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.

Population Genomics of Plasmodium malariae from Four African Countries.

Plasmodium malariae is geographically widespread but neglected and may become more prevalent as P. falciparum declines. We completed the largest genomic study of African P. malariae to-date by performing hybrid capture and sequencing of 77 isolates from Cameroon (n=7), the Democratic Republic of the Congo (n=16), Nigeria (n=4), and Tanzania (n=50) collected between 2015 and 2021. There is no evidence of geographic population structure. Nucleotide diversity was significantly lower than in co-localized P. falciparum isolates, while linkage disequilibrium was significantly higher. Genome-wide selection scans identified no erythrocyte invasion ligands or antimalarial resistance orthologs as top hits; however, targeted analyses of these loci revealed evidence of selective sweeps around four erythrocyte invasion ligands and six antimalarial resistance orthologs. Demographic inference modeling suggests that African P. malariae is recovering from a bottleneck. Altogether, these results suggest that P. malariae is genomically atypical among human Plasmodium spp. and panmictic in Africa.

Population genomic evidence of a putative 'far-west' African cryptic taxon in the Anopheles gambiae complex.

The two main Afrotropical malaria vectors - Anopheles coluzzii and An. gambiae - are genetically distinct and reproductively isolated across West Africa. However, populations at the western extreme of their range are assigned as "intermediate" between the two species by whole genome sequence (WGS) data, and as hybrid forms by conventional molecular diagnostics. By exploiting WGS data from 1190 specimens collected across west Africa via the Anopheles gambiae 1000 Genomes network, we identified a putative taxon in the far-west (provisionally named Bissau molecular form), which did not arise by admixture but rather may have originated at the same time as the split between An. coluzzii and An. gambiae. Intriguingly, this taxon lacks insecticide resistance mechanisms commonly observed in the two main species. These findings lead to a change of perspective on malaria vector species in the far-west region with potential for epidemiological implications, and a new challenge for genetic-based mosquito control approaches.

Seasonal variations in haematological and biochemical parameters of healthy Gambian adults: Retrospective study 2018-2022.

The objectives of this study were to determine the presence and effect of seasonal variations and provide insights into trend from 2018 to 2022 in a comprehensive set of routine haematological indices and biochemical measurements in Gambian adults with no known underlying health condition. We retrieved five years of data from an electronic database and analysed 493 full blood counts and 643 biochemical data from different individuals. In this study, we focused on data from individuals with no known underlying health condition who visited the clinical diagnostic laboratory for routine medical examinations or assessments.Our study found a positive association between seasonality (wet season as the reference) and Hb (HB: 0.014(0.015), P<0.05), White blood cells (WBC) (WBC: 0.243(0.163), p = 0.0014), and neutrophils (neutrophils: 0.271(0.131), P<0.05) with exception to red blood cells (RBC) (RBC: - 0.184(0.061), P< 0.003) that showed negative association. Despite the association, the seasonal effects on our derived reference intervals for haematological indices and biochemical measurements from wet season to dry season were not statistically significant (P>0.05). In addition, we observed in our heatmap result that some laboratory parameters, including HB, RBC, haematocrit (HCT), urea, liver enzymes, and potassium, showed seasonal variation patterns throughout the year, with median levels being normal to slightly low during the dry season and normal to high during the wet season. We also found no significant difference (P>0.05) among the median values for all parameters from 2018 to 2022. Additionally, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) parameters showed a consistent declining trend from 2018 to 2022. Our study found no seasonal effects on the derived reference intervals of haematological indices and biochemical measurements. However, we observed changes in patterns for certain parameters particularly HB, RBC, liver enzymes, and potassium based on seasonality.

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.

Mosquitocidal effect of ivermectin-treated nettings and sprayed walls on Anopheles gambiae s.s.

Ivermectin (IVM) has been proposed as a new tool for malaria control as it is toxic on vectors feeding on treated humans or cattle. Nevertheless, IVM may have a direct mosquitocidal effect when applied on bed nets or sprayed walls. The potential for IVM application as a new insecticide for long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) was tested in this proof-of-concept study in a laboratory and semi-field environment. Laboratory-reared, insecticide-susceptible Kisumu Anopheles gambiae were exposed to IVM on impregnated netting materials and sprayed plastered- and mud walls using cone bioassays. The results showed a direct mosquitocidal effect of IVM on this mosquito strain as all mosquitoes died by 24 h after exposure to IVM. The effect was slower on the IVM-sprayed walls compared to the treated nettings. Further work to evaluate possibility of IVM as a new insecticide formulation in LLINs and IRS will be required.

