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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 <1 asexual parasite/μl. DNA was also successfully amplified from (1) 70/71 (98.6%) archived positive RDTs (RDTs and microscopy positive) (2) 52/63 (82.5%) false negative RDTs (negative by RDTs but positive by microscopy) and (3) 4/24 (16.7%) false positive RDTs (positive by RDTs but negative by microscopy). Finally, 7(100%) negative RDTs (negative by RDTs and microscopy) were also negative by PCR., 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.

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]

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]