P. falciparum-host red blood cell (RBC) rosettes compared to non-O blood groups. Whether the non-O blood groups encoded by the specific ABO genotypes AO, BO, AA, BB and AB differ in their associations with severe malaria and rosetting is unknown. The A and B antigens are host RBC receptors for rosetting, hence we hypothesized that the higher levels of A and/or B antigen on RBCs from AA, BB and AB genotypes compared to AO/BO genotypes could lead to larger rosettes, increased microvascular obstruction and higher risk of malaria pathology. We used a case-control study of Kenyan children and in vitro adhesion assays to test the hypothesis that “double dose” non-O genotypes  are associated with increased risk of severe malaria and larger rosettes than “single dose” heterozygotes . In the case-control study, compared to OO, the double dose genotypes consistently had higher odds ratios (OR) for severe malaria than single dose genotypes, with AB (OR 1.93) and AO (OR 1.27) showing most marked difference . In vitro experiments with blood group A-preferring P. falciparum parasites showed that significantly larger rosettes were formed with AA and AB host RBCs compared to OO, whereas AO and BO genotypes rosettes were indistinguishable from OO. Overall, the data show that ABO genotype influences P. falciparum rosetting and support the hypothesis that double dose non-O genotypes confer a greater risk of severe malaria than AO/BO heterozygosity.Blood group O is associated with protection against severe malaria and reduced size and stability of  P. falciparum-infected RBCs to form clumps known as rosettes, that occlude microvasculature circulation and cause severe malaria pathology. Nevertheless, to date, these conclusions have largely been based on ABO blood groups determined by serological antibody typing. Genetic classification into AO, AA, BO, BB, AB and OO offers a finer and more specific classification of ABO blood groups, but associations with severe malaria and rosetting based on this method have not been described previously. In a case-control study of >5000 Kenyan children, we show that the “double dose” non-O genotypes AA, BB and AB are associated with an increased risk to severe malaria compared to the “single dose” non-O genotypes AO and BO, and that this is most significant for AB versus AO. In in vitro experiments, double dose non-O genotypes formed larger rosettes compared to single dose non-O genotypes, providing a potential explanation for their increased severe malaria risk.The most common human blood group, ABO, affects susceptibility to multiple diseases including malaria, whereby the non-O blood groups A, B and AB are associated with an increased risk of severe malaria in comparison to blood group O. This may occur because red blood cell (RBC) surface-expressed non-O blood group antigens mediate binding to  Plasmodium falciparum infected red blood cells (iRBCs), such as P. falciparum erythrocyte membrane protein 1 (PfEMP1), mediating iRBC binding to uninfected RBCs to form clusters called rosettes [P. falciparum iRBC surface expressed parasite ligands implicated in rosetting include the repetitive interspersed family proteins (RIFIN) interacting with blood group A [P. falciparum isolates vary in their propensity to form rosettes, high levels of rosetting contributing to impaired microvascular blood flow, and severe malaria [The ABO blood group was the first human blood group system to be discovered, and has since been widely studied . Three rrosettes –20. Othe group A  and the  group A . P. falc malaria –24. Rose malaria –19,25–27 malaria ,25,28–30ABO gene to derive ABO genotype , which have then been used to infer ABO phenotypes. While multiple polymorphisms underlie the genetic basis of the ABO system [ABO genotype and ABO phenotype has not been reported in previous population studies of malaria susceptibility, most of which have been conducted in sub-Saharan Africa.Previous studies linking ABO blood group to malaria susceptibility and rosetting have either used serological RBC agglutination assays to determine ABO blood group phenotype , or single nucleotide polymorphisms (SNPs) at the O system ,32, mostO system –15,33,34ABO genotype (rather than phenotype) and malaria susceptibility, or potential protective mechanisms such as their impacts on rosetting. Prior work shows a gene dosage effect between ABO genotype and the number of ABH antigens on RBCs. For example, AA and BB homozygotes have substantially higher levels of A and B antigens on RBCs than AO and BO heterozygotes respectively [AB heterozygotes have antigen levels that are more similar to those found in AA/BB homozygotes [2 blood group phenotype, in which fewer A antigen sites are displayed per RBC than cells of the common A1 phenotype [AO and BO genotypes differ in their rosette-forming ability compared to RBCs from AA/BB/AB genotypes with higher levels of A and B antigens has not yet been tested.Similarly, few studies have been conducted which have examined the association between ectively ,36, whilozygotes –39. The henotype ,17,40. HABO genotypes are associated with differing levels of susceptibility to severe childhood malaria. In addition, we examined the concordance between ABO genotype and phenotype. We also investigated whether ABO genotypes are associated with risk of uncomplicated malaria or asymptomatic infection in a longitudinal cohort and cross-sectional study, respectively. Finally, through in vitro studies, we investigated whether ABO genotype influences either P. falciparum rosetting or other parasite adhesion-related phenomena including binding to endothelial receptors and the display of PfEMP1 on the iRBC surface [O alleles (AA/BB/AB) might have a greater risk of severe malaria than non-O heterozygotes (AO/BO), due to the former having increased levels of A or B antigens on their RBCs, enabling iRBCs to form larger, more stable rosettes that cause greater microvascular obstruction and pathology.Here, we used a case-control study conducted in East Africa to investigate whether specific  surface . We hypoIndividual written informed consent was obtained from the parents or guardians of all study participants. All study protocols were approved by the Kenya Medical Research Institute (KEMRI) National Ethical Review Committee . RBCs and sera from Scottish blood donors were obtained following informed consent, with approval from the Scottish National Blood Transfusion Service Committee for the Governance of Blood and Tissue Samples for Nontherapeutic Use (reference SNBTS 12~35).All epidemiological and clinical studies were conducted in Kilifi County on the coast of Kenya . At the P. falciparum accompanied by any of the following complications: cerebral malaria , severe malarial anaemia  or respiratory distress  [This study has been described in detail elsewhere ,44,45. 2eathing) . In a reeathing) . For thieathing) . Therefoeathing) ,13,49, beathing) .P. falciparum carriage through four cross-sectional surveys carried out in March, July and October 2000 and June 2001. Exclusion criteria included death, migration from the study area for more than 2 months and consent withdrawal. Uncomplicated malaria in this cohort was defined as fever (axillary temperature of >37.5°C) with P. falciparum infection at any density, in the absence of any signs of severity [The Kilifi longitudinal cohort study, described in detail elsewhere , was estseverity .ABO genotyping was carried out by assessing SNPs rs8176719 and rs8176746, using the SEQUENOM iPLEX Gold (Sequenom) multiplex system following DNA amplification by whole genome amplification, as described previously [ABO gene on chromosome 9 encodes the 261G deletion giving rise to the O allele [ABO gene encodes C796A which distinguishes A and B alleles [ABO genotypes and infer blood groups as described previously in this population [O deletion (D) arose on the background of the A allele (C). Therefore, double heterozygotes GD X AC are assumed to have the haplotype GA and DC, giving rise to the BO genotype, rather than haplotype GC and DA that gives rise to the AO genotype  or sickle cell anaemia (HbSS), and for the 3.7Kb α-globin deletion at the HBA locus, which gives rise to the common African variant of α+thalassaemia, was conducted by PCR as described elsewhere [ABO genotype SNPs in control samples [ABO SNPs among controls , and as such, all SNPs were included in our analysis.Serological typing for ABO blood group phenotypes was carried out by slide haemagglutination assays using anti-A and anti-B monoclonal antibodies . For genotyping, DNA was extracted from fresh or frozen whole blood samples using either an ABI PRISM 6100 Nucleic acid prep station  or QIAamp DNA Blood Mini Kits  respectively. O allele . rs81767 alleles . The twogenotype . Genotyplsewhere ,55. Comp samples . SNPs di samples . In the ABO blood group genotype and serological phenotype was tested using Cohen’s Kappa statistic, for which scores of 1 and 0 signify complete agreement and no agreement, respectively.Agreement between 2 or Fisher’s exact tests were used to test for differences in the distribution of ABO genotypes between severe malaria  and community controls or across groups in different categorical variables including gender, ethnic group and HbS and α+thalassaemia genotypes. We tested for differences in age across ABO genotypes by use of the Kruskal Wallis test.The Pearson’s χ+thalassaemia and HbAS. The OO genotype was taken as the reference group to which the other blood group genotypes  were compared both individually and as ‘non-O’ genotypes combined. To test the hypothesis that double dose non-O genotypes  are associated with a higher risk of severe malaria than single dose non-O genotypes , ORs between single and double dose non-O genotypes from the logistic regression analysis above were compared using the Wald test. As an alternative approach, logistic regression analysis was carried out as described above, but instead comparing the ORs of severe malaria for double dose genotypes AA and AB to single dose AO as the reference genotype, and similarly comparing BB and AB to BO as the reference genotype.Odds ratios (ORs)  for severe malaria and its various clinical sub-types were determined by comparison of genotype frequencies among cases and controls, using a fixed-effects logistic-regression model. We conducted both a univariate and multivariate analysis with adjustments for self-reported ethnicity, gender, αABO genotype on the incidence of uncomplicated malaria was investigated using a random effects Poisson regression model with the reference OO genotype compared to all other ABO genotypes assuming a genotypic model of inheritance, or to non-O genotypes combined in a recessive model of inheritance using the rs8176719 SNP. Incidence Rate Ratios (IRR) were generated from both a univariate model and a multivariate model which were both adjusted for age, season, ethnic group, and HbS and α+thalassaemia genotypes. All analyses accounted for within-person clustering of events. For the cross-sectional survey, ORs investigating the impact of ABO genotypes on the prevalence of asymptomatic parasitaemia were generated