Detection of novel Plasmodium falciparum coronin gene mutations in a recrudescent ACT-treated patient in South-Western Nigeria.

Background: Routine surveillance for antimalarial drug resistance is critical to sustaining the efficacy of artemisinin-based Combination Therapies (ACTs). Plasmodium falciparum kelch-13 (Pfkelch-13) and non-Pfkelch-13 artemisinin (ART) resistance-associated mutations are uncommon in Africa. We investigated polymorphisms in Plasmodium falciparum actin-binding protein (Pfcoronin) associated with in vivo reduced sensitivity to ART in Nigeria. Methods: Fifty-two P. falciparum malaria subjects who met the inclusion criteria were followed up in a 28-day therapeutic efficacy study of artemether-lumefantrine in Lagos, Nigeria. Parasite detection was done by microscopy and molecular diagnostic approaches involving PCR amplification of genes for Pf18S rRNA, varATS, telomere-associated repetitive elements-2 (TARE-2). Pfcoronin and Pfkelch-13 genes were sequenced bi-directionally while clonality of infections was determined using 12 neutral P. falciparum microsatellite loci and msp2 analyses. Antimalarial drugs (sulfadoxine-pyrimethamine, amodiaquine, chloroquine and some quinolones) resistance variants (DHFR_51, DHFR_59, DHFR_108, DHFR_164, MDR1_86, MDR1_184, DHPS_581 and DHPS_613) were genotyped by high-resolution melting (HRM) analysis. Results: A total of 7 (26.92%) cases were identified either as early treatment failure, late parasitological failure or late clinical failure. Of the four post-treatment infections identified as recrudescence by msp2 genotypes, only one was classified as recrudescence by multilocus microsatellites genotyping. Microsatellite analysis revealed no significant difference in the mean allelic diversity, He, (P = 0.19, Mann-Whitney test). Allele sizes and frequency per locus implicated one isolate. Genetic analysis of this isolate identified two new Pfcoronin SNVs (I68G and L173F) in addition to the P76S earlier reported. Linkage-Disequilibrium as a standardized association index, IAS, between multiple P. falciparum loci revealed significant LD (IAS = 0.2865, P=0.02, Monte-Carlo simulation) around the neutral microsatellite loci. The pfdhfr/pfdhps/pfmdr1 drug resistance-associated haplotypes combinations, (108T/N/51I/164L/59R/581G/86Y/184F), were observed in two samples. Conclusion: Pfcoronin mutations identified in this study, with potential to impact parasite clearance, may guide investigations on emerging ART tolerance in Nigeria, and West African endemic countries.

Sub-microscopic Plasmodium falciparum infections and multiple drug resistant single nucleotide polymorphic alleles in pregnant women from southwestern Nigeria.

OBJECTIVES: The study evaluated sub-microscopic malaria infections in pregnancy using two malaria Rapid Diagnostic Tests (mRDTs), microscopy and RT-PCR and characterized Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and Plasmodium falciparum dihydropteroate synthase (Pfdhps) drug resistant markers in positive samples. METHODS: This was a cross sectional survey of 121 pregnant women. Participants were finger pricked, blood drops were collected for rapid diagnosis with P. falciparum histidine-rich protein 11 rapid diagnostic test kit and the ultra-sensitive Alere Pf malaria RDT, Blood smears for microscopy and dried blood spots on Whatman filter paper for molecular analysis were made. Real time PCR targeting the var acidic terminal sequence (varATS) gene of P. falciparum was carried out on a CFX 96 real time system thermocycler (BioRad) in discriminating malaria infections. For each run, laboratory strain of P. falciparum 3D7 and nuclease free water were used as positive and negative controls respectively. Additionally, High resolution melt analyses was employed for genotyping of the different drug resistance markers. RESULTS: Out of one hundred and twenty-one pregnant women sampled, the SD Bioline™ Malaria Ag P.f HRP2-based malaria rapid diagnostic test (mRDT) detected eight (0.06%) cases, the ultra-sensitive Alere™ malaria Ag P.f rapid diagnostic test mRDT had similar outcome in the same samples as detected by the HRP2-based mRDT. Microscopy and RT-PCR confirmed four out of the eight infections detected by both rapid diagnostic tests as true positive and RT-PCR further detected three false negative samples by the two mRDTs providing a sub-microscopic malaria prevalence of 3.3%. Single nucleotide polymorphism in Pfdhps gene associated with sulphadoxine resistance revealed the presence of S613 mutant genotypes in three of the seven positive isolates and isolates with mixed wild/mutant genotype at codon A613S. Furthermore, four mixed genotypes at the A581G codon were also recorded while the other Pfdhps codons (A436G, A437G and K540E) showed the presence of wild type alleles. In the Pfdhfr gene, there were mutations in 28.6%, 28.6%, and 85.7% at the I51, R59 and N108 codons respectively. Mixed wild and mutant type genotypes were also observed in 28.6% each of the N51I, and C59R codons. For the Pfcrt, two haplotypes CVMNK and CVIET were observed. The SVMNT was altogether absent. Triple mutant CVIET 1(14.3%) and triple mutant + wild genotype CVIET + CVMNK 1(14.3%) were observed. The Pfmdr1 haplotypes were single mutants YYND 1(14.3%); NFND 1(14.3%) and double mutants YFND 4(57.1%); YYDD 1(14.3%).

