Novel Plasmodium falciparum histidine-rich protein 2/3 repeat type in Ethiopian malaria infection: does this affect performance of HRP2-based malaria RDT?

BACKGROUND: Rapid diagnostic tests (RDTs) provide quick, easy, and convenient early diagnosis of malaria ensuring better case management particularly in resource-constrained settings. Nevertheless, the efficiency of HRP2-based RDT can be compromised by Plasmodium falciparum histidine-rich protein 2/3 gene deletion and genetic diversity. This study explored the genetic diversity of PfHRP2/3 in uncomplicated malaria cases from Ethiopia. METHODS: A cross-sectional study was conducted from June 2022 to March 2023 at Metehara, Zenzelema and Kolla Shele health centres, Ethiopia. Finger-prick blood samples were collected for RDT testing and microscopic examination. For molecular analysis, parasite genomic DNA was extracted from venous blood. Plasmodium falciparum was confirmed using VarATS real time PCR. Additionally, PfHRP2/3 was amplified, and DNA amplicons were sequenced using Oxford Nanopore technology. RESULTS: PfHRP2/3 sequences revealed small variations in the frequency and number of amino acid repeat types per isolate across the three health centres. Twelve and eight types of amino acid repeats were identified for PfHRP2 and PfHRP3, respectively, which had been previously characterized. Repeat type 1, 4 and 7 were present in both PfHRP2 and PfHRP3 amino acid sequences. Type 2 and 7 repeats were commonly dispersed in PfHRP2, while repeat types 16 and 17 were found only in PfHRP3. A novel 17 V repeat type variant, which has never been reported in Ethiopia, was identified in six PfHRP3 amino acid sequences. The majority of the isolates, as determined by the Baker's logistic regression model, belonged to group C, of which 86% of them were sensitive to PfHRP2-based RDT. Likewise, PfHRP2-based RDT detected 100% of the isolates in group A (product of type 2 × type 7 repeats ≥ 100) and 85.7% in group B (product of types 2 × type 7 repeats 50-99) at a parasitaemia level > 250 parasite/µl. CONCLUSION: This study highlights the significant diversity observed in PfHRP2 and PfHRP3 among clinical isolates of Plasmodium falciparum in Ethiopia. This emphasizes the necessity for monitoring of PfHRP2- based RDT efficacy and their repeat type distribution using a large sample size and isolates from various ecological settings.

Histidine-rich protein (hrp) 2-based RDT false-negatives and Plasmodium falciparum hrp 2 and 3 gene deletions in low, seasonal and intense perennial transmission zones in Cameroon: a cross - sectional study.

BACKGROUND: False negative rapid diagnostic tests (RDTs) accruing to the non-detection of Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) is threatening the diagnosis and management of malaria. Although regular monitoring is necessary to gauge the level of efficacy of the tool, studies in Cameroon remain limited. This study assessed Plasmodium spp. prevalence and Pfhrp2/3 gene deletions across ecological and transmission zones in Cameroon. METHODS: This is a cross-sectional, multi-site, community- and hospital- based study, in 21 health facilities and 14 communities covering all five ecological settings in low seasonal (LS) and intense perennial (IPT) malaria transmission zones between 2019 and 2021. Participants were screened for malaria parasite using Pfhrp2 RDT and light microscopic examination of thick peripheral blood smears. DNA was extracted from dried blood spot using chelex®-100 and P. falciparum confirmed using varATS real-time quantitative Polymerase Chain Reaction (qPCR), P. malariae and P. ovale by real-time qPCR of Plasmepsin gene, and P. vivax using a commercial kit. Isolates with amplified Pfcsp and Pfama-1 genes were assayed for Pfhrp 2/3 gene deletions by conventional PCR. RESULTS: A total of 3,373 participants enrolled, 1,786 Plasmodium spp. infected, with 77.4% P. falciparum. Discordant RDT and qPCR results (False negatives) were reported in 191 (15.7%) P. falciparum mono-infected samples from LS (29%, 42) and IPT (13.9%, 149). The Pfhrp2+/Pfhrp3 + genotype was most frequent, similar between LS (5.5%, 8/145) and IPT (6.0%, 65/1,076). Single Pfhrp2 and Pfhrp3 gene deletions occurred in LS (0.7%, 1/145 each) and IPT (3.6%, 39/1,076 vs. 2.9%, 31/1,076), respectively. Whilst a single sample harboured Pfhrp2-/Pfhrp3- genotype in LS, 2.4% (26/1,076) were double deleted at IPT. Pfhrp2+/Pfhrp3- (0.3%, 3/1,076) and Pfhrp2-/Pfhrp3+ (1.2%, 13/1,076) genotypes were only observed in IPT. Pfhrp2, Pfhrp3 deletions and Pfhrp2-/Pfhrp3- genotype accounted for 78.8% (26), 69.7% (23) and 63.6% (21) RDT false negatives, respectively. CONCLUSION: Plasmodium falciparum remains the most dominant and widely distributed Plasmodium species across transmission and ecological zones in Cameroon. Although the low prevalence of Pfhrp2/3 gene deletions supports the continued use of HRP2-based RDTs for routine malaria diagnosis, the high proportion of false-negatives due to gene deleted parasites necessitates continued surveillance to inform control and elimination efforts.

Gametocyte carriage after seasonal malaria chemoprevention in Plasmodium falciparum infected asymptomatic children.

