochondrial cytochrome b coding region. This evidence of diversity and selection calls for including P. malariae in malaria genomic surveillance towards improved tools and strategies for malaria elimination.

Abstract Background Malaria rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen are widely used for detection of active infection with this parasite and are the only practical malaria diagnostic test in some endemic settings. External validation of RDT results from field surveys can confirm appropriate RDT performance. Methods A community-based cross-sectional survey was conducted between July and November 2017 enrolling participants of all ages in households from 15 villages in four border regions of Tanzania: Geita, Kigoma, Mtwara and Ruvuma. All participants had an RDT performed in the field and provided a blood sample for later laboratory multiplex antigen detection of HRP2. In assessing the continuous HRP2 levels in participant blood versus RDT result, dose–response logistic regression provided quantitative estimates for HRP2 limit of detection (LOD). Results From the 15 study villages, 6941 persons were enrolled that had a RDT at time of enrollment and provided a DBS for later laboratory antigen detection. RDT positive prevalence for the HRP2 band by village ranged from 20.0 to 43.6%, but the magnitude of this prevalence did not have an effect on the estimated LOD of RDTs utilized in different villages. Overall, HRP2 single-target tests had a lower LOD at the 95% probability of positive RDT (4.3 ng/mL; 95% CI 3.4–5.4) when compared to pLDH/HRP2 dual target tests (5.4 ng/mL; 4.5–6.3), though this difference was not significant. With the exception of one village, all other 14 villages (93.3%) showed RDT LOD estimates at 90% probability of positive RDT between 0.5 and 12.0 ng/mL. Conclusions Both HRP2-only and pLDH/HRP2 combo RDTs utilized in a 2017 Tanzania cross-sectional survey of border regions generally performed well, and reliably detected HRP2 antigen in the low ng/mL range. Though single target tests had lower levels of HRP2 detection, both tests were within similar ranges among the 15 villages. Comparison of quantitative HRP2 detection limits among study sites can help interpret RDT testing results when generating population prevalence estimates for malaria infection.

Abstract A rare disease is generally defined as a condition which affects about 1 among 2000 people and currently, there are approximately 5000–8000 rare diseases (RDs) affecting over 400 million people world-wide. Although RDs may arise from different causes such as infections and environmental factors, about 80% are caused by genetic abnormalities. In Tanzania, there are no reports of the types of RDs, their incidence, distribution and numbers of individuals affected. In addition, there have been no strategies to map RDs in the country and develop a definition that fits the local context. Public awareness and understanding of RDs are very limited, and these lead to poor management and stigmatisation of patients. To address the ongoing problems, Tanzania joined other countries world-wide and global partners to commemorate the rare diseases day (RDD) for the first time in 2016 and subsequently every year. Unlike previous years where the RDD was organised by Ali Kimara Rare Diseases Foundation (AKRDF) with few partners, in 2020, a bigger event was co-hosted by Ali AKRDF and Tanzania Human Genetics Organization together with government representatives and other multiple partners. The organisers, government representatives and participants proposed a national “Call for Action” with the overall goal of improving the lives of patients/individuals with RDs. The call focuses and aims to address 17 strategic issues that are broadly categorised into four areas. These include generating demographic data of individuals with RDs; advocating for policies and guidelines for diagnosis, care, treatment and health financing; developing policies supporting public education, awareness and advocacy; and strengthening research, innovation and public–private partnerships. If adopted and implemented, the potential impacts of these recommendations will include improved access to adequate and high-quality health and education services, and policies and guidelines to address the current and future challenges facing individuals with RDs and their families.

Artemisinin resistance (AR) emerged in South East Asia 13 years ago and the identification of the resistance conferring molecular marker, Plasmodium falciparum Kelch 13 (Pfk13), 7 years ago has provided an invaluable tool for monitoring AR in malaria endemic countries. Molecular Pfk13 surveillance revealed the resistance foci in the Greater Mekong Subregion, an independent emergence in Guyana, South America, and a low frequency of mutations in Africa. The recent identification of the R561H Pfk13 AR associated mutation in Tanzania, Uganda and in Rwanda, where it has been associated with delayed parasite clearance, should be a concern for the continent. In this review, we provide a summary of Pfk13 resistance associated propeller domain mutation frequencies across Africa from 2012 to 2020, to examine how many other countries have identified these mutations. Only four African countries reported a recent identification of the M476I, P553L, R561H, P574L, C580Y and A675V Pfk13 mutations at low frequencies and with no reports of clinical treatment failure, except for Rwanda. These mutations present a threat to malaria control across the continent, since the greatest burden of malaria remains in Africa. A rise in the frequency of these mutations and their spread would reverse the gains made in the reduction of malaria over the last 20 years, given the lack of new antimalarial treatments in the event artemisinin-based combination therapies fail. The review highlights the frequency of Pfk13 propeller domain mutations across Africa, providing an up-to-date perspective of Pfk13 mutations, and appeals for an urgent and concerted effort to monitoring antimalarial resistance markers in Africa and the efficacy of antimalarials by re-establishing sentinel surveillance systems.