Prevalence of Malaria Infection in Pregnant Women Attending Antenatal Clinics in Southern Senegal.

Despite marked progress in Senegal, three regions in the southeast part continue to have a high burden of malaria, but there have been no recent studies assessing the prevalence of malaria associated with pregnancy. This study aimed to determine the prevalence of malaria infection in pregnant women attending antenatal clinics in Senegal. During the malaria transmission season of 2019, pregnant women attending 11 health care facilities for a scheduled visit and those presenting unwell with signs of malaria were invited to participate in a malaria screening study. A finger prick blood sample was taken for malaria diagnosis by rapid diagnosis test (RDT) and polymerase chain reaction (PCR). A total of 877 pregnant women were enrolled, 787 for a scheduled antenatal consultation and 90 for an unscheduled consultation with signs of malaria. The prevalence of Plasmodium falciparum among the first group was 48% by PCR and 20% by RDT, and that among the second group was 86% by PCR and 83% by RDT. RDT sensitivity in capturing asymptomatic, PCR-positive infections was 9.2% but ranged from 83% to 94% among febrile women. The prevalence of infection by PCR in women who reported having received at least three doses of sulfadoxine pyrimethamine (SP) was 41.9% compared with 58.9% in women who reported they had not received any SP doses (prevalence ratio adjusted for gravidity and gestational age, 0.54; 95% CI, 0.41-0.73). The burden of P. falciparum infections remains high among pregnant women, the majority of which are not captured by RDT. More effective measures to prevent malaria infection in pregnancy are needed.

Plasmodium falciparum population structure and genetic diversity of cell traversal protein for ookinetes and sporozoites (CelTOS) during malaria resurgences in Dielmo, Senegal.

The ability to accurately measure the intensity of malaria transmission in areas with low transmission is extremely important to guide elimination efforts. Plasmodium falciparum Cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an important conserved sporozoite antigen reported as one of the promising malaria vaccine candidates, and could be used to estimate malaria transmission intensity. This study aimed at determining whether the diversity of PfCelTOS gene reflects the changes in malaria transmission that occurred between 2007 and 2014 in Dielmo, a Senegalese village, before and after the implementation of insecticide treated bed nets (ITNs). Of the 109 samples positive for PfCelTOS PCR, 96 (88%) were successfully sequenced and analysed for polymorphisms and population diversity. The number of segregating sites was higher during the pre-intervention period (13) and the malaria resurgences (11) than during the intervention period (5). Similarly, the number and diversity of haplotypes were higher during the pre-intervention period (16 and 0.914, respectively) and the malaria resurgences (6 and 0.821, respectively) than during the intervention period (4 and 0.758, respectively). Moreover, the average number of nucleotide differences was higher during the pre-intervention (3.792) and during malaria resurgences (3.467) than during the intervention period (2.189). The 3D7 KSSFNEP haplotype was only observed during the intervention period. Only two haplotypes were shared in both the pre-intervention and intervention periods while four haplotypes were shared between the pre-intervention and the malaria resurgences. The Fst values indicate moderate differentiation between pre-intervention and intervention periods (0.17433), and between intervention and malaria resurgences period (0.19198) as well as between pre-intervention and malaria resurgences periods (0.06607). PfCelTOS genetic diversity reflected changes of malaria transmission, with higher polymorphisms recorded before the large-scale implementation of ITNs and during the malaria resurgences. PfCelTOS is also a candidate vaccine; mapping its diversity across multiple endemic environments will facilitate the design and optimisation of a broad and efficacious vaccine.