by use of logistic regression analysis. We conducted both a univariate analysis and multivariate analysis with adjustment for confounding by age, season, ethnic group and HbS genotype. The analysis also accounted for clustering of asymptomatic parasitaemia events within individuals.In the longitudinal cohort study, the impact of p values have been clearly reported and it is emphasized that no single study is conclusive and additional studies are needed to determine if results are replicable. This approach has been suggested previously for epidemiological data, to avoid potentially important findings being discarded (type II error) due to the stringency of multiple comparison adjustments [In the epidemiological analyses, adjustments for multiple testing were not performed; instead, all adjusted ORs, confidence intervals and ustments –59.in vitro assays were carried out using malaria negative RBC samples that were collected and processed during May 2009 and May 2010, as described in detail previously [All eviously , as parteviously ,52. Brieeviously , 61 befoP. falciparum clone IT/R29 [P. falciparum line used for the in vitro static adhesion assays binds to both ICAM-1 and CD36 endothelial receptors and expresses the ITvar16 PfEMP1 variant [Rosetting assays were carried out with the e IT/R29 –66 expree IT/R29 –67. The  variant ,63. IT/R variant . Due to  variant  while IT variant .in vitro experiments, mature pigmented-trophozoite stage iRBCs were purified to >90% parasitaemia from uninfected and ring-stage iRBCs by magnetic–activated cell sorting (MACS) [ABO genotypes, in duplicate flasks or duplicate wells in 96-well flat-bottomed plates, at a starting parasitaemia of 1.5% for ItG and 3% for IT/R29 and cultured for 24–48 hours to the mature pigmented-trophozoite stage [OO donor were included in all experiments to adjust for day-to-day variation in the assays.All parasites were maintained using standard culture methods  in bloodg (MACS) . The purte stage . RBCs frRosetting was assessed as described previously ,61. BrieP. falciparum parasites were assessed for binding to purified recombinant proteins CD36  and ICAM1-Fc  spotted on bacteriological petri dishes  at a concentration of 50μg/ml. Each donor sample was tested once in duplicate dishes run on the same day, with triplicate spots of each protein in each dish. For each spot, 6 images of adherent iRBCs were captured across random fields using an inverted microscope , giving 36 images per protein for each donor RBC. Images were processed and analysed using Image SXM software  [2 of surface area.Static adhesion assays were carried out as described in detail previously ,68. Brieool, UK)  and the P. falciparum clone has been described in detail previously [Assessment of PfEMP1 expression by flow cytometry, including gating strategies, in donor RBCs infected with the IT/R29 eviously . Brieflyeviously . ITvar9 ABO genotype on rosetting, cytoadhesion or PfEMP1 expression. Potential confounding variables including HbS and α+thalassaemia genotypes [+thalassaemia which showed significant associations on univariate analysis (p<0.05) and an interaction term between the two, and improved the overall model fit tested using the log-likelihood ratio test. Experimental day was included as a covariate to account for day-to-day variation when experiments were conducted over several days. Non-normally distributed CD36 and ICAM-1 binding data were normalized by square root-transformation. The final list of variables adjusted for in each analysis is shown in the footnote under each respective results table. To visualise the data, dot plots were generated showing individual data points for each donor, normalized to the mean of control donor genotype OO cells run on the same day to account for day-to-day variation. Non-O blood group genotypes were compared to the reference OO genotype using a Kruskal Wallis test with Dunn’s multiple comparisons. A p value of <0.05 was considered significant in all analyses. Statistical analyses were performed in Stata v13.1  or Prism v7.0  and graphs were generated using Prism v7.0.Multivariate regression analysis was used to test the effect of enotypes , mean coenotypes ,74 and Kenotypes ,44 were RBCs were obtained from 13 Scottish donors  following verbal consent. After the removal of plasma and white cells as described above, RBCs from the 13 donors, and an aliquot of the uninfected group O RBCs in which the parasites were cultured (hereafter known as ‘home O’) were labelled with 6-carboxyfluorescein diacetate (C-FDA) (150μg/ml in RPMI 1640 medium), at 2% haematocrit for 15 minutes, washed twice with RPMI 1640 and stored at 4°C. Blood group preference assays were carried out as described ,37. BrieABO genotype was determined in a case-control study on susceptibility to severe malaria involving >5000 Kenyan children, while ABO blood group phenotypes were assessed on a sub-group of 2761 control children. O was the most common blood group being found in 55.8% of controls, followed by A (23.1%) and B (18.2%), with AB being relatively rare (2.9%) (OO was the most common (54.8% of controls), followed by AO (21.0%) and BO (17.7%). The AA (2.1%), BB (1.4%) and AB (3.0%) genotypes were rare (p <0.0001), giving confidence that studies using ABO genotype to infer ABO blood group phenotype are based on solid assumptions. The highest agreement was seen for the OO genotype, with 99.3% of OO samples being phenotyped as blood group O  showed >94% genotype-phenotype concordance. The blood group B encoding genotypes (BO and BB) showed the lowest concordance at 93.4% and 92.7% respectively, with most of the discordant samples being typed as blood group O or A by serology (e (2.9%) . In termere rare . The ove group O . The bloserology .ABO genotype and susceptibility to severe malaria in the case-control study. Associations between ABO blood group and severe malaria for this full dataset, which also included cases who we have subsequently shown to have a low probability of severe malaria, have been reported previously [-O genotypes, which were not analysed in the previous study [ABO genotype distributions in relation to the variables included in this study are shown in OO genotype compared to the severe malaria cases , severe malarial anaemia (SMA) and respiratory distress (RD), and malaria-specific mortality using a logistic regression model both with, and without, adjustment for the confounders HbS, α+thalassaemia, gender, and ethnicity. When grouped together, the non-O genotypes were associated with a 49% greater risk of all severe malaria (adjusted ORs (aOR) 1.49; 95% CI 1.31–1.70; p<0.001), with similar values for each of the clinical sub-phenotypes individually , and were also associated with the highest risks for the specific severe malaria syndromes of CM and RD (BB) and SMA (AB)  .AO, BO) and double dose  non-O genotypes generated by logistic regression analysis above using the OO genotype as the reference group. When compared to OO genotype, AA and AB were associated with a 46%  and 93% greater risk of severe malaria overall respectively compared to 27% greater risk seen with AO. Similarly, BB (aOR 2.08) and AB (aOR 1.93) were associated with higher odds for all severe malaria that that seen with BO (aOR 1.65). However, only the AB vs AO comparison reached statistical significance  using the P. falciparum clone IT/R29 [ABO genotyped donors, the double dose genotype RBCs  formed significantly larger rosettes and showed a higher proportion of large rosettes compared to single dose  and OO genotype RBCs  rosetting is seen in  malaria ,77. If Anfection  Tables.P. falciparum rosetting and susceptibility to severe malaria are well-established [ABO genotypes have not been conducted. Our results show a significant effect of ABO genotype on rosette size, with double dose AA/AB genotype RBCs forming significantly larger rosettes than OO genotype RBCs with the A-preferring P. falciparum line IT/R29. In contrast, single dose AO and BO genotype RBCs did not differ from OO genotype RBCs in the rosetting assays. Previous work has shown that rosettes in non-O blood are not only larger but are also more stable and more difficult to disrupt with reagents such as heparin, compared to rosettes in group O RBCs, [ABO genotype, parasite virulence in terms of rosette size, and the primary pathological process of microvascular obstruction in severe malaria is suggested.Although associations between ABO blood group, ablished ,28,78–80 O RBCs, ,19,26,29 O RBCs, . Hence, ABO gene dose affects rosetting supports our prediction that AA/BB/AB individuals will be at higher risk of severe malaria than AO/BO individuals. In the case-control study we found that for most comparisons, AA/BB/AB individuals had higher ORs for severe malaria and specific severe malaria syndromes than AO and BO individuals, but we could not reject the null hypothesis on statistical grounds. Despite recruiting ~1400 severe malaria cases into our study, the frequency of the key AA, BB and AB genotypes in the dataset was low  which limited the power of our study. Similarly, the in vitro rosetting assays, which used RBCs collected from the same study area as the epidemiological work, were limited by small sample size in the double dose genotypes . Future studies with larger sample sizes in the key double dose genotypes will be needed to confirm our findings.Our finding that the in vitro rosetting results support previous suggestions that rosetting is a causal factor in the protective association between ABO blood group and severe malaria [ABO genotype and either endothelial receptor binding or PfEMP1 display. However, our study only used a single parasite clone  and two endothelial receptors. Future studies could further explore the role of ABO on cytoadhesion using other P. falciparum clones/variants and additional endothelial receptors. The only previous study to examine ABO and cytoadhesion found no effect of ABO serological phenotype on binding to multiple endothelial receptors, with decreased ICAM-1 adhesion in non-O blood groups being the only significant result [P. falciparum rosetting in O RBCs and consequent reduced microvascular obstruction, as a key mechanism by which ABO blood group influences the risk of severe malaria.Our  malaria ,16,25,29 malaria ,81. We et result , a findit result ,84 and pt result ,85,86. Ht result ), and weABO genotype and blood group phenotype in an African population. We report strong agreement between ABO genotype and phenotype, with only 79/2686 samples (2.9%) being discordant. The strongest concordance was for genotype OO with blood group O (99.3%), and the weakest for genotype BB with blood group B (92.3%). Two American studies using the same SNPs reported a genotype-phenotype concordance of 100% and 92% respectively, although the perfect concordance seen by Risch and colleagues could potentially have been a reflection of their very small sample size (n = 30) [AO, AA, BO and BB but serologically typed as blood group O, could be due to weak A and B subgroups that reduce A and B antigen density on the RBC surface [ABO genotype in heterozygotes. 