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Background: In 2021, nationwide malaria molecular surveillance revealed a high prevalence of a validated artemisinin resistance marker, the kelch13 (k13) Arg561His mutation, in the Kagera region of northwestern Tanzania. We aimed to investigate the efficacy of artemether-lumefantrine and artesunate-amodiaquine and to confirm the presence of artemisinin partial resistance (ART-R) in the Karagwe district of this region., Methods: This single-arm, therapeutic efficacy study was carried out at the Bukangara dispensary in the Karagwe district of the Kagera region in northwestern Tanzania. Eligible participants were aged between 6 months and 120 months, had confirmed Plasmodium falciparum asexual parasitaemia, and met other inclusion criteria according to WHO's standard protocol. Participants were enrolled, treated sequentially with either artemether-lumefantrine or artesunate-amodiaquine, and assessed clinically and parasitologically for 28 days as per WHO protocol. Parasitaemia was measured every 8 h until day 3, on day 7, and then during weekly follow-up visits until day 28. Mutations in the k13 gene and extended haplotypes with the mutations were analysed, and comparisons were made with previous samples collected in the same region of Kagera and in Rwanda and southeast Asia. The primary endpoint was PCR-corrected cure rate., Findings: Between April 29 and Sept 1, 2022, 343 patients were screened, of whom 176 were enrolled: 88 in each treatment group. The PCR-corrected cure rate was 98% (95% CI 91-100) in the artemether-lumefantrine group and 100% (96-100) in the artesunate-amodiaquine group. Persistent parasitaemia on day 3 occurred in 11 (13%) of 88 patients in the artemether-lumefantrine group and 17 (19%) of 88 patients in the artesunate-amodiaquine group. Arg561His mutations on day 0 and parasitaemia on day 3 were reported in eight (9%) of 87 patients in the artemether-lumefantrine group and ten (12%) of 86 patients in the artesunate-amodiaquine group. The median parasite clearance half-life in patients harbouring parasites with Arg561His mutation was 6·1 h in the artemether-lumefantrine group and 6·0 h in the artesunate-amodiaquine group. Parasites with the Arg561His mutation were not similar to those from southeast Asia and Rwanda but had similar haplotypes to parasites reported in the same Tanzanian region of Kagera in 2021., Interpretation: This study confirms the presence of ART-R in Tanzania, although artemether-lumefantrine and artesunate-amodiaquine showed high efficacy. A context-specific response strategy and vigilance to detect the reduced efficacy of current antimalarial treatments and ART-R in other parts of the country are urgently needed., Funding: The Bill & Melinda Gates Foundation and the US National Institutes of Health., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: The emergence of the artemisinin partial resistance (ART-R) mutation in the Plasmodium falciparum kelch13 gene (k13), Arg561His, in Rwanda and the regional presence of polymorphisms affecting sulfadoxine-pyrimethamine have raised concern in neighbouring Tanzania. The goal of this study was to assess the status of antimalarial resistance in Tanzania, with a focus on the border with Rwanda, to understand the distribution of the Arg561His mutation, partner drug resistance, and resistance to chemoprevention drugs., Methods: In this cross-sectional survey, capillary dried blood spots were collected from malaria positive asymptomatic individuals in the community and symptomatic individuals in health facilities aged 6 months and older, in 13 regions of mainland Tanzania from Jan 31 to June 26, 2021. Exclusion criteria included residence of the areas other than the target sites, presenting to the health facility for care and treatment of conditions other than malaria, and not providing informed consent. Samples were assessed for antimalarial resistance polymorphisms and genetic relatedness using molecular inversion probes targeting P falciparum and short-read whole-genome sequencing. The primary outcome was the prevalence of molecular markers of antimalarial resistance at the region level, as well as at the district level in Kagera, a region in the northwest of the country at the border with Rwanda., Findings: 6855 (88·1%) of 7782 capillary dried blood spot samples collected were successfully genotyped. The overall prevalence of k13 Arg561His in Kagera was 7·7% (90% CI 6·0-9·4; 50 of 649), with the highest prevalence in the districts near the Rwandan border (22·8% [31 of 136] in Karagwe, 14·4% [17 of 118]) in Kyerwa, and 1·4% [two of 144] in Ngara). k13 Arg561His was uncommon in the other regions. Haplotype analysis suggested that some of these parasites are related to isolates collected in Rwanda in 2015, supporting regional spread of Arg561His. However, a novel k13 Arg561His haplotype was observed, potentially indicating a second origin in the region. Other validated k13 resistance polymorphisms (one Arg622Ile and two Ala675Val isolates) were also identified. A region of prevalent dihydrofolate reductase Ile164Leu mutation, associated with sulfadoxine-pyrimethamine resistance, was also identified in Kagera (15·2% [12·6-17·8%]; 80 of 526). The mutant crt Lys76Thr mutation, associated with chloroquine and amodiaquine resistance, was uncommon, occurring only in 75 of 2861 genotyped isolates, whereases the wild-type mdr1 Asn86Tyr allele, associated with reduced sensitivity to lumefantrine, was found in 99·7% (3819 of 3830) of samples countrywide., Interpretation: These findings show that the k13 Arg561His mutation is common in northwest Tanzania and that multiple emergences of ART-R, similar as to what was seen in southeast Asia, have occurred. Mutations associated with high levels of sulfadoxine-pyrimethamine resistance are common. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Understanding how multiple emergences interact with drivers of regional spread is essential for combating ART-R in Africa., Funding: This study was funded by the Bill & Melinda Gates Foundation and the National Institutes of Health., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Recent data indicate that non-Plasmodium falciparum species may be more prevalent than thought in sub-Saharan Africa. Although Plasmodium malariae, Plasmodium ovale spp., and Plasmodium vivax are less severe than P. falciparum, treatment and control are more challenging, and their geographic distributions are not well characterized., Methods: We randomly selected 3284 of 12 845 samples collected from cross-sectional surveys in 100 health facilities across 10 regions of Mainland Tanzania and performed quantitative real-time PCR to determine presence and parasitemia of each malaria species., Results: P. falciparum was most prevalent, but P. malariae and P. ovale were found in all but 1 region, with high levels (>5%) of P. ovale in 7 regions. The highest P. malariae positivity rate was 4.5% in Mara and 8 regions had positivity rates ≥1%. We only detected 3 P. vivax infections, all in Kilimanjaro. While most nonfalciparum malaria-positive samples were coinfected with P. falciparum, 23.6% (n = 13 of 55) of P. malariae and 14.7% (n = 24 of 163) of P. ovale spp. were monoinfections., Conclusions: P. falciparum remains by far the largest threat, but our data indicate that malaria elimination efforts in Tanzania will require increased surveillance and improved understanding of the biology of nonfalciparum species., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