Rebound of multiple infections and prevalence of anti-malarial resistance associated markers following malaria upsurges in Dielmo village, Senegal, West Africa.

BACKGROUND: Thanks to the scale up of malaria control interventions, the malaria burden in Senegal has decreased substantially to the point that the National Malaria Control Programme plans to achieve malaria elimination by 2030. To guide such efforts, measuring and monitoring parasite population evolution and anti-malarial drugs resistance is extremely important. Information on the prevalence of parasite mutations related to drug resistance can provide a first signal of emergence, introduction and selection that can help with refining drug interventions. The aim of this study was to analyse the prevalence of anti-malarial drug resistance-associated markers before and after the implementation of artemisinin-based combination therapy (ACT) from 2005 to 2014 in Dielmo, a model site for malaria intervention studies in Senegal. METHODS: Samples from both malaria patients and Plasmodium falciparum asymptomatic carriers were analysed with high resolution melting (HRM) technique to genotype P. falciparum chloroquine resistance transporter (Pfcrt) gene haplotypes and multidrug-resistant protein 1 (Pfmdr1) gene at codons N86 and Y184. RESULTS: Among the 539 samples analysed, 474, 486, and 511 were successfully genotyped for Pfmdr1 N86, Y184, and Pfcrt, respectively. The prevalence of drug resistance markers was high, particularly during the malaria upsurges. Following the scale-up in bed net distribution, only the mutant (86F-like) variant of Pfmdr1 86 was present while during the malaria upsurges the predominance of two types 86Y-86N (43%) and 86F-like (56%) were observed. Most infections (87%) carried the wild type Y-allele at Pfmdr1 184 during the period of nets scale-up while during the malaria upsurges only 16% of infections had wild type and 79% of infections had mixed (mutant/wild) type. The frequency of the mixed genotypes SVMNT-like_CVMNK and SVMNT-like_CVIET within Pfcrt gene was particularly low during bednet scale up. Their frequency increased significantly (P < 0.001) during the malaria upsurges. CONCLUSION: This data demonstrated the effect of multiple interventions on the dynamics of drug resistance-associated mutations in the main malaria parasite P. falciparum in an endemic village in Senegal. Monitoring drug resistance markers should be conducted periodically to detect threats of emergence or resurgence that could compromise the efficacy of anti-malarial drugs.

Malaria in adults after the start of Covid-19 pandemic: an analysis of admission trends, demographics, and outcomes in a tertiary hospital in the Gambia.

BACKGROUND: Malaria remains a major public health concern in The Gambia. The study assessed the trend of malaria admissions and outcome of adult patients admitted after the start of the COVID-19 pandemic in a tertiary hospital in The Gambia. METHODS: This was a retrospective hospital-based study and data was collected from the 18th October 2020 to 28th February 2023. Demographic data, clinical features, investigations, treatment, and outcomes were recorded. RESULTS: A total of 499 malaria cases were admitted to the hospital over the 29 months of the study period. Data from 320 (67.2% of the total cases) adult patients admitted into the internal medicine department were analysed. The median age was 22 years, range (15-90) and 189 (59.1%) cases were youth with a youth (15-24 years) to older adult (> 24 years) ratio of 1.4:1. The majority of the patients were male 199 (62.2) with a male to female ratio of 1.6:1. The total number of malaria cases admitted into the internal medicine department increased from 103 cases in 2021 to 182 cases in 2022and admission peaked in November in both years. The total number of admitted malaria cases during the peak of the malaria season also increased from 92 patients between September 2021 and December 2021 to 132 patients from September 2022 to December 2022.There was also an increase in both severe and uncomplicated malaria during the same period. The total mortality was 31 (9.7%) and the rate was similar in 2021 9 (8.7%) and 2022 15 (8.4%). Patients with impaired consciousness were more likely to die when compared to those without impaired consciousness [19 (23.6%) vs 12 (5%), p ≤ 0.001]. Patients with acute kidney injury were also more likely to die when compared with those without acute kidney injury [10 (20.4%) vs 15 (7.7%), p = 0.009]. CONCLUSION: The findings show an emerging and consistent trend of malaria admissions and the outcome in the youth and older adult population after the start of the COVID-19 pandemic in The Gambia. This, therefore, suggests the need for the implementation of targeted malaria prevention interventions in this population to further prevent the spread of the disease to the more vulnerable population.