62/79 (78%) of discrepant samples were heterozygous for the O deletional allele (“GD” in BO genotype (GA and DC haplotype) or AO genotype from the rare GC and DA haplotype. The O deletion is thought to have arisen on the background of an A allele [BO genotype. However, O deletions arising on a B allele background have been described [An additional aim of our study was to investigate the correlation between (n = 30) ,34. Some surface –89, whic surface –93. Anot “GD” in . The douescribed , and couAO/BO heterozygotes differ from AA/AB/BB individuals in relation to P. falciparum rosetting and severe malaria risk. Alternative mechanisms of protection for blood group O and the OO genotype, such as binding to endothelial receptors ICAM-1 and CD36 and effects on PfEMP1 display were not supported by the data. Additional studies examining the effects of ABO genotype, as well as weak A and B blood groups, may give further insights into the complex host-parasite interactions in severe malaria.In conclusion, our combined epidemiological and laboratory studies support the hypothesis that S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S1 Figp<0.01).ABO blood group preference assay of the IT/R29 parasite clone was assessed using fluorescently-labelled RBC from 13 donors . “Home O” indicates the blood group O donor that was used to culture the parasites. The y-axis shows the difference between the percentage of labelled cells found in rosettes, compared to the percentage found in the mix. Experiments were carried out in triplicate, and the mean and SEM are shown for each donor. For statistical analysis, the triplicate values for each donor were averaged and treated as a single data point, such that n = 4 for groups O and A, and n = 5 for group B. The blood groups were compared using a Kruskal Wallis test with Dunn’s multiple comparisons (** (TIFF)Click here for additional data file.S2 Fig(A) IT/R29 mean rosette size (number of uninfected RBCs per rosette) (B) IT/R29 frequency of large rosettes (more than 4 uninfected RBCs per rosette). Purified IT/R29 infected RBCs (iRBCs) were allowed to invade into RBCs from 60 donors  and rosette size and frequency of large rosettes were assessed after one complete life-cycle by fluorescence microscopy. Samples were tested over two consecutive experimental days (day 1 = 30 and day 2 = 30) in duplicates. Horizontal bars represent the median rosette size and median frequency of large rosettes for each genotype. The number of donors per genotype are shown in parenthesis. Sample genotype was masked during counting to avoid observer bias. **AB differed from OO for both mean rosette size and frequency of large rosettes, p<0.01 Kruskal-Wallis test with Dunn’s multiple comparisons.(TIFF)Click here for additional data file.S3 Fig(A) Relative binding to CD36 recombinant protein. (B) Relative binding to ICAM-1 recombinant protein. Purified ITg infected RBCs (iRBCs) were allowed to invade into RBCs from 112 donors  and static adhesion to immobilized recombinant proteins was tested after one complete life-cycle. Samples were tested over seven experimental days , with each donor being tested once. For each RBC sample, adhesion was tested in two dishes with triplicate protein spots in each dish and the data presented as the mean iRBC bound/mm2 for each donor. Because baseline binding using a single donor varies from day to day, the binding data for each sample were normalized to that of the mean binding for the control iRBCs (OO) run on the same day . Horizontal bars represent median relative adhesion for each genotype. Number of samples per genotype are shown in parenthesis. A Kruskal-Wallis test gave p = 0.310 for CD36 adhesion and p = 0.814 for ICAM-1 adhesion.(TIFF)Click here for additional data file.S4 Fig(A) Relative ITvar9 PfEMP1 expression. (B) Relative ITvar9 positive iRBCs. Purified IT/R29 infected RBCs (iRBCs) were allowed to invade into RBCs from 60 donors  and PfEMP1 expression was assessed after one complete life-cycle by flow cytometry with antibodies specific for the ITvar9 PfEMP1 variant. Samples were tested over two consecutive experimental days (day 1 = 30 and day 2 = 30). Median fluorescent intensity and proportion of ITvar9 positive iRBC data for all samples were normalized to that of the mean MFI and proportion of ITvar9 positive iRBCs for the control pRBCs (OO) (14 and 9 reference OO genotype samples run on day 1 and 2 respectively) run on the same day. Horizontal bars represent median relative MFI and proportion of ITvar9 positive iRBCs for each genotype. Kruskal-Wallis test p = 0.076 for MFI and p = 0.285 for % positive iRBCs.(TIFF)Click here for additional data file. 23 Feb 2023Dear Dr Herbert Opi, Thank you very much for submitting your Research Article entitled 'Non-O ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malaria' to PLOS Genetics.The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. 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All points should be backed up by data provided with the submission.Please be aware that our Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.While revising your submission, please upload your figure files to the Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Susana Campino, PhDAcademic EditorPLOS GeneticsHua TangSection EditorPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: Non-ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malariaThis work described tackles a key question in malaria pathogenesis, does ABO blood type influence clinical features of disease? The work is focused partially around determining an association between Non-O dosage and clinical outcome/risk of severe disease and partially around investigating an association between ABO blood type and rosetting, a feature of malaria infection where parasitized RBCs will “coat” themselves in uninfected RBCs.Overall, this work is interesting and exciting to the field. The results are conveyed with clarity, and the caveats regarding the work well highlighted. Of note is the association study which ultimately stops short of demonstrating an association between O-dosage and the outcome of infection, though replicates a link between O vs non-O, and the link between rosette size and non-O dosage.The major unique factor of the work, to my eye, is the focus on non-O dosage. The advance described is arguably subtle, showing a tendancy for non-O dosage to increase adverse outcome in the association study. As noted, a lack of sample size and numbers in some ABO genotype groups reduced the power of the study.As I understand the work, there is a link between O dosage and either rosetting or clinical outcome, beyond which has already been reported (i.e. O vs the rest). This is the result of hard won and clearly described data and analysis and is supportive of a role whereby (1) non-O genotype dosage influences the clinical outcome of malaria disease and (2) rosetting phenotypes vary between ABO genotypes. While this appears to be a subtle advance, the careful work described here is robust and should be a springboard for larger studies with better power.Given the small numbers in Figure 1, and the focus of the paper around non-O dosage it was somewhat surprising to not see the grouping by number of non-O alleles.The derivation of the IT clones for growth in A+ cells could have been given greater discussion. The IT/R29 clone, it appears that two clones are used in the study (R29 expressing ITvar9 PfEMP1 and another (ItG), clone expressing ITvar16). How may the way these lines were derived impact the findings of the study? These lines, as I understand, were derived through selection under specific conditions. How stable are the phenotypes in each of these clones, and was the phenotype of each line confirmed using controls?A minor aside; Kappa score is described as 100% to 0% in the methods, and 0.95 listed in the results. I assume that either the score should be described as a proportion of 0 to 1 or the results should be 95%Reviewer #2: Manuscript Number: PGENETICS-D-22-01112Full Title: Non-O ABO blood group genotypes differ in their associations with Plasmodiumfalciparum rosetting and severe malariaSummary: This observational study of Kenyan children from a hospital-based case-control study and a community-based cohort examines the associations between genetically-determined ABO genotypes and risk of severe malaria. These associations are supported by in vitro assays of rosette formation in RBCs of different ABO types infected with laboratory strains of P falciparum. While this study does not necessarily present new biology about malaria pathogenesis, it does improve the precision of our understanding of the relationship between ABO genotypes and rosetting phenomena. While the experimental and epidemiological methods are sound, more attention to the interpretation of multiple testing needs to be provided.Major comments:1. Rosettes are proposed to contribute to severe malaria pathogenesis by obstructing microvessels. Presumably, rosettes would be susceptible to removal from the circulation by the spleen. Were there any associations between rosetting genotypes and measures of anemia?2. It is disconcerting to observe non-random associations between ABO genotypes encoded on chromosome 9 and HBA and HBB genotypes encoded on chromosomes 16 and 11 in the population cohort described in Table S5. Are these spurious associations? If not, what is the reason for these associations? Why the age differences by ABO genotypes?3. In the case-control study described in Table 3, why is ABO genotype associated with sex and age? Perhaps these associations are driven by cases due to relationships with susceptibility to malaria? If so, perhaps the cases and controls need to be analyzed separately, either way an explanation for these unexpected associations needs to be offered.4. Some acknowledgement, discussion, or statistical adjustment should be made regarding multiple tests performed and the interpretation of p values.Minor comments:5. While the statement in line 352 may be true, “To the best of our knowledge, whether host RBC ABO genotype influences P. falciparum rosetting has not been investigated previously.” It is really a stretch especially when you show such a high concordance between ABO genotype and phenotype.Typos, etc:6. The subheading in line 298 could be stated in the past tense.Reviewer #3: This study aimed to analyze the association between ABO blood group genotype and severe malaria in Sub-Saharan Africa. Expanding on previous knowledge that individuals with blood type O are less vulnerable to severe malaria than non-O blood types, the authors hypothesized that “double-dose” genotypes  would be more vulnerable to severe malaria compared to “single-dose” genotypes . This is an interesting and important hypothesis that could possibly improve the tailoring of severe malaria treatment and prevention. The manuscript is well-written and straight-forward to follow. The most valuable contribution is the data analysis from a large case-control study in Kenya, finding that children with double-dose genotypes versus single-dose genotypes had higher odds of severe malaria over children with the OO