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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.
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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 (< 0.95), there was high polyclonality (ranging from 69.23% in Nachingwea to 56.9% in Muheza). No population structure was detected in the Pfcsp gene in the five regions (mean FST = 0.0068). The average nucleotide diversity (π), nucleotide differentiation (K) and haplotype diversity (Hd) in the five regions were 4.19, 0.973 and 0.0035, respectively. The C-terminal region of Pfcsp showed high nucleotide diversity at Th2R and Th3R regions. Positive values for the Tajima's D were observed in the Th2R and Th3R regions consistent with balancing selection. The Pfcsp C-terminal sequences revealed 50 different haplotypes (H_1 to H_50), with only 2% of sequences matching the 3D7 strain haplotype (H_50). Conversely, with the NF54 strain, the Pfcsp C-terminal sequences revealed 49 different haplotypes (H_1 to H_49), with only 0.4% of the sequences matching the NF54 strain (Hap_49). CONCLUSIONS: The findings demonstrate high diversity of the Pfcsp gene with limited population differentiation. The Pfcsp gene showed positive Tajima's D values, consistent with balancing selection for variants within Th2R and Th3R regions. The study observed differences between the intended haplotypes incorporated into the design of RTS,S and R21 vaccines and those present in natural parasite populations. Therefore, additional research is warranted, incorporating other regions and more recent data to comprehensively assess trends in genetic diversity within this important gene. Such insights will inform the choice of alleles to be included in the future vaccines.
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Plasmodium falciparum , Polimorfismo Genético , Proteínas Protozoarias , Selección Genética , Humanos , Enfermedades Endémicas , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , TanzaníaRESUMEN
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.
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Antígenos de Grupos Sanguíneos , Malaria Falciparum , Humanos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Eliminación de Gen , Tanzanía/epidemiología , Pruebas Diagnósticas de Rutina/métodos , Antígenos de Protozoos/genética , Malaria Falciparum/diagnóstico , Malaria Falciparum/epidemiología , Malaria Falciparum/genética , Instituciones de Salud , ADNRESUMEN
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 this gene raises 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 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 (< 0.95), there was high polyclonality (ranged from 69.23% in Nachingwea to 56.9% in Muheza). No population structure was detected in the Pfcsp gene in the five regions (mean FST= 0.0068). The average nucleotide diversity (π), nucleotide differentiation (K) and haplotype diversity (Hd) in the five regions were 4.19, 0.973 and 0.0035, respectively. The C-terminal region of Pfcsp showed high nucleotide diversity at Th2R and Th3R regions. Positive values for the Tajima's D were observed in the Th2R and Th3R regions consistent with balancing selection. The Pfcsp C-terminal sequences had 50 different haplotypes (H_1 to H_50) and only 2% of sequences matched the 3D7 strain haplotype (H_50). Conclusions: The findings demonstrate high diversity of the Pfcsp gene with limited population differentiation. The Pfcsp gene showed positive Tajima's D values for parasite populations, consistent with balancing selection for variants within Th2R and Th3R regions. This data is consistent with other studies conducted across Africa and worldwide, which demonstrate low 3D7 haplotypes and little population structure. Therefore, additional research is warranted, incorporating other regions and more recent data to comprehensively assess trends in genetic diversity within this important gene. Such insights will inform the choice of alleles to be included in the future vaccines.
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Background: Emergence of artemisinin partial resistance (ART-R) in Plasmodium falciparum is a growing threat to the efficacy of artemisinin combination therapies (ACT) and the efforts for malaria elimination. The emergence of Plasmodium falciparum Kelch13 (K13) R561H in Rwanda raised concern about the impact in neighboring Tanzania. In addition, regional concern over resistance affecting sulfadoxine-pyrimethamine (SP), which is used for chemoprevention strategies, is high. Methods: To enhance longitudinal monitoring, the Molecular Surveillance of Malaria in Tanzania (MSMT) project was launched in 2020 with the goal of assessing and mapping antimalarial resistance. Community and clinic samples were assessed for resistance polymorphisms using a molecular inversion probe platform. Findings: Genotyping of 6,278 samples collected countrywide in 2021 revealed a focus of K13 561H mutants in northwestern Tanzania (Kagera) with prevalence of 7.7% (50/649). A small number of 561H mutants (about 1%) were found as far as 800 km away in Tabora, Manyara, and Njombe. Genomic analysis suggests some of these parasites are highly related to isolates collected in Rwanda in 2015, supporting regional spread of 561H. However, a novel haplotype was also observed, likely indicating a second origin in the region. Other validated resistance polymorphisms (622I and 675V) were also identified. A focus of high sulfadoxine-pyrimethamine drug resistance was also identified in Kagera with a prevalence of dihydrofolate reductase 164L of 15% (80/526). Interpretation: These findings demonstrate the K13 561H mutation is entrenched in the region and that multiple origins of ART-R, similar as to what was seen in Southeast Asia, have occurred. Mutations associated with high levels of SP resistance are increasing. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Funding: This study was funded by the Bill and Melinda Gates Foundation and the National Institutes of Health.
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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.
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Enfermedades Transmisibles , Malaria , Sistemas CRISPR-Cas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Malaria/diagnóstico , Malaria/epidemiología , Malaria/prevención & control , TanzaníaRESUMEN
We compared the prevalence of antibiotic-resistant Escherichia coli isolates from household-level producers of broiler (commercial source breeds) and local chickens in the Arusha District of Tanzania. Households were composed of a single dwelling or residence with independent, penned broiler flocks. Free-range, scavenging chickens were mixed breed and loosely associated with individual households. A total of 1,800 E. coli isolates (1,200 from broiler and 600 from scavenging local chickens) from 75 chickens were tested for their susceptibility against 11 antibiotics by using breakpoint assays. Isolates from broiler chickens harbored a higher prevalence of antibiotic-resistant E. coli relative to scavenging local chickens, including sulfamethoxazole (80.3 versus 34%), followed by trimethoprim (69.3 versus 27.7%), tetracycline (56.8 versus 20%), streptomycin (52.7 versus 24.7%), amoxicillin (49.6 versus 17%), ampicillin (49.1 versus 16.8%), ciprofloxacin (21.9 versus 1.7%), and chloramphenicol (1.5 versus 1.2%). Except for resistance to chloramphenicol, scavenging local chickens harbored fewer resistant E. coli isolates (P < 0.05). Broiler chickens harbored more isolates that were resistant to ≥7 antibiotics (P < 0.05). The higher prevalence of antibiotic-resistant E. coli from broiler chickens correlated with the reported therapeutic and prophylactic use of antibiotics in this poultry population. We suggest that improved biosecurity measures and increased vaccination efforts would reduce reliance on antibiotics by these households.