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.

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.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients enrolled at 100 health facilities throughout Tanzania: February to July 2021.

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.

Prevalence of mutations associated with artemisinin partial resistance and sulfadoxine-pyrimethamine resistance in 13 regions in Tanzania in 2021: a cross-sectional survey.

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.