Abstract Background Tanzania started implementing single screening and treatment (SST) for all pregnant women attending their first antenatal care (ANC) visits in 2014, using malaria rapid diagnostic tests (RDTs) and treating those who test positive according to the national guidelines. However, there is a paucity of data to show the acceptability of SST to both pregnant women and health care workers (HCWs), taking into consideration the shortage of workers and the added burden of this policy to the health system. This study assessed the perceptions and opinions of health service users and providers to determine the acceptability of SST policy. Methods Pregnant women and HCWs in eight health facilities in two districts of Lindi region (Kilwa and Lindi) were interviewed using semi-structured questionnaires with open and close-ended questions. Both qualitative and quantitative data were collected, including demographic characteristics, women’s experience, their perception on SST and challenges they face when receiving services for malaria offered at ANC. Experience of HCWs regarding the implementation of SST as part of routine services and the challenges encountered when providing ANC services for malaria in pregnancy (MIP) were also assessed. Results Of the 143 pregnant women interviewed, 97% viewed testing favourably and would wish to be tested for malaria again, while 95% were satisfied with services and reasons for testing during the first ANC visit. Nearly all (99%) would recommend their fellow pregnant women to be tested for malaria and all women recommended that the Ministry of Health should continue the SST strategy. This was despite the fact that 76% of the women experienced pain and 16% had anxiety as a result of finger prick. Sixteen HCWs (mostly nurses) were interviewed; they also viewed SST implementation favourably and reported feeling empowered to use RDTs for malaria screening. The main challenge identified by HCWs was that nurses are not allowed to prescribe anti-malarials to women who test positive and need to refer them to the outpatient department for treatment. Conclusion SST was considered an acceptable approach to control MIP by HCWs and pregnant women, and they recommended the continuation of the policy. In addition, consideration should be given to implementing a task-shifting policy to allow nurses to dispense anti-malarials to pregnant women.

Recent developments in molecular biology and genomics have revolutionized biology and medicine mainly in the developed world. The application of next generation sequencing (NGS) and CRISPR-Cas tools is now poised to support endemic countries in the detection, monitoring and control of endemic diseases and future epidemics, as well as with emerging and re-emerging pathogens. Most low and middle income countries (LMICs) with the highest burden of infectious diseases still largely lack the capacity to generate and perform bioinformatic analysis of genomic data. These countries have also not deployed tools based on CRISPR-Cas technologies. For LMICs including Tanzania, it is critical to focus not only on the process of generation and analysis of data generated using such tools, but also on the utilization of the findings for policy and decision making. Here we discuss the promise and challenges of NGS and CRISPR-Cas in the context of malaria as Africa moves towards malaria elimination. These innovative tools are urgently needed to strengthen the current diagnostic and surveillance systems. We discuss ongoing efforts to deploy these tools for malaria detection and molecular surveillance highlighting potential opportunities presented by these innovative technologies as well as challenges in adopting them. Their deployment will also offer an opportunity to broadly build in-country capacity in pathogen genomics and bioinformatics, and to effectively engage with multiple stakeholders as well as policy makers, overcoming current workforce and infrastructure challenges. Overall, these ongoing initiatives will build the malaria molecular surveillance capacity of African researchers and their institutions, and allow them to generate genomics data and perform bioinformatics analysis in-country in order to provide critical information that will be used for real-time policy and decision-making to support malaria elimination on the continent.

Abstract Background In areas of high transmission, malaria in pregnancy (MiP) primarily causes asymptomatic infections; these infections nonetheless increase the risk of adverse maternal and fetal outcomes. In 2014, Tanzania initiated a single screening and treatment (SST) strategy for all pregnant women at their first antenatal care (ANC) visit using malaria rapid diagnostic tests (RDT) for surveillance purposes. However, there is paucity of data on the effectiveness of SST in the prevention of MiP. The objective of this study was to estimate the number of asymptomatic infections among pregnant women detected by SST, which would have been missed in the absence of the policy. Methods Data from pregnant women attending their first ANC visits between October 2017 and June 2018, including gestational age, history of fever, and RDT results, were abstracted from ANC registers in eight health centres in two randomly selected districts, Kilwa and Lindi, in Lindi Region. The proportion of symptomatic (with history of fever in the past 48 h) and asymptomatic pregnant women with positive RDTs were calculated and stratified by trimester (first, second and third). The study areas were categorized as low transmission with prevalence

