Genetic polymorphism and evidence of signatures of selection in the Plasmodium falciparum circumsporozoite protein gene in Tanzanian regions with different malaria endemicity.

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.

Prevalence of non-falciparum malaria infections among asymptomatic individuals in four regions of Mainland Tanzania.

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.

Genetic polymorphism and evidence of signatures of selection in the Plasmodium falciparum circumsporozoite protein gene in Tanzanian regions with different malaria endemicity.

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.

Malaria Species Positivity Rates Among Symptomatic Individuals Across Regions of Differing Transmission Intensities in Mainland Tanzania.

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.

Country wide surveillance reveals prevalent artemisinin partial resistance mutations with evidence for multiple origins and expansion of high level sulfadoxine-pyrimethamine resistance mutations in northwest Tanzania.

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.

Malaria species positivity rates among symptomatic individuals across regions of differing transmission intensities in Mainland Tanzania.

Recent data indicate that non- Plasmodium falciparum species may be more prevalent than previously realized in sub-Saharan Africa, the region where 95% of the world's malaria cases occur. Although Plasmodium malariae, Plasmodium ovale spp., and Plasmodium vivax are generally less severe than P. falciparum , treatment and control are more challenging, and their geographic distributions are not well characterized. In order to characterize the distribution of malaria species in Mainland Tanzania (which has a high burden and geographically heterogeneous transmission levels), we randomly selected 3,284 samples from 12,845 samples to determine presence and parasitemia of different malaria species. The samples were collected from cross-sectional surveys in 100 health facilities across ten regions and analyzed via quantitative real-time PCR to characterize regional positivity rates for each species. P. falciparum was most prevalent, but P. malariae and P. ovale were found in all regions except Dar es Salaam, with high levels (>5%) of P. ovale in seven regions (70%). The highest positivity rate of P. malariae was 4.5% in Mara region and eight regions (80%) had positivity rates ≥1%. We also detected three P. vivax infections in the very low-transmission Kilimanjaro region. While most samples that tested positive for non-falciparum malaria were co-infected with P. falciparum , 23.6% (n = 13/55) of P. malariae and 14.7% (n = 24/163) of P. ovale spp. samples were mono-infections. 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 non-falciparum species.

Malaria species prevalence among asymptomatic individuals in four regions of Mainland Tanzania.

Recent studies point to the need to incorporate non-falciparum species detection into malaria surveillance activities in sub-Saharan Africa, where 95% of malaria cases occur. Although Plasmodium falciparum infection is typically more severe, diagnosis, treatment, and control for P. malariae, P. ovale spp., and P. vivax may be more challenging. The prevalence of these species throughout sub-Saharan Africa is poorly defined. Tanzania has geographically heterogeneous transmission levels but an overall high malaria burden. In order to estimate the prevalence of malaria species in Mainland Tanzania, 1,428 samples were randomly selected from 6,005 asymptomatic isolates collected in cross-sectional community surveys across four regions and analyzed via qPCR to detect each Plasmodium species. P. falciparum was most prevalent, with P. malariae and P. ovale spp. detected at lower prevalence (<5%) in all four regions. P. vivax was not detected. Malaria elimination efforts in Tanzania will need to account for these non-falciparum species.

Potential Opportunities and Challenges of Deploying Next Generation Sequencing and CRISPR-Cas Systems to Support Diagnostics and Surveillance Towards Malaria Control and Elimination in Africa.

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.