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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 < 0.001). The prevalence and odds of infections were significantly higher in males (aOR = 1.28, 95% CI 1.08 –1.51, p = 0.003), school children (aged 5–≤10 years (aOR = 3.88, 95% CI 3.07–4.91, p < 0.001) and 10–≤15 years (aOR = 4.06, 95% CI 3.22–5.13, p < 0.001)) and among individuals who were not using bed nets (aOR = 1.22, 95% CI 1.03–1.46, p = 0.024). The odds of malaria infections were also higher in individuals with lower SES (aOR = 1.42, 95% CI 1.17–1.72, p < 0.001), and living in houses without windows (aOR = 2.08, 95% CI 1.46–2.96, p < 0.001), partially open (aOR = 1.33, 95% CI 1.11–1.58, p = 0.002) or fully open windows (aOR = 1.30, 95%CI 1.05–1.61, p = 0.015). Conclusion: The five villages had a high prevalence of malaria infections and heterogeneity at micro-geographic levels. Groups with higher odds of malaria infections included school children, males, and individuals with low SES, living in poorly constructed houses or non-bed net users. These are important baseline data from an area with high prevalence of parasites with ART-R and will be useful in planning interventions for these groups, and in future studies to monitor the trends and potential spread of such parasites, and in designing a response to ART-R. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Abstract Background Malaria prevalence has recently declined markedly in many parts of Tanzania and other sub-Saharan African countries due to scaling-up of control interventions including more efficient treatment regimens (e.g. artemisinin-based combination therapy) and insecticide-treated bed nets. Although continued molecular surveillance of malaria parasites is important to early identify emerging anti-malarial drug resistance, it is becoming increasingly difficult to obtain parasite samples from ongoing studies, such as routine drug efficacy trials. To explore other sources of parasite DNA, this study was conducted to examine if sufficient DNA could be successfully extracted from malaria rapid diagnostic tests (RDTs), used and collected as part of routine case management services in health facilities, and thus forming the basis for molecular analyses, surveillance and quality control (QC) testing of RDTs. Methods One hyper-parasitaemic blood sample (131,260 asexual parasites/μl) was serially diluted in triplicates with whole blood and blotted on RDTs. DNA was extracted from the RDT dilution series, either immediately or after storage for one month at room temperature. The extracted DNA was amplified using a nested PCR method for Plasmodium species detection. Additionally, 165 archived RDTs obtained from ongoing malaria studies were analysed to determine the amplification success and test applicability of RDT for QC testing. Results DNA was successfully extracted and amplified from the three sets of RDT dilution series and the minimum detection limit of PCR was Conclusion This study showed that DNA extracted from archived RDTs can be successfully amplified by PCR and used for detection of malaria parasites. Since Tanzania is planning to introduce RDTs in all health facilities (and possibly also at community level), availability of archived RDTs will provide an alternative source of DNA for genetic studies such as continued surveillance of parasite resistance to anti-malarial drugs. The DNA obtained from RDTs can also be used for QC testing by detecting malaria parasites using PCR in places without facilities for microscopy.

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. [ABSTRACT FROM AUTHOR]

Background: Asymptomatic malaria infections largely remain undetected and act as a reservoir for continuous transmission. Objectives: To determine the prevalence of sub-microscopic asymptomatic malaria infections and anaemia in two rural 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 RDT, quantification of malaria parasitaemia by microscopy, as well as dried blood spot (DBS) for determining sub-microscopic 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. Three out of 206 (1.5%) RDT/microscopy negative samples were P. falciparum positive by PCR. Of the 211 RDT and 81 microscopies 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). Conclusion: 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 risk factor for anaemia in the community and act as a source of continued transmission of malaria in the study area. [ABSTRACT FROM AUTHOR]

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

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.)

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).)