Abstract Background Histidine-rich protein 2 (HRP2)-based malaria rapid diagnostic tests (RDTs) are effective and widely used for the detection of wild-type Plasmodium falciparum infections. Although recent studies have reported false negative HRP2 RDT results due to pfhrp2 and pfhrp3 gene deletions in different countries, there is a paucity of data on the deletions of these genes in Tanzania. Methods A community-based cross-sectional survey was conducted between July and November 2017 in four regions: Geita, Kigoma, Mtwara and Ruvuma. All participants had microscopy and RDT performed in the field and provided a blood sample for laboratory multiplex antigen detection (for Plasmodium lactate dehydrogenase, aldolase, and P. falciparum HRP2). Samples showing RDT false negativity or aberrant relationship of HRP2 to pan-Plasmodium antigens were genotyped to detect the presence/absence of pfhrp2/3 genes. Results Of all samples screened by the multiplex antigen assay (n = 7543), 2417 (32.0%) were positive for any Plasmodium antigens while 5126 (68.0%) were negative for all antigens. The vast majority of the antigen positive samples contained HRP2 (2411, 99.8%), but 6 (0.2%) had only pLDH and/or aldolase without HRP2. Overall, 13 samples had an atypical relationship between a pan-Plasmodium antigen and HRP2, but were positive by PCR. An additional 16 samples with negative HRP2 RDT results but P. falciparum positive by microscopy were also chosen for pfhrp2/3 genotyping. The summation of false negative RDT results and laboratory antigen results provided 35 total samples with confirmed P. falciparum DNA for pfhrp2/3 genotyping. Of the 35 samples, 4 (11.4%) failed to consistently amplify positive control genes; pfmsp1 and pfmsp2 and were excluded from the analysis. The pfhrp2 and pfhrp3 genes were successfully amplified in the remaining 31 (88.6%) samples, confirming an absence of deletions in these genes. Conclusions This study provides evidence that P. falciparum parasites in the study area have no deletions of both pfhrp2 and pfhrp3 genes. Although single gene deletions could have been missed by the multiplex antigen assay, the findings support the continued use of HRP2-based RDTs in Tanzania for routine malaria diagnosis. There is a need for the surveillance to monitor the status of pfhrp2 and/or pfhrp3 deletions in the future.

Abstract Objective Good quality microscopy is critical for accurate detection and confirmation of malaria parasite infections. Microscopy relies on the skills of technicians to prepare and read slides, high quality reagents, and a good program of internal and external quality control (EQA), which are lacking in most malaria endemic settings. This study was undertaken between January 2016 and December 2018 to pilot an EQA of microscopy for improved diagnosis of malaria and patient care in Tanzanian Military health facilities. Results Of all blood smears crosschecked (n = 4000) at baseline, only 38.5% were incorrectly diagnosed by laboratory staff with false positive and negative rates of 46.7% and 16.4%, respectively. During the implementation of EQA, false positive and negative results decreased due to increased quality index of slide preparation and reading through supportive supervision, and retraining of laboratory personnel. There was a gradual increase of quarterly and annual total quality index for all laboratories, from 60% in 2016 to 78% in 2017 and 90% in 2018. The mean proficiency testing performance scores also increased from 75% in 2016 to 82% in 2017 and to 90% in 2018. Poor blood smear preparation and staining contributed to high false positive and negative rates while EQA helped in improvement of diagnostics.

The genome of the malaria parasite Plasmodium falciparum contains a record of past evolutionary forces. Here, using 2537 parasite sequences from the Democratic Republic of the Congo, the authors demonstrate how drug pressure and human movement have shaped the present-day parasite population.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Abstract Parasite resistance against anti-malarial drugs is a major threat to the ongoing malaria control and elimination strategies. This is especially true since resistance to the currently recommended artemisinins and partner drugs has been confirmed in South East Asia (SEA) and new anti-malarial compounds are not expected to be available in the near future. Spread from SEA or independent emergence of artemisinin resistance in sub-Saharan Africa (SSA) could reverse the achievements in malaria control that have been attained in the past two decades and derail the ongoing elimination strategies. The current surveillance of clinical efficacy and resistance to anti-malarial drugs is based on efficacy trials to assess the clinical performance of anti-malarials, in vivo/ex vivo assessment of parasite susceptibility to anti-malarials and prevalence of known molecular markers of drug resistance. Whereas clinical efficacy trials are restricted by cost and the complex logistics of patient follow-up, molecular detection of genetic mutations associated with resistance or reduced susceptibility to anti-malarials is by contrast a simple and powerful tool for early detection and monitoring of the prevalence of resistant parasites at population level. This provides needed information before clinical failure emerges, allowing policy makers to anticipate problems and respond. The various methods previously used in detection of molecular markers of drug resistance share some limitations: low-throughput, and high costs per sample and demanding infrastructure. However, recent technological advances including next-generation sequencing (NGS) methodologies promise greatly increased throughput and reduced costs, essentially providing unprecedented potential to address different research and operational questions of relevance for drug policy. This review assesses the potential role of NGS to provide comprehensive information that could guide drug policies in malaria endemic countries and looks at the foreseeable challenges facing the establishment of NGS approaches for routine surveillance of parasite resistance to anti-malarials in SSA.