genotype. Importantly, this was also true for separate severe malaria syndromes. However, it will be crucial to see if these results remain robust after age-adjustment. It was also important to learn that ABO genotype may not always concur with phenotype, which should be of interest to the broader field of genetics and genomics.The authors included additional epidemiological and mechanistic analyses with a more limited contribution to the overall findings. These analyses had small sample sizes and possible statistical overfitting, raising concerns that this in vitro evidence was not substantial enough to support the article’s conclusions.Major comments1. The authors’ hypothesis is that double-dose genotypes  are more vulnerable to severe malaria than single-dose genotypes . The authors use Wald tests to determine that the double-dose genotypes had higher odds of severe malaria over OO compared to the single-dose genotypes, but they do not find statistical significance. Why not compare the odds of severe malaria between double-dose and single-dose genotypes directly?2. In the case-control study, are there age differences between the cases and controls? I would like to see the demographic characteristics stratified by cases/controls. It seems that age is not controlled for and, given that there are age differences between the genotypes, it may be an important confounding factor to consider. Ref 14 suggests that there may be a significant age difference between cases and controls. The authors did control for age in the cohort and cross-sectional analyses.1. Were there any episodes of severe malaria in the controls? This information should be stated.3. In Table 4, the authors used the same controls for each severe malaria phenotype comparison. Again, this makes me wonder whether the control pools are age-comparable to different subsets of cases.4. In Table 5, the accompanying results text, and the discussion, the authors should be clear that the reference group is still OO in all odds ratios presented. Rather than comparing single-dose to double-dose genotypes directly, they are comparing the magnitude of each genotype to OO.5. The sample sizes of RBCs for the in vitro rosetting and receptor adhesion assays were very small for the AA, BB, and AB genotypes, reducing the reliability of the statistical findings. The authors should qualify their conclusions about static adhesion and PfEMP1 expression, as their findings are limited to two PfEMP1 variants (particularly Lines 415-416).1. Consider changing Line 423 to “a key mechanism”, not “the key mechanism”6. Related to above, the authors adjusted the in vitro signals for several covariates, but I am not sure that the sample size can support so many parameters without overfitting the model.7. While the ABO findings in the cohort study are interesting, they would be more informative if there was specific information about rosetting in the symptomatic and asymptomatic episodes. As such, the conclusions from this section appear limited and somewhat peripheral to the rest of the study.8. Are there instances of clinically significant ABO genotype-phenotype discrepancies? As this is a motivating concern underlying this study, any examples should be cited and explained in the Introduction and/or Discussion.9. The Discussion should comment on the specific findings for particular severe malaria syndromes and ABO genotypes.Minor comments• Background1. It should be mentioned that RIFINs and STEVORs also mediate rosetting, especially RIFINs in blood type A.2. The cohort and cross-sectional analyses were not introduced.• Results1. I don’t understand what the authors were trying to show by demonstrating Hardy-Weinberg Equilibrium among the controls in the case-control study. This should be clarified.2. Reading the text, I was wondering what an example of ABO genotype-phenotype discordancy may look like. It was made clearer in Table 2 and the discussion, but an example in the text would help.3. In Table 3, I think that column percentages would support the authors’ argument better than row percentages.4. The authors should consider providing interpretation of the odds ratios from Table 4. For example, they could indicate that non-O genotypes had 49% higher odds of severe malaria compared to OO. Simply stating that “the odds ratio is 1.49” does not help the reader’s understanding.5. Since the authors used a parasite strain that preferentially forms rosettes with blood type A, for their rosetting experiments, it seems like the only important result is that AA > AO. Could they derive information about B-antigens if they did not use an A-preferring parasite?6. The authors should expand on the null findings for the cytoadhesion and cohort study sections in the Discussion, with specific acknowledgment of limitations and next steps.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesNo: The datasets from the case-control study and cohort study are not included.Reviewer #3: **********what does this mean?). 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Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!Yours sincerely,Susana Campino, PhDAcademic EditorPLOS GeneticsHua TangSection EditorPLOS Geneticswww.plosgenetics.orgTwitter: @PLOSGenetics----------------------------------------------------Comments from the reviewers (if applicable):Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #2: Thank you for the thorough responses and revisions.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #2: Yes**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  12 Sep 2023PGENETICS-D-22-01112R1 Non-O ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malaria Dear Dr Opi, We are pleased to inform you that your manuscript entitled "Non-O ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malaria" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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Plasmodium falciparum malaria. During the blood stage of P. falciparum infection, infected red blood cells adhere to the vascular endothelium, leading to widespread microcirculatory obstruction in critical tissues, including the splanchnic vasculature. This process may cause intestinal injury and gut leakage. Epidemiological studies indicate higher rates of concurrent bacteraemia in severe malaria cases. Furthermore, severe malaria patients exhibit alterations in the composition and diversity of the intestinal microbiota, although the exact contribution to pathophysiology remains unclear. Mouse studies have demonstrated that the gut microbiota composition can impact susceptibility to Plasmodium infections. In patients with severe malaria, the microbiota shows an enrichment of pathobionts, including pathogens that are known to cause concomitant bloodstream infections. Microbial metabolites have also been detected in the plasma of severe malaria patients, potentially contributing to metabolic acidosis and other clinical complications. However, establishing causal relationships requires intervention studies targeting the gut microbiota.The pathophysiology of severe falciparum malaria involves a complex interaction between the host, parasite, and gut microbes. In this review, we focus on understanding parasite-induced intestinal injury and changes in the human intestinal microbiota composition in patients with  Plasmodium falciparum, one of the 5 malaria parasite species affecting humans. A crucial aspect of the pathophysiology of severe falciparum malaria is the extensive sequestration of parasitized red blood cells in the microcirculation, which impairs microcirculatory blood flow and leads to dysfunction of vital organs [P. falciparum histidine-rich protein 2 (PfHRP2) [Malaria remains a significant global health burden, with an estimated 247 million cases and 620.000 deaths in 2021 [l organs . The degClostridium difficile enteritis [Plasmodium infections, with an association observed with disease severity. The causal relationship between gut microbiota composition and disease severity in humans remains unclear, but it may offer new insights into the pathophysiology of severe malaria and potential targets for intervention.The microbiome consists of a diverse group of bacteria, archaea, fungi, protozoa, and viruses. Among the human-associated microbial communities, the gut microbiota is the largest and most heterogeneous . The gutnteritis , and in nteritis ,14. Prevnteritis –18, in pnteritis . SeveralP. falciparum malaria. These consequences include the translocation of bacteria and their metabolites across the gut barrier and alterations in gut immunological functions. Furthermore, we briefly discuss potential interventions targeting the gut microbiota.In this review, we focus on the evidence from clinical studies in patients with malaria for intestinal injury, changes in microbiota composition, and their potential consequences in In a parallel review, Mandal and Schmidt discuss the evidence and insights from laboratory and experimental studies into the interaction between the host gut microbiome and malaria.Patients with malaria commonly experience gastrointestinal symptoms such as nausea, vomiting, abdominal pain, and diarrhea. Intestinal damage occurs through a complex pathological cascade, primarily driven by the extensive sequestration of parasitized red blood cells in the splanchnic microcirculation of severe malaria patients. For a summary of the mechanisms involved, see The splanchnic circulation supplies blood to abdominal organs, including the liver, spleen, stomach, pancreas, small intestine, and large intestine. It is perfused by 3 branches of the abdominal aorta: the coeliac artery, superior mesenteric artery, and inferior mesenteric artery.The splanchnic blood flow is primarily influenced by systemic vascular resistance and cardiac output. Under normal conditions, splanchnic blood flow accounts for about 25% to 30% of cardiac output, but it can vary depending on factors such as recent feeding or physiological stress .Hemodynamic shock is rare in severe malaria, as both systemic vascular resistance and cardiac output are typically maintained at adequate levels despite infections with a large parasite load. This is different from the macrovascular changes seen in bacterial sepsis. The key feature of septic shock is significant peripheral arteriolar vasodilation. This leads to low systemic vascular resistance, high cardiac output, severe hypotension, and shock, with subsequent inadequate tissue perfusion. Studies have shown that systemic vascular resistance remains at adequate levels in both uncomplicated and severe malaria, possibly due to the vasoconstrictive effects of cell-free hemoglobin released during red cell hemolysis . Mildly Sequestration of parasitized red blood cells, however, significantly affects the blood flow in the intestines’ microcirculation. Autopsy studies have shown that, given the total blood volume of the splanchnic circulation, the intestines represent a significant proportion of the total body sequestration ,22. VideAdditional mechanisms that may contribute to intestinal injury include ischemia-reperfusion injury, intestinal inflammation, and mast cell activation. Mast