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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.

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 (He = 0.83, range = 0.80–0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (FST) 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 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 Recently, Gansané and colleagues published an article on inadequate efficacy of two different forms of artemisinin-based combination therapy (ACT) in Burkina Faso. The development of Plasmodium falciparum resistance to different ACT partner drugs at levels that could affect the efficacy of two ACT would both be startling and a cause for great concern. In reviewing the available data collected since 2008 on ACT efficacy in Burkina Faso, the analysis shows that the reported efficacy of the tested ACT varies greatly. Most of the studies have considerable methodological deviations and challenges, especially in PCR correction done to distinguish between recrudescence and re-infection, and in the failure to omit re-infections in the calculation of efficacy rates. So far, there is no convincing evidence in the articles reviewed that multidrug resistance has emerged in Burkina Faso. However, the potential consequence of failing ACT means that the results published by Gansané et al. urgently need to be confirmed. Furthermore, articles reporting on efficacy data need to include an examination of the potential consequences of any methodological deviations.

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites. [ABSTRACT FROM AUTHOR]

Recently, Gansané and colleagues published an article on inadequate efficacy of two different forms of artemisinin-based combination therapy (ACT) in Burkina Faso. The development of Plasmodium falciparum resistance to different ACT partner drugs at levels that could affect the efficacy of two ACT would both be startling and a cause for great concern. In reviewing the available data collected since 2008 on ACT efficacy in Burkina Faso, the analysis shows that the reported efficacy of the tested ACT varies greatly. Most of the studies have considerable methodological deviations and challenges, especially in PCR correction done to distinguish between recrudescence and re-infection, and in the failure to omit re-infections in the calculation of efficacy rates. So far, there is no convincing evidence in the articles reviewed that multidrug resistance has emerged in Burkina Faso. However, the potential consequence of failing ACT means that the results published by Gansané et al. urgently need to be confirmed. Furthermore, articles reporting on efficacy data need to include an examination of the potential consequences of any methodological deviations. [ABSTRACT FROM AUTHOR]

Abstract Background In a drug-resistant, malaria elimination setting like Western Cambodia, field research is essential for the development of novel anti-malarial regimens and the public health solutions necessary to monitor the spread of resistance and eliminate infection. Such field studies often face a variety of similar implementation challenges, but these are rarely captured in a systematic way or used to optimize future study designs that might overcome similar challenges. Field-based research staff often have extensive experience and can provide valuable insight regarding these issues, but their perspectives and experiences are rarely documented and seldom integrated into future research protocols. This mixed-methods analysis sought to gain an understanding of the daily challenges encountered by research field staff in the artemisinin-resistant, malaria elimination setting of Western Cambodia. In doing so, this study seeks to understand how the experiences and opinions of field staff can be captured, and used to inform future study designs. Methods Twenty-two reports from six field-based malaria studies conducted in Western Cambodia were reviewed using content analysis to identify challenges to conducting the research. Informal Interviews, Focus Group Discussions and In-depth Interviews were also conducted among field research staff. Thematic analysis of the data was undertaken using Nvivo 9® software. Triangulation and critical case analysis was also used. Results There was a lack of formalized avenues through which field workers could report challenges experienced when conducting the malaria studies. Field research staff faced significant logistical barriers to participant recruitment and data collection, including a lack of available transportation to cover long distances, and the fact that mobile and migrant populations (MMPs) are usually excluded from studies because of challenges in follow-up. Cultural barriers to communication also hindered participant recruitment and created unexpected delays. Field staff often paid a physical, emotional and financial cost, going beyond their duty in order to keep the study running. Conclusions Formal monthly reports filled out by field study staff could be a key tool for capturing field study staff experiences effectively, but require specific report fields to encourage staff to outline their challenges and to propose potential solutions. Forging strong bonds with communities and their leaders may improve communication, and decrease barriers to participant recruitment. Study designs that make it feasible for MMPs to participate should be pursued; in addition to increasing the potential participant pool, this will ensure that the most malaria-endemic demographic is taken into account in research studies. Overlaps between clinical care and research create ethical dilemmas for study staff, a fact that warrants careful consideration. Lessons learned from study field staff should be used to create a set of locally-relevant recommendations to inform future study designs.

Background: In a drug-resistant, malaria elimination setting like Western Cambodia, field research is essential for the development of novel anti-malarial regimens and the public health solutions necessary to monitor the spread of resistance and eliminate infection. Such field studies often face a variety of similar implementation challenges, but these are rarely captured in a systematic way or used to optimize future study designs that might overcome similar challenges. Field-based research staff often have extensive experience and can provide valuable insight regarding these issues, but their perspectives and experiences are rarely documented and seldom integrated into future research protocols. This mixed-methods analysis sought to gain an understanding of the daily challenges encountered by research field staff in the artemisinin-resistant, malaria elimination setting of Western Cambodia. In doing so, this study seeks to understand how the experiences and opinions of field staff can be captured, and used to inform future study designs. Methods: Twenty-two reports from six field-based malaria studies conducted in Western Cambodia were reviewed using content analysis to identify challenges to conducting the research. Informal Interviews, Focus Group Discussions and In-depth Interviews were also conducted among field research staff. Thematic analysis of the data was undertaken using Nvivo 9 ® software. Triangulation and critical case analysis was also used. Results: There was a lack of formalized avenues through which field workers could report challenges experienced when conducting the malaria studies. Field research staff faced significant logistical barriers to participant recruitment and data collection, including a lack of available transportation to cover long distances, and the fact that mobile and migrant populations (MMPs) are usually excluded from studies because of challenges in follow-up. Cultural barriers to communication also hindered participant recruitment and created unexpected delays. Field staff often paid a physical, emotional and financial cost, going beyond their duty in order to keep the study running. Conclusions: Formal monthly reports filled out by field study staff could be a key tool for capturing field study staff experiences effectively, but require specific report fields to encourage staff to outline their challenges and to propose potential solutions. Forging strong bonds with communities and their leaders may improve communication, and decrease barriers to participant recruitment. Study designs that make it feasible for MMPs to participate should be pursued; in addition to increasing the potential participant pool, this will ensure that the most malaria-endemic demographic is taken into account in research studies. Overlaps between clinical care and research create ethical dilemmas for study staff, a fact that warrants careful consideration. Lessons learned from study field staff should be used to create a set of locally-relevant recommendations to inform future study designs. [ABSTRACT FROM AUTHOR]