Abstract Next-generation sequencing (NGS) technologies are increasingly being used to address a diverse range of biological and epidemiological questions. The current understanding of malaria transmission dynamics and parasite movement mainly relies on the analyses of epidemiologic data, e.g. case counts and self-reported travel history data. However, travel history data are often not routinely collected or are incomplete, lacking the necessary level of accuracy. Although genetic data from routinely collected field samples provides an unprecedented opportunity to track the spread of malaria parasites, it remains an underutilized resource for surveillance due to lack of local awareness and capacity, limited access to sensitive laboratory methods and associated computational tools and difficulty in interpreting genetic epidemiology data. In this review, the potential roles of NGS in better understanding of transmission patterns, accurately tracking parasite movement and addressing the emerging challenges of imported malaria in low transmission settings of sub-Saharan Africa are discussed. Furthermore, this review highlights the insights gained from malaria genomic research and challenges associated with integrating malaria genomics into existing surveillance tools to inform control and elimination strategies.

Abstract Targeted Next Generation Sequencing (TNGS) is an efficient and economical Next Generation Sequencing (NGS) platform and the preferred choice when specific genomic regions are of interest. So far, only institutions located in middle and high-income countries have developed and implemented the technology, however, the efficiency and cost savings, as opposed to more traditional sequencing methodologies (e.g. Sanger sequencing) make the approach potentially well suited for resource-constrained regions as well. In April 2018, scientists from the Plasmodium Diversity Network Africa (PDNA) and collaborators met during the 7th Pan African Multilateral Initiative of Malaria (MIM) conference held in Dakar, Senegal to explore the feasibility of applying TNGS to genetic studies and malaria surveillance in Africa. The group of scientists reviewed the current experience with TNGS platforms in sub-Saharan Africa (SSA) and identified potential roles the technology might play to accelerate malaria research, scientific discoveries and improved public health in SSA. Research funding, infrastructure and human resources were highlighted as challenges that will have to be mitigated to enable African scientists to drive the implementation of TNGS in SSA. Current roles of important stakeholders and strategies to strengthen existing networks to effectively harness this powerful technology for malaria research of public health importance were discussed.

Abstract Background The World Health Organization recommends regular therapeutic efficacy studies (TES) to monitor the performance of first and second-line anti-malarials. In 2016, efficacy and safety of artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria were assessed through a TES conducted between April and October 2016 at four sentinel sites of Kibaha, Mkuzi, Mlimba, and Ujiji in Tanzania. The study also assessed molecular markers of artemisinin and lumefantrine (partner drug) resistance. Methods Eligible patients were enrolled at the four sites, treated with standard doses of AL, and monitored for 28 days with clinical and laboratory assessments. The main outcomes were PCR corrected cure rates, day 3 positivity rates, safety of AL, and prevalence of single nucleotide polymorphisms in Plasmodium falciparum kelch 13 (Pfk13) (codon positions: 440–600) and P. falciparum multi-drug resistance 1 (Pfmdr1) genes (codons: N86Y, Y184F and D1246Y), markers of artemisinin and lumefantrine resistance, respectively. Results Of 344 patients enrolled, three withdrew, six were lost to follow-up; and results were analysed for 335 (97.4%) patients. Two patients had treatment failure (one early treatment failure and one recrudescent infection) after PCR correction, yielding an adequate clinical and parasitological response of > 98%. Day 3 positivity rates ranged from 0 to 5.7%. Common adverse events included cough, abdominal pain, vomiting, and diarrhoea. Two patients had serious adverse events; one died after the first dose of AL and another required hospitalization after the second dose of AL (on day 0) but recovered completely. Of 344 samples collected at enrolment (day 0), 92.7% and 100% were successfully sequenced for Pfk13 and Pfmdr1 genes, respectively. Six (1.9%) had non-synonymous mutations in Pfk13, none of which had been previously associated with artemisinin resistance. For Pfmdr1, the NFD haplotype (codons N86, 184F and D1246) was detected in 134 (39.0%) samples; ranging from 33.0% in Mlimba to 45.5% at Mkuzi. The difference among the four sites was not significant (p = 0.578). All samples had a single copy of the Pfmdr1 gene. Conclusion The study indicated high efficacy of AL and the safety profile was consistent with previous reports. There were no known artemisinin-resistance Pfk13 mutations, but there was a high prevalence of a Pfmdr1 haplotype associated with reduced sensitivity to lumefantrine (but no reduced efficacy was observed in the subjects). Continued TES and monitoring of markers of resistance to artemisinin and partner drugs is critical for early detection of resistant parasites and to inform evidence-based malaria treatment policies. Trial Registration ClinicalTrials.gov NCT03387631