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., (© 2024. The Author(s).)

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 (< 5%) in all four regions; P. vivax was not detected in any sample., Conclusions: The results of this study indicate that malaria elimination efforts in Tanzania will need to account for and enhance surveillance of these non-falciparum species., (© 2024. The Author(s).)

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 (R S  = 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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Background: Asymptomatic malaria infections largely remain undetected and act as a reservoir for continuous transmission. The study assessed the prevalence of submicroscopic asymptomatic malaria infections and anaemia in two rural low (300 m above sea level) and highland (700 m asl) settings of Korogwe District north-eastern Tanzania., Methods: A cross-sectional malariometric survey involving individuals aged 0-19 years was conducted in June 2018 in the two rural villages. Venous blood was collected from eligible study participants for estimation of haemoglobin level, detection of malaria by rapid diagnostic test (RDT), quantification of malaria parasitaemia by microscopy, as well as dried blood spot (DBS) for determining submicroscopic infections by PCR targeting the small subunit of the ribosomal ribonucleic acid (ssrRNA) of human Plasmodium., Results: Out of 565 individuals tested, 211 (37.3%) were malaria positive based on RDT, whereas only 81 (14.3%) were positive by microscopy. There was no significant difference in the prevalence between the highland and the lowland village, p = 0.19 and p = 0.78 microscopy and RDT, respectively. Three out of 206 (1.5%) RDT/microscopy negative samples were P. falciparum positive by PCR. Of the 211 RDT and 81 microscopy positive, 130 (61.6%) and 33 (40.7%), respectively, were defined as being asymptomatic. Of the 565 individuals, 135 (23.9%) were anaemic (haemoglobin < 11 g/dL) out of which 5.2% were severely anaemic. The risk of being anaemic was significantly higher among individuals with asymptomatic malaria as compared to those without malaria as confirmed by RDT (AOR = 2.06 (95% CI 1.32-3.20) while based on microscopic results there was no significant differences observed (AOR = 2.09, 95% CI 0.98-4.47). Age and altitude had no effect on the risk of anaemia even after adjusting for asymptomatic malaria., Conclusions: Asymptomatic malaria is associated with an increased risk of having anaemia in the study communities. The findings highlight the need for targeted interventions focusing on asymptomatic infections which is an important risks factor for anaemia in the community and act as a source of continued transmission of malaria in the study area., (© 2021. The Author(s).)

High-throughput Plasmodium genomic data is increasingly useful in assessing prevalence of clinically important mutations and malaria transmission patterns. Understanding parasite diversity is important for identification of specific human or parasite populations that can be targeted by control programmes, and to monitor the spread of mutations associated with drug resistance. An up-to-date understanding of regional parasite population dynamics is also critical to monitor the impact of control efforts. However, this data is largely absent from high-burden nations in Africa, and to date, no such analysis has been conducted for malaria parasites in Tanzania countrywide. To this end, over 1,000 P. falciparum clinical isolates were collected in 2017 from 13 sites in seven administrative regions across Tanzania, and parasites were genotyped at 1,800 variable positions genome-wide using molecular inversion probes. Population structure was detectable among Tanzanian P. falciparum parasites, approximately separating parasites from the northern and southern districts and identifying genetically admixed populations in the north. Isolates from nearby districts were more likely to be genetically related compared to parasites sampled from more distant districts. Known drug resistance mutations were seen at increased frequency in northern districts (including two infections carrying pfk13-R561H), and additional variants with undetermined significance for antimalarial resistance also varied by geography. Malaria Indicator Survey (2017) data corresponded with genetic findings, including average region-level complexity-of-infection and malaria prevalence estimates. The parasite populations identified here provide important information on extant spatial patterns of genetic diversity of Tanzanian parasites, to which future surveys of genetic relatedness can be compared., (© 2020 John Wiley & Sons Ltd.)