cell activation can damage the gut barrier, both physically and immunologically, through the release of Th2 cytokines that affect the defence against bacteria that may translocate from the intestine ,24. ThisIntestinal injury in patients with malaria can progress to impaired intestinal barrier function, as evidenced by increased intestinal permeability and abnormal markers of intestinal integrity, Streptococcus pneumoniae, nontyphoidal Salmonella, and Escherichia coli [Impairment of the gut barrier can increase the likelihood of translocation of enteric bacteria into the bloodstream, leading to concomitant bacteraemia and sepsis. In children with severe malaria in Africa, concurrent invasive bacterial infections are described, involving bacteria such as hia coli –30. Addihia coli ,32.C. difficile enteritis following broad-spectrum antibiotic treatment [The composition of the gut microbiota can potentially influence the progression of malaria infection through various mechanisms. One important mechanism is colonization resistance, which refers to the ability of the gut microbiota to prevent the overgrowth of harmful bacteria by employing different mechanisms, including the reduction of gut pH –43. Pertreatment . The gutreatment . Additioreatment .Enterococcus and Escherichia/Shigella species in their gut microbiota, pathogens that are known to be able to cause bloodstream infections [Bacteroides, Streptococcus spp., and Lactobacillus spp., in the gut microbiota was associated with the severity of metabolic acidosis, which is a strong predictor of fatal outcomes in severe malaria [In an observational study conducted in Bangladesh, the gut microbiota composition of adult patients with severe and uncomplicated falciparum malaria was compared to healthy volunteers . The stufections . Further malaria ,31. HowePlasmodium species. In Swiss Webster and C57BL/6 (B6) mice infected with Plasmodium yoelii, a reduction in the Firmicutes/Bacteroidetes ratio and a decrease in Proteobacteria were observed [Plasmodium berghei revealed a decrease in Firmicutes and, specifically in one mouse strain, an increase in Proteobacteria and Verrucomicrobia [P. yoelii, suggesting a potential role of bile acids in shaping the gut microbiota [Furthermore, mouse models have shown changes in the gut microbiota composition following infection with different observed . Anothermicrobia . In C57Bcrobiota . These fThe role of the gut microbiota in influencing the immune response to malaria has been studied primarily in mouse models, with some evidence suggesting a similar effect in human malaria. Here, we summarize important findings from recent experimental studies, but for more insights into the interaction between the host gut microbiome and malaria, see the parallel review by Mandal and Schmidt, where the experimental evidence is further elaborated.Plasmodium berghei or Plasmodium yoelii infection, including BALB/c and C57BL/6 mice, have been used to investigate the relationship between the gut microbiota and malaria pathogenesis. Studies have shown that mice colonized with the gut pathobiont Escherichia coli O86:B7, which expresses α-galactosyl, produce antibodies  that cross-react with Plasmodium sporozoites, and could mediate clinical protection against malaria infection [Mouse models of nfection .Plasmodium species. In one study, fecal content from mice with different susceptibility to P. yoelii infection was transplanted into germ-free mice, demonstrating that resistance to infection could be transferred through fecal transplant [Plasmodium correlated with the abundance of Lactobacillus and Bifidobacterium bacteria [The composition of the gut microbiota in mouse models appears to impact parasite burden and fatality rates following infection with various ansplant . In thisbacteria .Differences in parasite burden and bacterial community composition have been observed between different strains of mice. For example, Taconic mice showed lower peak parasite burden and faster recovery compared to Charles River mice, suggesting that the host–microbiota interaction plays a role in parasite burden rather than genetics or environmental factors . The stuAncylostoma duodenale, Necator americanus), ascaris (Ascaris lumbricoides), and whipworm (Trichuris trichiura), have also been associated with malaria susceptibility [Intestinal helminth infections, such as hookworm  coinfections was associated with increased densities of both P. vivax . Other sasitemia ,53, whilasitemia . Most stasitemia . Another malaria ,56. Howe malaria .P. falciparum strains. It has also been suggested that IgE-anti-IgE immune complexes resulting from helminth infections reduce the severity of falciparum malaria and can mediate tolerance to the malaria parasite through the CD23/NO pathway [A. lumbricoides infection and the risk of P. vivax malaria [P. falciparum but not P. vivax parasitemia [Proposed mechanisms for the protective effect of ascaris infection include endothelial cell receptor down-regulation and the production of IgE-anti-IgE immune complexes that reduce the severity of falciparum malaria. Endothelial cell receptor down-regulation reduces parasite erythrocyte cytoadherence or selective splenic parasite clearance, thus reducing the proportion of virulent  pathway . Studies malaria , while hasitemia ,59. OverPlasmodium infections. These interventions include probiotics, selective digestive tract decontamination, fecal transplants, and antibiotics.Therapies aimed at modifying the gut microbiota composition could be potential interventions to influence susceptibility to and severity of Lactobacilli spp., Bifidobacteria spp., Saccharomyces boulardii, and Bacillus coagulans. Probiotics and synbiotics have been suggested to reduce pathobionts through colonization resistance, prevent bacterial translocation, degrade toxins, and modulate the immune response [Probiotics are live microorganisms that can modify the gut microbiota. When combined with prebiotics, which support their growth, they are known as synbiotics ,61. Commresponse .Plasmodium infections remain largely unexplored in human studies. However, experimental mouse studies have shown promising results and have suggested a positive effect on time to death, reduction of bacteraemia, and improved gut wall integrity [Lactobacillus plantarum plus fructooligosaccharide on the prevalence of sepsis in neonates and infants. Although an earlier meta-analysis suggested a beneficial effect of probiotics on the prevention of ventilator-associated pneumonia (VAP) [Lactobacillus rhamnosus GG (LGG) versus placebo in preventing VAP did not confirm these findings [While probiotics and synbiotics have been primarily studied for their potential in sepsis, their effects on ntegrity . A largeia (VAP) , a recenfindings .There are no human studies on the use of probiotics in patients with malaria.Lactobacillus and Bifidobacterium have demonstrated a beneficial effect in reducing P. yoelii parasitemia in another experimental mouse study [Lactobacillus casei reduced the severity of Plasmodium chabaudi infection [Plasmodium berghei, the efficacy of Lactobacillus casei as adjuvant therapy to chloroquine, an antimalarial drug, was evaluated and showed a reduction in peripheral blood parasitemia with probiotic treatment [Probiotics containing se study . The admnfection . In a moreatment .Staphylococcus aureus, in the oropharynx and intestines [Selective digestive tract decontamination (SDD) involves the use of non-absorbable antimicrobials applied daily in the oropharynx and gastrointestinal tract . SDD hastestines . SDD hasC. difficile infections (CDI) [Fecal microbiota transplantation (FMT) involves the administration of a solution of fecal material from a healthy donor into the intestinal tract of a recipient through a feeding tube to restore the gut microbiota . FMT is ns (CDI) . It has ns (CDI) . HoweverAntibiotics represent a potential intervention for the treatment of bacterial coinfections in severe malaria. World Health Organization management guidelines recommend empirical broad-spectrum antibacterial therapy for all children diagnosed with severe falciparum malaria in malaria-endemic areas .Plasmodium infections. In humans, malaria leads to changes in the gut microbiota, including an increase in pathogens associated with severe disease and bacterial infections. Studies in patients with malaria show parasite-induced intestinal injury. Subsequently, impaired intestinal barrier function could allow translocation of gut microbiota and microbial metabolites into the bloodstream, potentially leading to concomitant sepsis. Observational studies in humans provide associations between the microbiota and disease severity or protection. Possible interventions include probiotic therapy, selective digestive tract decontamination, or fecal transplantation therapy. However, intervention studies are needed to establish causal relationships.Intestinal injury and the gut microbiota appear to play a role in the severity and outcome of falciparum malaria. Mouse studies show that altering the microbiota affects susceptibility to


We assess the relationship between various hemoglobin variants and some hematological parameters packed cell volume, white blood cells  and parasitemia level of patients with malaria in the southwestern, Uganda.Patient were enrolled by rapid diagnostic tests (RDTs), confirmed by microscopy, and laboratory outcomes were determined.P. falciparum parasites were 95 (61.29%) having hemoglobin variants HbAA and HbAS; 75 (78.95%) and 13 (13.68%), respectively. The laboratory outcomes showed mean, PCV (32.19 ± 4.83), WBC (5831.66 ± 2888.29) and P. falciparum parasitaemia density , while the hemoglobin variants mean values AA , AC (15908 ± 10173.48), AS , SC  and SS(1652 ± 0.000) were significantly different from the total population  parasite-density.Patients positive for malaria RDTs were 155, microscopic-confirmed Patients with hemoglobin variants HbAA had a significantly higher parasite-carrying capacity and PCV levels. In endemic countries, malaria continues to be one of the leading causes of illness and mortality, mainly affecting young children ,2. Malarfalciparum malaria in African children, with a reducing risk of about 70% in homozygosity HbC and 90% in heterozygosity HbS individuals [P. falciparum infection While P. falciparum have advanced a vast repertoire of genetic mutations to elude the human immune system and resist antimalarial medications [Malaria is a complicated illness that is influenced by numerous unclear host genetic variables . The hemividuals . Howeverividuals ,13. Howeications .It has been found that among patients with malaria, changes in hemoglobin and platelet counts occurred more frequently than changes in WBC counts . SimilarP. falciparum, such mutations may give inherent resistance to falciparum malaria and cause hemoglobinopathies imprinted on the human genome [P. falciparum parasitaemia density among patients with malaria. Such research is relevant in understanding the development mechanism of resistance to approved and effective antimalarial drugs. Therefore, in this study, we evaluate the association