Abstract Background The Tanzanian National Malaria Control Programme (NMCP) and its partners have been implementing regular therapeutic efficacy studies (TES) to monitor the performance of different drugs used or with potential use in Tanzania. However, most of the recent TES focused on artemether–lumefantrine, which is the first-line anti-malarial for the treatment of uncomplicated falciparum malaria. Data on the performance of other artemisinin-based combinations is urgently needed to support timely review and changes of treatment guidelines in case of drug resistance to the current regimen. This study was conducted at two NMCP sentinel sites (Kibaha, Pwani and Ujiji, Kigoma) to assess the efficacy and safety of artesunate–amodiaquine (ASAQ) and dihydroartemisinin–piperaquine (DP), which are the current alternative artemisinin-based combinations in Tanzania. Methods This was a single-arm prospective evaluation of the clinical and parasitological responses of ASAQ and DP for directly observed treatment of uncomplicated falciparum malaria. Children aged 6 months to 10 years and meeting the inclusion criteria were enrolled and treated with either ASAQ or DP. In each site, patients were enrolled sequentially; thus, enrolment of patients for the assessment of one artemisinin-based combination was completed before patients were recruited for assessment of the second drugs. Follow-up was done for 28 or 42 days for ASAQ and DP, respectively. The primary outcome was PCR corrected cure rates while the secondary outcome was occurrence of adverse events (AEs) or serious adverse events (SAEs). Results Of the 724 patients screened at both sites, 333 (46.0%) were enrolled and 326 (97.9%) either completed the 28/42 days of follow-up, or attained any of the treatment outcomes. PCR uncorrected adequate clinical and parasitological response (ACPR) for DP on day 42 was 98.8% and 75.9% at Kibaha and Ujiji, respectively. After PCR correction, DP’s ACPR was 100% at both sites. For ASAQ, no parasite recurrence occurred giving 100% ACPR on day 28. Only one patient in the DP arm (1.1%) from Ujiji had parasites on day 3. Of the patients recruited (n = 333), 175 (52.6%) had AEs with 223 episodes (at both sites) in the two treatment groups. There was no SAE and the commonly reported AE episodes (with > 5%) included, cough, running nose, abdominal pain, diarrhoea and fever. Conclusion Both artemisinin-based combinations had high cure rates with PCR corrected ACPR of 100%. The two drugs had adequate safety with no SAE and all AEs were mild, and not associated with the anti-malarials. Continued TES is critical to monitor the performance of nationally recommended artemisinin-based combination therapy and supporting evidence-based review of malaria treatment policies. Trial registration This study is registered at ClinicalTrials.gov, No. NCT03431714