Recent increase in low complexity polygenomic infections and sialic acid-independent invasion pathways in Plasmodium falciparum from Western Gambia.

BACKGROUND: The malaria parasite Plasmodium falciparum utilizes multiple alternative receptor-ligand interactions for the invasion of human erythrocytes. While some P. falciparum clones make use of sialic acid (SA) residues on the surface of the human glycophorin receptors to invade the erythrocyte, others use alternative receptors independent of sialic acid residues. We hypothesized that over the years, intensified malaria control interventions and declining prevalence in The Gambia have resulted in a selection of parasites with a dominant invasion pathways and ligand expression profiles. METHODS: Blood samples were collected from 65 malaria-infected participants with uncomplicated malaria across 3 years (2015, 2016, and 2021). Genetic diversity was determined by genotyping the merozoite surface protein 2 (msp2) polymorphic gene of P. falciparum. Erythrocyte invasion phenotypes were determined using neuraminidase, trypsin, and chymotrypsin enzymes, known to cleave different receptors from the surface of the erythrocyte. Schizont-stage transcript levels were obtained for a panel of 6 P. falciparum invasion ligand genes (eba175, eba181, Rh2b, Rh4, Rh5, and clag2) using 48 successfully cultured isolates. RESULTS: Though the allelic heterozygosity of msp2 repeat region decreased as expected with reduced transmission, there was an increase in infections with more than a single msp2 allelotype from 2015 to 2021. The invasion phenotypes of these isolates were mostly SA independent with a continuous increase from 2015 to 2021. Isolates from 2021 were highly inhibited by chymotrypsin treatment compared to isolates from 2015 and 2016. Higher invasion inhibition for 2021 isolates was further obtained following erythrocyte treatment with a combination of chymotrypsin and trypsin. The transcript levels of invasion ligand genes varied across years. However, levels of clag2, a rhoptry-associated protein, were higher in 2015 and 2016 isolates than in 2021 isolates, while Rh5 levels were higher in 2021 compared to other years. CONCLUSIONS: Overall, these findings suggest increasing mixed infections with an increase in the use of sialic-acid independent invasion pathways by P. falciparum clinical isolates in the Western part of Gambia.

Laboratory indices of hospitalized sickle cell disease patients, prevalence and antimicrobial susceptibility of pathogenic bacterial isolates at MRCG ward in the Gambia.

BACKGROUND: The aim of this study was to determine the prevalence of invasive bacterial infections and their antimicrobial resistance patterns in sickle cell disease (SCD) patients admitted at the Medical Research Council the Gambia (MRCG) Ward in the era of PCV and Hib vaccination in the Gambia. METHODS AND RESULTS: This study was conducted in the clinical laboratory department of MRCG. We retrospectively generated haematological, and blood culture data from our electronic medical records from 2015 to 2022 of SCD patients admitted to MRCG Ward. Of 380 SCD patients, blood culture was requested only for 159. Of the 159 admitted SCD, 11 patients had qualified positive blood cultures. Five different types of bacterial pathogens were isolated from these positive blood cultures: 4 Staphylococcus aureus, 3 Streptococcus pneumoniae, 2 Salmonella species, 1 Enterococcus species, and 1 Shigella boydii. No episode of bacteremia caused by Haemophilus influenzae type b was identified. The molecular serotyping of the Streptococcus pneumoniae isolates revealed non-vaccine serotypes 10 A, 12 F and 12 F. Penicillin resistance was recorded in two of the three Streptococcus pneumoniae. The Staphylococcus aureus isolates were penicillin resistant but cefoxitin sensitive, hence no methicillin (oxacillin) resistant Staphylococcus aureus was reported. Generally, the isolated pathogens were all sensitive to chloramphenicol, and vancomycin. The haematological indices were not significantly varied between SCD patients with and without microbiologically confirmed bacterial infection. CONCLUSION: Streptococcus pneumoniae and Staphylococcus aureus were the most common cause of bacteremia in these admitted SCD patients. The presence of non-typhoidal Salmonella and Shigella infection coupled with penicillin resistance should be considered during penicillin prophylaxis and empirical treatment regimens for SCD patients and future SCD management policies in the Gambia. The haematological parameters may not be reliable biomarkers in differentiating bacterial from non-bacterial infections in SCD patients.