between different hemoglobin genotype and the laboratory outcomes of patients with malaria in some selected local health facilities in South West of Uganda.Some studies indicated that humans hemoglobin variants co-evolved with n genome . One metn genome . GeneticThis study was conducted among patient present with malaria infections in three medical centers  South Western Uganda. These health centers where randomly selected based on their importance location and the most attend health that met the medical care/need of about 75% of the population in the region.Patients who were between the ages of 1 month and 65 years, presenting with malaria symptoms such as fever, chill, vomiting, body aches and headaches that agreed to participate via informed consent were recruited. Only patient that tested positive to malaria rapid diagnostic test (RDTs) kit   were qualify for inclusion, while those that show negative for the RDT test were excluded.The patients' characteristics, such as age, gender, malaria symptoms, patient positive by RDTs, and patient positive by microscopy, were recorded in data collection form in an Excel sheet.Blood samples were collected at the clinic from the patients with malaria symptoms and based on preliminary screening that is positive to RDTs. Each patient's 3 ml of venous blood was withdrawn into an EDTA (ethylene diamine tetra acetic acid) container. The tube was appropriately mixed and maintained at 4° for further analysis.Frosted end slides were used to prepared a smear. The thick and thin films were slides placed on a slide preparation template. The thin film was fixed in absolute methanol for 1 min and allowed to air dried before staining. The dried smears were stained with 3% Giemsa  for 40–60 min. The area of the already stained slide that needed to be studied received about two drops of immersion oil. The slide was positioned on the mechanical stage and examined with a 100 × objective light microscope. First, the thick blood film was inspected to count and identify the parasites. Leukocytes and plasmodium parasites were also counted using separate tally counters . Two traSpecial simple capillary glass tubes with an internal diameter of 1 mm and a length of 70 mm were employed. One end of the capillary tubes was sealed with plasticine, and the capillary tubes were two-thirds filled by capillary action with well-mixed venous blood. Using a Haematocrit 210 centrifuge, the sealed capillary tube was spun for five minutes at 1500 r.p.m. . The packed cell volume (PCV) was calculated using a hand-held Microhaematocrit Reader  and expressed as a percentage (%) ,20.For WBC count, 380 μl of Turks solution was added to 20 μl of blood sample and carefully mixed. A cover slip was used to charge the white blood cell (WBC) cell/μl Neuber counting chamber, and the well was used to pour the diluent into the chamber's cells. The 16 cells' WBC were counted and recorded ,22.The Helena BioSciences method was used to prepared the cellulose acetate membrane. Each of the outer parts of the electrophoresis chamber received 100 ml of tris-borate-EDTA buffer. To ensure that each support bridge made contact with the buffer and that there were no air bubbles underneath the wicks, two wicks were moistened in the buffer and draped over each one. A cover was placed over the chambers to stop evaporation. In the Zip-Zone well plate, 5 μl of each haemolysate sample test and control were added. The 8unit applicator was used to apply the samples to a cellulose acetate membrane (plate)  that had been placed in the Zip-Zone alignment plate. The electrophoresis chamber was immediately plugged in, the cellulose acetate side facing down, and the cellulose acetate membrane was inserted within. The plate was then electrophoresed for 25 minutes. A method called acid citrate agar electrophoresis was employed to separate some of the co-migrating hemoglobins ,20.Means percentage was use to analyzed the frequency of patient's clinical characteristic of the study population. The significant epidemiological distribution and risk estimate of malaria hemoglobin variants at 95% confidence interval were evaluated. One-way ANOVA (Turkey test) was used to determine the association between hemoglobin variants and patient laboratory outcome. The statistically significant differences were observed at p < 0.05. All analysis were done in RStudio version 3.5.1 software.A total of 155 patients that tested positive to malaria RDTs were recruited, 37.42% were between the ages of 1–10 years, female 81 (52.26%), malaria symptoms; fever 140 (90.32%), body pain and tiredness 142 (91.61%). Out of the 155 positives for RDT, 95(61.29%) were positive to microscopy test, the demographic characteristics are shown in P. falciparum malaria infection, with odd ratio 1.00, 95% CI (69.89–86.26) attributed risk of (aR = 57.89) and relative risk of (RR = 3.75). Also, the HbSS and HbSC variant distribution was 1 (1.05), respectively. The hemoglobin HbAA has a relative risk of (RR = 3.75) detailed in The epidemiological distribution of malaria infection among the Hb variants showed that (HbAA) 115 (74.20%), (HbAC) 8 (5.20%) and (HbAS) 28 (18.10%) and  2 (1.30%) each among the RDT positive patients detailed in P. falciparum parasitaemia density were 34.19 ± 4.83, 5831.66 ± 2888.29 and 32,605.45 ± 54,079, respectively.The malaria patient's laboratory outcomes showed that the mean ± SEM of PCV, WBC, A comparison of how the different variants were associated with the laboratory outcomes showed that for PCV, HbAS, HbSS and HbSC had mean values of (32.59 ± 4.97), (24.5 ±0.71), and 25 ± 2.83), with the , respectively. There was no significant difference (p = 0.0905) in the mean values of patient with Hb AA and AC  compared with the PCV mean value (33.89 ± 5.08) of the total population. The mean PCV value of the other hemoglobin variants and their association are shown in For WBC, The HbAC, HbSS and HbSC had the mean values of (4195.71 ± 1592.09),  and (8310 ± 1654.63), respectively. There was significant difference  between the men value of HbSS, SC, AC and the WBC mean value (6138.59 ± 3040.3) of the total population. The other WBC value for other hemoglobin variants with their association are shown in For parasite density, the HbAA, AC, AS, SC and SS has mean mean values of , , ,  and (1652 ± 0.000), respectively. There was a significant difference ) in the mean value of all the hemoglobin variants compared with that of the total population . The various mean parasite densities of other hemoglobin genotype are shown in There is an evolving knowledge on the relationship between different hemoglobin variants and malaria parasites . To expaP. falciparum malaria parasites is significant to the study population. Furthermore, the chance of a patient with HbAS becoming malaria undesirable compared with a patient with HbAA is about 4%. The population with HbAS had a lower prevalence of malaria than with HbAA, and this was correlated with a considerably lower level of parasitemia. This finding is in agreement with a number of studies indicate that both hemoglobin genotype C and S offer protection against severe malaria invasion [The relatively low PCV of about 32%, which is less than the standard lowest limits of 36–38%, indicates a potential anaemia condition which is a common finding in malaria, especially children; similar findings have been reported in studies from Keyan and Nigeria ,15,29. Hinvasion . Althouginvasion . HoweverP. falciparum parasitaemia density. This is expected as malaria is known to be characterized by hematological abnormalities, which are said to be most prominent in infections with P. falciparum. A number of investigations have revealed either a lower prevalence or a lower density of parasitaemia compared with with HbAA [Our results also indicate variation in the association between hemoglobin variants and patient PCV, WBC, ith HbAA . The lowith HbAA ,36,37. Aith HbAA ,39. An oith HbAA .This study's limitations included the absence of prior medical histories for conditions such Hb disorders, anemia, bacterial or viral infections, which could potentially skew analysis and alter the interpretation of the findings. Also, the use of estimation methods as set by WHO to that of the hematology analyzers that was not available at the time of this study.P. falciparum parasitaemia in HbAS compared with HbAA patients may corroborate the protective effects that hemoglobinopathies give. The low level of P. falciparum parasitaemia in older age groups supports the idea that immunity develops naturally over time in endemic areas. In order to build strategic policies for the creation of efficient malaria control programs aimed at eradicating and/or lowering this life-threatening disease to the bare minimum, this study will help to better understand the endemicity of malaria in the study area.This study has demonstrated that individuals who had the HbAC, HbAS, HbSS, or HbSS variants, has a variable susceptibility to malaria with the high prevalence of the HbAA phenotype to malaria parasite infection. However, the prevalence of Understanding malaria epidemiology and its association with hemoglobin variants may improve treatment policy.Patients with malaria confirmed by microscopy assay were recruited to determine the association between various hemoglobin variants and PCV, WBC, parasitaemia density.Findings showed that patients with hemoglobin variants HbAA have a significantly higher parasite-carrying capacity.P. falciparum parasitaemia in HbAS may corroborate with the protective effects offered by hemoglobinopathies.The low level of