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Abstract Background Artemisinin-based combination therapy (ACT) is the first-line anti-malarial treatment of uncomplicated malaria in most malaria endemic countries, including Tanzania. Unfortunately, there have been reports of artemisinin resistance and ACT failure from South East Asia highlighting the need to monitor therapeutic efficacy of ACT in these countries as recommended by World Health Organization. Methods Open-label single arm studies in mainland Tanzania were conducted in nine sentinel sites in 2011, 2012 and 2015 to assess the efficacy and safety of artemether/lumefantrine (AL) and artesunate/amodiaquine (ASAQ) using 28 days follow-up and dihydroartemisinin/piperaquine (DHAPQ) using 42 days follow-up. Mutations in the propeller domain of the Plasmodium falciparum kelch 13 (k13) gene and amplification of the P. falciparum plasmepsin 2 (pm2) gene, associated with artemisinin and piperaquine (PQ) resistance, were also investigated. Results Of the 428 patients enrolled, 328 patients provided study endpoint. For AL, the PCR corrected per-protocol analysis showed adequate clinical and parasitological response (ACPR) of 90.3% (n = 28; 95% CI 74.2–98.0) in Kyela 2012, 95.7% (n = 22; 95% CI 78.1–99.0) in Chamwino, 100% in Muheza (n = 29; 95% CI 88.1–100), 100% in Nagaga (n = 39; 95% CI 91.0–100) and Kyela 2015 (n = 60; 95% CI 94.0–100). For ASAQ, PCR corrected ACPR of 98% (n = 49; 95% CI 89.4–99.9) and 100% (n = 25; 95% CI 86.3–100) were observed in 2011 in Ujiji and Kibaha, respectively. For DHAPQ, the ACPR was 100% (n = 71; 95% CI 94.9–100). Of the 235 samples with genetic interpretable results, only 7 (3%) had non-synonymous k13 mutations. None of these are candidate or validated markers of artemisinin resistance and all patients carrying these alleles cleared the parasites on day 3. Of the DHAPQ group, 10% (3/29) of the samples with interpretable results had pm2 multiple copies and none of them was associated with treatment failure. Conclusion All the tested ACT in mainland Tanzania were highly efficacious and none of validated k13 mutants associated with artemisinin resistance was observed. However, three isolates with multiple copy numbers of pm2 gene associated with PQ resistance among the limited samples tested successfully calls for further investigation. Trial registration Number ACTRN12615000159550. Registered 18th February 2015, https://www.anzctr.org.au/trial/MyTrial.aspx

Abstract Background Artemether–lumefantrine (AL) is the recommended first-line artemisinin-based combination therapy (ACT) for the treatment of uncomplicated falciparum malaria in most of the malaria-endemic countries, including Tanzania. Recently, dihydroartemisinin–piperaquine (DP) has been recommended as the alternative anti-malarial to ensure effective case management in Tanzania. This study assessed the parasite clearance rate and efficacy of AL and DP among patients aged 6 months to 10 years with uncomplicated falciparum malaria in two sites with different malaria transmission intensity. Methods This was an open-label, randomized trial that was conducted at two sites of Muheza Designated District Hospital and Ujiji Health Centre in Tanga and Kigoma regions, respectively. Patients meeting inclusion criteria were enrolled, treated with either AL or DP and followed up for 28 (extended to 42) and 42 (63) days for AL and DP, respectively. Parasite clearance time was monitored in the first 72 h post treatment and the clearance rate constant and half-life were calculated using an established parasite clearance estimator. The primary outcome was parasitological cure on days 28 and 42 for AL and DP, respectively, while secondary outcome was extended parasitological cure on days 42 and 63 for AL and DP, respectively. Results Of the 509 children enrolled (192 at Muheza and 317 at Ujiji), there was no early treatment failure and PCR uncorrected cure rates on day 28 in the AL group were 77.2 and 71.2% at Muheza and Ujiji, respectively. In the DP arm, the PCR uncorrected cure rate on day 42 was 73.6% at Muheza and 72.5% at Ujiji. With extended follow-up (to day 42 for AL and 63 for DP) cure rates were lower at Ujiji compared to Muheza (AL: 60.2 and 46.1%, p = 0.063; DP: 57.6 and 40.3% in Muheza and Ujiji, respectively, p = 0.021). The PCR corrected cure rate ranged from 94.6 to 100% for all the treatment groups at both sites. Parasite clearance rate constant was similar in the two groups and at both sites (

Abstract Background Although the recent decline of malaria burden in some African countries has been attributed to a scale-up of interventions, such as bed nets (insecticide-treated bed nets, ITNs/long-lasting insecticidal nets, LLINs), the contribution of other factors to these changes has not been rigorously assessed. This study assessed the trends of Plasmodium falciparum prevalence in Magoda (1992–2017) and Mpapayu (1998–2017) villages of Muheza district, North-eastern Tanzania, in relation to changes in the levels of different interventions and rainfall patterns. Methods Individuals aged 0–19 years were recruited in cross-sectional surveys to determine the prevalence of P. falciparum infections in relation to different malaria interventions deployed, particularly bed nets and anti-malarial drugs. Trends and patterns of rainfall in Muheza for 35 years (from 1981 to 2016) were assessed to determine changes in the amount and pattern of rainfall and their possible impacts on P. falciparum prevalence besides of those ascribed to interventions. Results High prevalence (84–54%) was reported between 1992 and 2000 in Magoda, and 1998 and 2000 in Mpapayu, but it declined sharply from 2001 to 2004 (from 52.0 to 25.0%), followed by a progressive decline between 2008 and 2012 (to ≤ 7% in both villages). However, the prevalence increased significantly from 2013 to 2016 reaching ≥ 20.0% in 2016 (both villages), but declined in the two villages to ≤ 13% in 2017. Overall and age specific P. falciparum prevalence decreased in both villages over the years but with a peak prevalence shifting from children aged 5–9 years to those aged 10–19 years from 2008 onwards. Bed net coverage increased from 98% in 2001 and was ≥ 85.0% in 2004 in both villages; followed by fluctuations with coverage ranging from 35.0 to ≤ 98% between 2008 and 2017. The 12-month weighted anomaly standardized precipitation index showed a marked rainfall deficit in 1990–1996 and 1999–2010 coinciding with declining prevalence and despite relatively high bed net coverage from 2000. From 1992, the risk of infection decreased steadily up to 2013 when the lowest risk was observed (RR = 0.07; 95% CI 0.06–0.08, P