BACKGROUND: Treatment of clinical Plasmodium falciparum malaria with sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) is associated with increased post-treatment gametocyte carriage. The effect of seasonal malaria chemoprevention (SMC) with SP and AQ on gametocyte carriage was assessed in asymptomatic P. falciparum infected children. METHODS: The study was carried out in eastern Gambia. Asymptomatic P. falciparum malaria infected children aged 24-59 months old who were eligible to receive SMC (SMC group) and children 5-8 years that were not eligible to receive SMC (comparison group) were recruited. Gametocytaemia was determined by molecular methods before and after SMC administration. Gametocyte carriage between the groups was compared using the chi-squared test and within-person using conditional logistic regression. RESULTS: During the 2017 and 2018 malaria transmission seasons, 65 and 75 children were recruited in the SMC and comparison groups, respectively. Before SMC administration, gametocyte prevalence was 10.7% (7/65) in the SMC group and 13.3% (10/75) in the comparison group (p = 0.64). At day 13 (IQR 12, 13) after SMC administration, this was 9.4% (5/53) in children who received at least the first dose of SMC treatment and 12.7% (9/71) for those in the comparison group (p = 0.57). Similarly, there was no difference in prevalence of gametocytes between children that adhered to all 3-day doses of SMC treatment 15.6% (5/32) and those in the comparison group (p = 0.68). In the SMC group, within-group gametocyte carriage was similar before and after SMC administration in children that received at least the first dose of SMC treatment (OR 0.6, 95% CI 0.14-2.51; p = 0.48) and in those that adhered to all 3-day doses of SMC treatment (OR 1.0, 95% CI 0.20-4.95; p = 1.0). CONCLUSION: In this study with relative low gametocyte prevalence prior to SMC treatment, no evidence was observed that SMC treatment increased gametocyte carriage in asymptomatic P. falciparum malaria infected children.

High cases of submicroscopic Plasmodium falciparum infections in a suburban population of Lagos, Nigeria.

BACKGROUND: Asymptomatic malaria parasites are significant sources of infections for onward malaria transmission. Conventional tools for malaria diagnosis such as microscopy and rapid diagnostic test kits (RDT) have relatively low sensitivity, hence the need for alternative tools for active screening of such low-density infections. METHODS: This study tested var acidic terminal sequence-based (varATS) quantitative polymerase chain reaction (qPCR) for screening asymptomatic Plasmodium falciparum infections among dwellers of a sub-urban community in Lagos, Nigeria. Clinically healthy participants were screened for malaria using microscopy, RDT and varATS qPCR techniques. Participants were stratified into three age groups: 1-5, 6-14 and > 14 years old. RESULTS: Of the 316 participants screened for asymptomatic malaria infection, 78 (24.68%) were positive by microscopy, 99 (31.33%) were positive by RDT and 112 (35.44%) by varATS qPCR. Participants aged 6-14 years had the highest prevalence of asymptomatic malaria, with geometric means of ~ 116 parasites/µL and ~ 6689 parasites/µL as detected by microscopy and varATS, respectively. CONCLUSION: This study has revealed high prevalence of asymptomatic malaria in the study population, with varATS detecting additional sub-microscopic infections. The highest concentration of asymptomatic malaria was observed among school-age children between 6 and 14 years old. A large-scale screening to identify other potential hotspots of asymptomatic parasites in the country is recommended.

Household-level effects of seasonal malaria chemoprevention in the Gambia.

BACKGROUND: In 2022 the WHO recommended the discretionary expansion of the eligible age range for seasonal malaria chemoprevention (SMC) to children older than 4 years. Older children are at lower risk of clinical disease and severe malaria so there has been uncertainty about the cost-benefit for national control programmes. However, emerging evidence from laboratory studies suggests protecting school-age children reduces the infectious reservoir for malaria and may significantly impact on transmission. This study aimed to assess whether these effects were detectable in the context of a routinely delivered SMC programme. METHODS: In 2021 the Gambia extended the maximum eligible age for SMC from 4 to 9 years. We conducted a prospective population cohort study over the 2021 malaria transmission season covering 2210 inhabitants of 10 communities in the Upper River Region, and used a household-level mixed modelling approach to quantify impacts of SMC on malaria transmission. RESULTS: We demonstrate that the hazard of clinical malaria in older participants aged 10+ years ineligible for SMC decreases by 20% for each additional SMC round per child 0-9 years in the same household. Older inhabitants also benefit from reduced risk of asymptomatic infections in high SMC coverage households. Spatial autoregression tests show impacts are highly localised, with no detectable spillover from nearby households. CONCLUSIONS: Evidence for the transmission-reducing effects of extended-age SMC from routine programmes implemented at scale has been previously limited. Here we demonstrate benefits to the entire household, indicating such programmes may be more cost-effective than previously estimated.

Seasonal malaria chemoprevention (SMC) is the provision of monthly, preventative, anti-malaria medication to young children at times when they are most at risk of severe disease. Recently the World Health Organisation recommended expanding SMC to children older than 4 years. Older children with malaria typically remain symptomless so the advantages were unclear. However, laboratory evidence suggests this group continues to transmit malaria to others. We conducted a population study in 2021 in 10 communities in the Gambia where SMC was extended to all children up to 9 years of age for the first time. We found household members aged over 9 years were less likely to get clinical disease when most young children in the same household did receive SMC. This suggests an added protection of SMC for those who do not receive it, potentially increasing cost-effectiveness.