Plasmodium falciparum diversity, usually derived from the highly polymorphic genes, such as msp1, msp2 and glurp as well as microsatellites. Conventional methods of deriving MOI lack fine resolution needed to discriminate minor clones. This study used amplicon sequencing (AmpliSeq) of P. falciparum msp1 (Pfmsp1) to measure spatial and temporal genetic diversity of P. falciparum.Multiplicity of infection (MOI) is an important measure of P. falciparum positive blood samples collected from areas of differing malaria endemicities between 2010 and 2019 were used. Pfmsp1 gene was amplified and amplicon libraries sequenced on Illumina MiSeq. Sequences were aligned against a reference sequence (NC_004330.2) and clustered to detect fragment length polymorphism and amino acid variations.264 p = 0.03) than the > 5–14 (= 25.3 ± 5 SD), and those > 15 (= 25.1 ± 6 SD). Of the alleles detected, 553 (54.5%) were K1, 250 (24.7%) MAD20 and 211 (20.8%) RO33 that grouped into 19 K1 allelic families (108–270 bp), 14 MAD20 (108–216 bp) and one RO33 (153 bp). AmpliSeq revealed nucleotide polymorphisms in alleles that had similar sizes, thus increasing the K1 to 104, 58 for MAD20 and 14 for RO33. By AmpliSeq, the mean MOI was 4.8  for the malaria endemic Lake Victoria region, 4.4  for the epidemic prone Kisii Highland and 3.4  for the seasonal malaria Semi-Arid region. MOI decreased with age: 4.5  for children < 5 years, compared to 3.9  for ages 5 to 14 and 2.7  for those > 15. Females’ MOI  was not different from males 4.0 . In all regions, the number of alleles were high in the 2014–2015 period, more so in the Lake Victoria and the seasonal transmission arid regions.Children < 5 years had higher parasitaemia (median = 23.5 ± 5 SD, P. falciparum population structure.These findings highlight the added advantages of AmpliSeq in haplotype discrimination and the associated improvement in unravelling complexity of The online version contains supplementary material available at 10.1186/s12936-023-04700-5. Plasmodium transmitted through bites of infected female Anopheles mosquitoes. From 2000 to 2016, the World Health Organization (WHO) recorded significant progress in combating malaria in endemic areas. However, data from the 2021 WHO World Malaria Report showed that the progress in reducing global malaria cases is stalling. Malaria cases numbered 241 million, up from 227 million in 2020, 627 000 people died of malaria of which 80% were children younger than 5 years and Africa had 95% of global malaria cases [P. falciparum [Malaria is a life-threatening infectious disease caused by parasites of the genus P. falciparum to evade the host immune response and develop resistance to anti-malarial drugs [Early molecular studies revealed that the parasites exist as a pool of genetic “clones” within a single host, and such multi-clones contributes to the ability of al drugs –5. It haal drugs , and mayal drugs . RTS,S/Aal drugs .P. falciparum and plays a crucial role in the initial low affinity attachment of parasite to red blood cell membrane during erythrocyte invasion [MSP1 is the most abundant surface antigen in the blood stage of invasion . MSP1 coinvasion , 10, repFor malaria research, the term MOI (multiplicity of infection) is defined as the number of distinct clones per individual infection. In a given population, the calculated average MOI from the individuals values, has been proposed as a valuable metric for studying infection dynamics, including of transmission intensity , and theP. falciparum msp1 (Pfmsp1) was used to illustrate the utility of amplicon deep sequencing (AmpliSeq) in determining the malaria parasite clonal diversity beyond what can be provided by conventional approaches that use allele sizes. SeekDeep, a bioinformatics pipeline designed for analysis of haplotype frequency from amplicon deep sequencing data has been used successfully in several studies investigating malaria population genetics [In this study, genetics , 15, 16.P. falciparum 3D7 was used to initiate and maintain a malaria culture as described by [+ human RBC diluted to 5% haematocrit in complete RPMI 1640 media supplemented with 0.2% bicarbonate, 25 mM HEPES, 50 µg/mL gentamicin and 10% heat inactivated human serum. Culture was maintained in 25 cm2 corning flasks  with daily replacement of growth medium. To enrich for early ring stages parasites, culture was synchronized with 5% D-sorbitol in distilled water, which lyses RBCs containing late ring stages and other mature parasites [Pfmsp1 sequence was used as a cutoff for selecting the field samples with adequate parasite density that would good sequence data.A laboratory strain of ribed by , with miarasites . This trPfmsp1 (NC_004330.2) from nucleotides 1201627 to 1201710  was amplified in a primary PCR using 5′-CTAGAAGCTTTAGAAGATGCAGTATTG-3′ as forward primer and 5'-CTTAAATAGTATTCTAATTCAAGTGGATCA-3' as reverse primer [Pfmsp1 gene. The primers also included the Illumina adapter overhang. Briefly, in the primary PCR, 3 μl of DNA template, 0.625 µM of each primer and 1X NEB Next HIFI master mix were used in a 25 μl reaction that included initial denaturation at 95 °C for 5 min, followed by 25 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 2 min and extension at 72 °C for 2 min, then a single annealing step at 58 °C for 2 min and final extension at 72 °C for 5 min. In the secondary PCR, 2.5 μl of DNA template, 0.2 µM of each primer, 1X NEB Next HIFI master mix were used in a 25 μl reaction. Cycling conditions included initial denaturation at 95 °C for 5 min, followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, then a final extension at 72 °C for 5 min. Amplicons were visualized on 1% agarose gel stained with gel red .QIAamp DNA Blood Mini Kit (Qiagen) was used extract DNA from 200 μl of cultured parasites and study samples as recommended by the manufacturer. A region of e primer . A subseAmplicons were cleaned using AmpureXP beads  followed by a dual indexing PCR to allow multiplexing of samples. For this, a 50 μL reaction consisting of 5 μL of purified amplicons, 5 μL of each Nextera XT i7 and i5 Index Primer , 25 μL of NEBNext High-Fidelity 2X PCR Master Mix  and 10 μL of PCR grade water  was made and amplified by PCR at 95 °C for 3 min, followed by 12 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. The indexed amplicon libraries were purified with AMPure XP beads according to the manufacturer’s instructions , and then quantified on Qubit Fluorometer 2.0 using Qubit dsDNA HS assay kit according to the manufacturer’s protocol . Libraries were normalized to a concentration of 4 nM and then pooled. The pooled samples were denatured and diluted to a final concentration of 12 pM, then spiked with 5% PhiX  as a sequencing control and then sequenced on MiSeq platform  using MiSeq 600 cycle reagent kit V3 .Pfmsp1 were determined using SeekDeep v2.6.0 [extractor module in SeekDeep with the paired-end feature. After quality filtering, the reads were merged, chimeras removed and the sequences clustered at the sample level by qluster, and finally assembled based on the msp1 reference gene (NC_004330.2) to generate msp1 haplotypes. The assembled haplotypes were analyzed by processCluster algorithm which compared sample haplotypes and generated individual and population-level haplotypes and statistics. A final mapping of all sequence reads to selected reference sequences was performed with the CLC Genomics workbench  and queried against the nucleotide database (GenBank) using the Nucleotide Basic Local Alignment Search Tool (BLASTn) [Pfmsp1. The MOI, defined as the number of distinct msp1 haplotypes in an individual infection (varying by length and at the nucleotide level) was determined for the different alleles . The number of distinct genotypes for the K1, MAD20 and RO33 in each sample were added and the sum regarded as the MOI for that individual. The calculated group average was regarded as mean MOI. The expected heterozygosity was calculated from the frequencies of the different alleles within the population according to the formula: He = 1–Σ(pi^2), where He is the expected heterozygosity, pi is the frequency of the i-th allele in the population.Haplotypes of p v2.6.0 . Briefly(BLASTn) . A haploGraphPad prism and R software were used for visualization and statistical analyses. Box plots comparing the identity between groups were created with GraphPad Prism 9 software . Paired P. falciparum to as low as 0.6 parasite/µl. By Amplicon sequencing of Pfmsp1, lowest parasite density that yielded usable Pfmsp1 sequence was 9 parasites/µL.18S rRNA qPCR of cultured parasites consistently detected P. falciparum parasitaemia ≥ 9 parasites/µL were selected for inclusion in the study, 122 (49%) were from females, and the median age was 7 years (interquartile range (IQR): 1–66). 83 (30%) were younger than 5 years, 99 (36%) between 5 and 14 years and 92 (34%) > 15 years. A total of 91 (33%) samples were from the malaria endemic lake region, 48 (18%) from the epidemic prone highlands region and 108 (39%) from arid regions that have seasonal malaria. qPCR Ct values were used as surrogate for malaria parasite density  years). These differences were only significant for under 5 years and 5 to 14 year age groups (p = 0.0245).A total of 247 samples with Pfmsp1 sequences from 274 samples, 247 (84%) passed the Q30 scores. The mean number of reads per sample was 30,998 . Based on size, 1,014 alleles (size range: 108–270 bp) were obtained of which, 553 (54.5%) were K1, 250 (24.7%) were MAD20 and 211 (20.8%) were RO33 that grouped into 19 K1 allelic families (108–270 bp), 14 MAD20 (108–216 bp) and one RO33 (153 bp) , a measure of the probability of being infected by two parasites with different alleles at a given locus in all the regions was high (> 0.98).As shown in Table In general, the temporal distribution of alleles was least stable in the malaria epidemic prone highland region of Kisii compared to the endemic Lake Victoria region or the seasonal transmission arid region. Overall, alleles frequency were low in the 2010–2014 period, and increased thereafter, more so in the Lake Victoria and the seasonal transmission arid regions , MAD20 (24.7%) and RO33 (20.8%) and grouped to 34 allelic families . By including sequence polymorphisms internal to the sequences, the overall increase in the number of allelic families was by 5x (34 to 176): 5.5x for K1 (19 to 104), 4.1x for MAD20 (from 14 to 58) and 14x for RO33 (from 1 to 14). Clearly, the use of size to deduce clonal multiplicity underestimates the number of clones in an infection. These findings corroborates previous studies that used AmpliSeq for estimating MOI [Pfmsp1 haplotyping for the population was high, with a an expected heterozygosity value of 0.98.AmpliSeq that combines the size and internal sequence polymorphism improved the power to detect multi clonal infections. As shown in Fig. ting MOI –33. As hting MOI . This isting MOI , Brazil ting MOI , and Gabting MOI  and unliting MOI , 41, Thating MOI , Pakistating MOI , and Colting MOI , Senegalting MOI . Pfmsp1 Pfmsp1 block 2  was very high (> 0.98) and was independent of transmission pattern. Similar findings have been reported before [MOI has been found to be high in children compared to adults and increases with transmission intensity , 33, 50.d before .Pfmsp1 sequences that are available in public databases. Our study adds 176 distinct allelic sequences to this database. The Pfmsp1 antigen has been highly studied as a malaria vaccine candidate and to date, data has demonstrated that MSP1 based vaccine protection against clinical malaria is strain-specific and, therefore, a clear understanding of MSP1 diversity is critical to developing an effective malaria vaccine [P. falciparum population structure. One of the limitation of the study is that the sampling was only possible in patients with parasitaemia cutoff of 10 parasites/μL, thus under representing haplotypes from patients with low parasite density, thereby failing to capture the full diversity of haplotypes present in the population. There is probably no way to solve this shortcoming since technology used is not sensitive enough to sequence very low parasite load. Nevertheless, the analytical depth of AmpliSeq give high confidence that the data obtained is robust and provide a credible overview of the P. falciparum population structure in the study populations and regions.There are over 500 different  vaccine . HaplotyAdditional file 1.Pfmsp1 block 2 nucleotide sequences showing the KI (sheet 1), MAD20 (sheet 2) and RO33 (sheet 3) allelic families, including the repeat motifs found in K1 and MAD20 (shown in bolded blue fonts) and the polymorphic nucleotides (shown in bold red fonts).