Abstract Background Although death records are useful for planning and monitoring health interventions, such information is limited in most developing countries. Verbal autopsy (VA) interviews are alternatively used to determine causes of death in places without or with incomplete hospital records. This study was conducted to determine all causes and cause-specific mortality in Korogwe health and demographic surveillance system (HDSS) undertaken in Korogwe district, northeastern Tanzania. Methods The study was conducted from January 2006 to December 2012 in 14 villages under Korogwe HDSS. Vital events such as births, deaths and migrations were routinely updated quarterly. A standard VA questionnaire was administered to parents/close relatives of the deceased to determine cause of death. Results Overall, 1325 deaths of individuals with median age of 46 years were recorded in a population with 170,471.4 person years observed (PY). Crude mortality rate was 7.8 per 1000 PY (95% CI 7.2–8.4) and the highest rate was observed in infants (77.9 per 1000 PY; 95% CI 67.4–90.0). The overall mortality increased between 2006 and 2007, followed by a slight decline up to 2011, with the highest decrease observed in 2012. Causes of deaths were established in 942 (71.1%) deaths and malaria (198 deaths, 21.0%) was the leading cause of death in all age groups except adults (15–59 years). HIV/AIDS (17.6%, n = 365) was the leading cause of death in individuals aged 15–59 years followed by malaria (13.9%) and tuberculosis. Non-communicable diseases (NCDs) including stroke, hypertension, cancer, and cardiac failure caused majority of deaths in elderly (60 years and above) accounting for 37.1% (n = 348) of all deaths, although malaria was the single leading cause of death in this group (16.6%). Conclusion The study showed a significant decline of deaths in the Korogwe HDSS site and malaria was the main cause of death in all age groups (except adults, aged 15–59 years) while HIV/AIDS and NCDs were the main causes in adults and elderly, respectively. Further surveillance is required to monitor and document changes in cause-specific mortality as malaria transmission continues to decline in this and other parts of Tanzania.

Abstract Background Early detection of febrile illnesses at community level is essential for improved malaria case management and control. Currently, mobile phone-based technology has been commonly used to collect and transfer health information and services in different settings. This study assessed the applicability of mobile phone-based technology in real-time reporting of fever cases and management of malaria by village health workers (VHWs) in north-eastern Tanzania. Methods The community mobile phone-based disease surveillance and treatment for malaria (ComDSTM) platform, combined with mobile phones and web applications, was developed and implemented in three villages and one dispensary in Muheza district from November 2013 to October 2014. A baseline census was conducted in May 2013. The data were uploaded on a web-based database and updated during follow-up home visits by VHWs. Active and passive case detection (ACD, PCD) of febrile cases were done by VHWs and cases found positive by malaria rapid diagnostic test (RDT) were given the first dose of artemether–lumefantrine (AL) at the dispensary. Each patient was visited at home by VHWs daily for the first 3 days to supervise intake of anti-malarial and on day 7 to monitor the recovery process. The data were captured and transmitted to the database using mobile phones. Results The baseline population in the three villages was 2934 in 678 households. A total of 1907 febrile cases were recorded by VHWs and 1828 (95.9%) were captured using mobile phones. At the dispensary, 1778 (93.2%) febrile cases were registered and of these, 84.2% were captured through PCD. Positivity rates were 48.2 and 45.8% by RDT and microscopy, respectively. Nine cases had treatment failure reported on day 7 post-treatment and adherence to treatment was 98%. One patient with severe febrile illness was referred to Muheza district hospital. Conclusion The study showed that mobile phone-based technology can be successfully used by VHWs in surveillance and timely reporting of fever episodes and monitoring of treatment failure in remote areas. Further optimization and scaling-up will be required to utilize the tools for improved malaria case management and drug resistance surveillance.