Complexity of Plasmodium falciparum infection and genetic variations associated with differences in parasite clearance time in two Malian villages.

Background: Effective approaches to fight against malaria include disease prevention, an early diagnosis of malaria cases, and rapid management of confirmed cases by treatment with effective antimalarials. Artemisinin-based combination therapies are first-line treatments for uncomplicated malaria in endemic areas. However, cases of resistance to artemisinin have already been described in South-East Asia resulting in prolonged parasite clearance time after treatment. In Mali, though mutations in the K13 gene associated with delayed clearance in Asia are absent, a significant difference in parasite clearance time following treatment with artesunate was observed between two malaria endemic sites, Bougoula-Hameau and Faladje. Hypothetically, differences in complexity of Plasmodium falciparum infections may be accounted for this difference. Hence, the aims of this study were to assess the complexity of infection (COI) and genetic diversity of P. falciparum parasites during malaria treatment in Bougoula-Hameau and Faladje in Mali. Methods: Thirty (30) patients per village were randomly selected from 221 patients enrolled in a prospective artesunate monotherapy study conducted in Faladje and Bougoula-Hameau in 2016. All parasitemic blood samples of patients from enrollment to last positive slide were retained to assess malaria parasite COI and polymorphisms. DNA were extracted with a Qiagen kit and Pfcsp and Pfama1 encoding gene were amplified by nested PCR and sequenced using the Illumina platform. The parasite clearance time (PCT) was determined using the parasite clearance estimator of Worldwide Antimarial Resistance Network (WWARN). Data were analyzed with R®. Results: The median number of genetically distinct parasite clones was similar at enrollment, 7 (IQR of 5-9) in Faladje and 6 (IQR of 4-10) in Bougoula-Hameau (p-value = 0.1). On the first day after treatment initiation, the COI was higher in Faladje (6; CI:4-8) than in Bougoula-Hameau (4; CI:4-6) with a p-value =0. 02. Overall, COI was high with higher PCT. Finally, there was a low genetic diversity between Faladje and Bougoula-Hameau. Conclusion: This study demonstrated that the difference in PCT observed between the two villages could be due to differences in the complexity of infection of these two villages.

Stepwise in vitro screening of MMV pathogen box compounds against Plasmodium falciparum to identify potent antimalarial candidates.

Development of resistance to deployed antimalarial drugs is inevitable and needs prompt and continuous discovery of novel candidate drugs. Therefore, the antimalarial activity of 125 compounds from the Medicine for Malaria Ventures (MMV) pathogen box was determined. Combining standard IC50 and normalised growth rate inhibition (GR50) analyses, we found 16 and 22 compounds had higher potencies than CQ respectively. Seven compounds with relatively high potencies (low GR50 and IC50) against P. falciparum 3D7 were further analysed. Three of these were tested on 10 natural P. falciparum isolates from The Gambia using our newly developed parasite survival rate assay (PSRA). According to the IC50, GR50 and PSRA analyses, compound MMV667494 was most potent and highly cytotoxic to parasites. MMV010576 was slow acting but more potent than dihydroartemisinin (DHA) 72 h after exposure. MMV634140 was potent against the laboratory-adapted 3D7 isolate, but 4 out of 10 natural Gambian isolates survived and replicated slowly despite 72 h of exposure to the compound, suggesting potential drug tolerance and risk of resistance development. These results emphasise the usefulness of in vitro testing as a starting point for drug discovery. Improved approaches to data analyses and the use of natural isolates will facilitate the prioritisation of compounds for further clinical development.

Clinical isolates of uncomplicated falciparum malaria from high and low malaria transmission areas show distinct pfcrt and pfmdr1 polymorphisms in western Ethiopia.