Plasmodium vivax, primaquine is the sole potent drug against mature/infectious Plasmodium falciparum gametocytes. It may prevent onward transmission and help contain the spread of artemisinin resistance. However, higher dose of primaquine is associated with the risk of acute haemolytic anaemia in individuals with a deficiency in glucose-6-phosphate dehydrogenase. In many P. falciparum endemic areas there is paucity of information about the distribution of individuals at risk of primaquine-induced haemolysis at higher dose 45 mg of primaquine.The use of primaquine for mass drug administration (MDA) is being considered as a key strategy for malaria elimination. In addition to being the only drug active against the dormant and relapsing forms of A retrospective cross-sectional study was carried out using archived samples to establish the prevalence of G6PD deficiency in a malaria hotspot area in Misungwi district, located in Mwanza region, Tanzania. Blood samples collected from individuals recruited between August and November 2010 were genotyped for G6PD deficiency and submicroscopic parasites carriage using polymerase chain reaction.P. falciparum positive by PCR. G6PD heterozygous deficiency were associated with carriage of submicroscopic P. falciparum (p = 0.029).A total of 263 individuals aged between 0 and 87 were recruited. The overall prevalence of the X-linked G6PD A− mutation was 83.7% (220/263) wild type, 8% (21/263) heterozygous and 8.4% (22/263) homozygous or hemizygous. Although, assessment of the enzymatic activity to assign the phenotypes according to severity and clinical manifestation as per WHO was not carried out, the overall genotype and allele frequency for the G6PD deficiency was 16.4% and 13. 2%, respectively. There was no statistically significant difference in among the different G6PD genotypes (p > 0.05). Out of 248 samples analysed for submicroscopic parasites carriage, 58.1% (144/248) were This study showed that 16.4% of the population in this part of North-western Tanzania carry the G6PD A− mutation, within the range of 15–32% seen in other parts of Africa. G6PD gene mutation is widespread and heterogeneous across the study area where primaquine would be valuable for malaria control and elimination. The maps and population estimates presented here reflect potential risk of higher dose of primaquine being associated with the risk of acute haemolytic anaemia (AHA) in individuals with a deficiency in glucose-6-phosphate dehydrogenase and call further research on mapping of G6PD deficiency in Tanzania. Therefore, screening and education programmes for G6PD deficiency is warranted in a programme of malaria elimination using a higher primaquine dose. Plasmodium falciparum in areas approaching malaria elimination  years  was not independently assessed to assign individuals either deficient or normal. However, based on genotype, individuals were assigned as normal, heterozygous, homozygous and hemizygous. Hence, with the exception of individuals who were assigned as normal, the rest of the individuals were termed as deficient although not all individuals termed as heterozygous will be deficient./homozygous G6PD deficiency  of those tested were G6PD deficiency heterozygous and 8.4% (22/263) were hemi1/263 of p > 0.05). For males hemizygote frequency was highest (17.7%) among those in age group 0–4 years and the highest carriage of G6PD hemizygote frequency (12.9%) was observed at Budutu village but all these were not statistically significant (p > 0.05) (Table p = 0.007).Among heterozygous individuals observed were found at the highest frequency (15.5%) among 5–14 years age and the highest carriage of heterozygous (18.2%) was at Mwaholo village and the lowest 3.6%) at Budutu village. However, the result reveals that there was no statistically significant difference in heterozygous G6PD deficiency carriage between villages and age (.6% at Bup > 0.05)  Table .P. falciparum positive by PCR whereas a total of 25 samples (10.1%) and 3 samples (1.2%) were positive for P. malariae and P. ovale, respectively. None of the samples were positive for P. vivax. Furthermore, 16 (6.0%) samples were positive for both P. falciparum and P. malariae. Three samples (1.2%) were positive for both P. falciparum and P. ovale. None of the samples were positive for all P. falciparum, P. malariae and P. ovale.Out of 248 samples analysed for submicroscopic parasites carriage, a total of 144 (58.1%) individuals were P. falciparum carriage on G6PD homo/hemizygous deficiency and gender (p > 0.05). However, individuals with G6PD heterozygous deficiency were associated with carriage of submicroscopic P. falciparum (p = 0.029)  to assign individuals either deficient or normal was not independently assessed. However, based on genotype individuals were assigned as normal, heterozygous, homozygous and hemizygous. Therefore, with the exception of individuals who were assigned as normal, the rest of the individuals were referred to deficient although not all individuals termed as heterozygous will be deficient. The study reveals that the prevalence of the G6PD deficiency is 16.4%, in which genotype frequency was 8.0% and 8.4% for female heterozygous, and male hemizygous/female homozygous respectively. The overall prevalence is very similar to what has been found elsewhere in sub-Saharan Africa, where it ranged from 0 to 25% , 49 confUnlike to a study which was carried out in Nigeria where high prevalence of G6PD deficiency was particularly among children 2–5 years old , there wStudy in this area has indicated that over half of individuals have malaria in form of submicroscopic . The obsP. falciparum development is hindered in G6PD deficient red cells [P. falciparum carriage in children aged between 6 and 12 years [In this study, heterozygous G6PD deficiency was associated with carriage of submicroscopic parasites. It has already been reported in other studies that female heterozygotes are protected from severe malaria , 54, whied cells , slowinged cells . However12 years . From thPlasmodium, and also offers activity against P. falciparum gametocytes, thereby blocking transmission to mosquitoes should be considered [This high prevalence of G6PD deficiency in the area raises concern when planning for MDA for clearing of the malaria parasites in endemic areas using primaquine if a higher primaquine dose is considered , 57–60. nsidered , 61.Although studies in different malaria endemic countries including Tanzania , 62 haveTanzania is focusing on reducing malaria transmission and exploring the possibilities towards the malaria pre-elimination and elimination , the useThis study has some limitations. The enzymatic activity was not assessed, haemoglobin level and other polymorphisms in the study area, and this is a topic which warrants future research.G6PD deficiency is prevalent and spatially heterogeneous across in the study area. Likewise, submicroscopic infections, which are possible infectious reservoirs of parasites, are common in the area. The government of Tanzania is implementing recommended preventive and curative interventions to malaria with the possibility of moving towards the malaria elimination. One possibility is the use of primaquine, although SLDPQ is well tolerated and safe in G6PD deficiency, a higher PQ dosage may be needed. Higher dosage of PQ is associated with acute haemolytic anaemia in G6PD deficient individuals. Awareness is needed regarding the use of a higher dose of primaquine for G6PD deficiency individuals if required.