Abstract Background Sickle Cell Trait (SCT) has been shown to be protective against malaria. A growing literature suggests that malaria exposure can reduce educational attainment. This study assessed the relationship and interactions between malaria, SCT and educational attainment in north-eastern Tanzania. Methods Seven hundred sixty seven children were selected from a list of individuals screened for SCT. Febrile illness and malaria incidence were monitored from January 2006 to December 2013 by community health workers. Education outcomes were extracted from the Korogwe Health and Demographic Surveillance system in 2015. The primary independent variables were malaria and SCT. The association between SCT and the number of fever and malaria episodes from 2006 to 2013 was analyzed. Main outcomes of interest were school enrolment and educational attainment in 2015. Results SCT was not associated with school enrolment (adjusted OR 1.42, 95% CI [0.593,3.412]) or highest grade attained (adjusted grade difference 0.0597, 95% CI [−0.567, 0.686]). SCT was associated with a 29% reduction in malaria incidence (adjusted IRR 0.71, 95% CI [0.526, 0.959]) but not with fever incidence (adjusted IRR 0.905, 95% CI [0.709-1.154]). In subgroup analysis of individuals with SCT, malaria exposure was associated with reduced school enrollment (adjusted OR 0.431, 95% CI [0.212, 0.877]). Conclusions SCT appears to reduce incidence of malaria. Overall, children with SCT do not appear to attend more years of school; however children who get malaria despite SCT appear to have lower levels of enrolment in education than their peers.

Abstract Background Malaria in pregnancy (MiP) remains a major public health challenge in areas of high malaria transmission. Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended to prevent the adverse consequences of MiP. The effectiveness of SP for IPTp may be reduced in areas where the dhps581 mutation (a key marker of high level SP resistance) is found; this mutation was previously reported to be common in the Tanga Region of northern Tanzania, but there are limited data from other areas. The frequency of molecular markers of SP resistance was investigated in malaria parasites from febrile patients at health centres (HC) in seven regions comprising the Lake and Southern Zones of mainland Tanzania as part of the ongoing efforts to generate national-wide data of SP resistance. Methods A cross-sectional survey was conducted in the outpatient departments of 14 HCs in seven regions from April to June, 2015. 1750 dried blood spot (DBS) samples were collected (117 to 160 per facility) from consenting patients with positive rapid diagnostic tests for malaria, and no recent (within past 2 months) exposure to SP or related drugs. DNA was extracted from the DBS, pooled by HC, and underwent pooled targeted amplicon deep sequencing to yield estimates of mutated parasite allele frequency at each locus of interest. Results The dhps540 mutation was common across all 14 sites, ranging from 55 to 98.4% of sequences obtained. Frequency of the dhps581 mutation ranged from 0 to 2.4%, except at Kayanga HC (Kagera Region, Lake Zone) where 24.9% of sequences obtained were mutated. The dhfr164 mutation was detected only at Kanyanga HC (0.06%). Conclusion By pooling DNA extracts, the allele frequency of mutations in 14 sites could be directly determined on a single deep-sequencing run. The dhps540 mutant was very common at all locations. Surprisingly, the dhps581 was common at one health center, but rare in all the others, suggesting that there is geographic micro-heterogeneity in mutant distribution and that accurate surveillance requires inclusion of multiple sites. A better understanding of the effect of the dhps581 mutant on the efficacy of IPTp-SP is needed.

Antimalarial drug resistance is an evolving global health security threat to malaria control. Early detection of Plasmodium falciparum resistance through therapeutic efficacy studies and associated genetic analyses may facilitate timely implementation of intervention strategies. The US President’s Malaria Initiative–supported Antimalarial Resistance Monitoring in Africa Network has assisted numerous laboratories in partner countries in acquiring the knowledge and capability to independently monitor for molecular markers of antimalarial drug resistance.

Super-resistant Plasmodium falciparum threatens the effectiveness of sulfadoxine–pyrimethamine in intermittent preventive treatment for malaria during pregnancy. It is characterized by the A581G Pfdhps mutation on a background of the double-mutant Pfdhps and the triple-mutant Pfdhfr. Using samples collected during 2004–2008, we investigated the evolutionary origin of the A581G mutation by characterizing microsatellite diversity flanking Pfdhps triple-mutant (437G+540E+581G) alleles from 3 locations in eastern Africa and comparing it with double-mutant (437G+540E) alleles from the same area. In Ethiopia, both alleles derived from 1 lineage that was distinct from those in Uganda and Tanzania. Uganda and Tanzania triple mutants derived from the previously characterized southeastern Africa double-mutant lineage. The A581G mutation has occurred multiple times on local Pfdhps double-mutant backgrounds; however, a novel microsatellite allele incorporated into the Tanzania lineage since 2004 illustrates the local expansion of emergent triple-mutant lineages.