BACKGROUND: Pfcrt gene has been associated with chloroquine resistance and the pfmdr1 gene can alter malaria parasite susceptibility to lumefantrine, mefloquine, and chloroquine. In the absence of chloroquine (CQ) and extensive use of artemether-lumefantrine (AL) from 2004 to 2020 to treat uncomplicated falciparum malaria, pfcrt haplotype, and pfmdr1 single nucleotide polymorphisms (SNPs) were determined in two sites of West Ethiopia with a gradient of malaria transmission. METHODS: 230 microscopically confirmed P. falciparum isolates were collected from Assosa (high transmission area) and Gida Ayana (low transmission area) sites, of which 225 of them tested positive by PCR. High-Resolution Melting Assay (HRM) was used to determine the prevalence of pfcrt haplotypes and pfmdr1 SNPs. Furthermore, the pfmdr1 gene copy number (CNV) was determined using real-time PCR. A P-value of less or equal to 0.05 was considered significant. RESULTS: Of the 225 samples, 95.5%, 94.4%, 86.7%, 91.1%, and 94.2% were successfully genotyped with HRM for pfcrt haplotype, pfmdr1-86, pfmdr1-184, pfmdr1-1042 and pfmdr1-1246, respectively. The mutant pfcrt haplotypes were detected among 33.5% (52/155) and 80% (48/60) of isolates collected from the Assosa and Gida Ayana sites, respectively. Plasmodium falciparum with chloroquine-resistant haplotypes was more prevalent in the Gida Ayana area compared with the Assosa area (COR = 8.4, P = 0.00). Pfmdr1-N86Y wild type and 184F mutations were found in 79.8% (166/208) and 73.4% (146/199) samples, respectively. No single mutation was observed at the pfmdr1-1042 locus; however, 89.6% (190/212) of parasites in West Ethiopia carry the wild-type D1246Y variants. Eight pfmdr1 haplotypes at codons N86Y-Y184F-D1246Y were identified with the dominant NFD 61% (122/200). There was no difference in the distribution of pfmdr1 SNPs, haplotypes, and CNV between the two study sites (P > 0.05). CONCLUSION: Plasmodium falciparum with the pfcrt wild-type haplotype was prevalent in high malaria transmission site than in low transmission area. The NFD haplotype was the predominant haplotype of the N86Y-Y184F-D1246Y. A continuous investigation is needed to closely monitor the changes in the pfmdr1 SNPs, which are associated with the selection of parasite populations by ACT.

Knowledge, attitude, and adherence to malaria control guidelines and the prevalence of Plasmodium species infection in localities across transmission and ecological zones in Cameroon.

Background: Despite a scale up of control interventions over the years, malaria remains a major public health and economic concern in Cameroon, contributing considerably to hospitalization and deaths. The effectiveness of control strategies depends on the extent of adherence by the population to national guidelines. This study assessed the influence of human knowledge, attitudes, and practices related to malaria and its control on the prevalence of malaria parasite infection, with implications for the elimination of the disease. Methodology: This is a cross-sectional community and hospital-based study, covering the five ecological and three malaria transmission zones in Cameroon. A pre-tested semi-structured questionnaire was used to document socio-demographic and clinical parameters as well as knowledge, attitudes, and practices toward malaria control and management. Consenting participants were screened for malaria parasite with rapid diagnostic test (mRDT) of the peripheral blood. Association between qualitative variables was determined using the chi-square test and logistic regression analysis. Results: A total of 3,360 participants were enrolled, 45.0% (1,513) of whom were mRDT positive, with 14.0% (451/3,216) and 29.6% (951/3,216) having asymptomatic parasitaemia and malaria, respectively. Although most participants knew the cause, symptoms, and control strategies, with 53.6% (1,000/1,867) expertly knowledgeable about malaria overall, only 0.1% (2/1,763) individuals were fully adherent to malaria control measures. Conclusion: The risk of malaria in Cameroon remains high, with the population considerably knowledgeable about the disease but poorly adherent to national malaria control guidelines. Concerted and more effective strategies aimed at improving knowledge about malaria and adherences to control interventions are necessary to ultimately eliminate the disease.

Genomic variation during culture adaptation of genetically complex Plasmodium falciparum clinical isolates.

Experimental studies on the biology of malaria parasites have mostly been based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates in analyses focusing on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections, which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points over several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and time points. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity, although proportions of genotypes varied over time. Drug resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, several of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of genomic identity by descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within endemic populations.

Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1.

Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.