Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a candidate malaria vaccine antigen expressed on merozoites and sporozoites. PfAMA1’s polymorphic nature impacts vaccine-induced protection. To address polymorphism, three Diversity Covering (DiCo) protein sequences were designed and tested in a staggered phase Ia/b trial. A cohort of malaria-naive adults received PfAMA1-DiCo adjuvanted with Alhydrogel® or GLA-SE and a cohort of malaria-exposed adults received placebo or GLA-SE adjuvanted PfAMA1 DiCo at weeks 0, 4 and 26. IgG and GIA levels measured 4 weeks after the third vaccination are similar in malaria-naive volunteers and placebo-immunised malaria-exposed adults, and have a similar breadth. Vaccination of malaria-exposed adults results in significant antibody level increases to the DiCo variants, but not to naturally occurring PfAMA1 variants. Moreover, GIA levels do not increase following vaccination. Future research will need to focus on stronger adjuvants and/or adapted vaccination regimens, to induce potentially protective responses in the target group of the vaccine. Unfortunately, it remains a major cause of morbidity and mortality, especially in children in sub-Saharan Africa. Effective vaccines would be a valuable addition to the arsenal needed to further reduce the impact of malaria. Currently only one moderately efficacious malaria vaccine , addressing the pre-erythrocytic stage, is implemented in three national (pilot) programmes2. One of these blood-stage candidates is the ectodomain of Plasmodium falciparum Apical Membrane Antigen 1 (AMA1)4. AMA1 plays an essential role in the red blood cell invasion cascade7 and anti-AMA1 IgG inhibits red blood cell invasion9. It is also expressed on sporozoites and may thus also confer protection against hepatocyte invasion10. However, it is fair to state that the expectations based on the above observations are not substantiated by clinical phase II studies11, nor by vaccination-challenge studies using the Controlled Human Malaria Infection model12. However, PfAMA1 could still be considered as a candidate as part of a multi-stage, multi-component malaria vaccine. The ultimate target population for such a vaccine would be infants in malaria-endemic countries.A strategy to improve malaria vaccine efficacy would be the inclusion of additional blood-stage antigens, of which a number are currently in clinical development 13. Owing to this sequence diversity, about half of the IgG response induced following vaccination cross-reacts functionally with heterologous variants14. The three PfAMA1 Diversity Covering (DiCo) proteins have been designed to, in combination, cover naturally occurring AMA1 amino acid polymorphisms4. Vaccination of rabbits and monkeys with a mixture of three DiCo proteins yielded broadly cross-reactive antibodies capable of inhibiting the in vitro growth of several laboratory malaria strains15. This broadening of the antibody response was shown to be due to an increased amount of cross-reactive antibodies, likely due to the dilution of strain-specific epitopes in the three vaccine antigens19.PfAMA1 shows considerable sequence diversity; with a PfAMA1 database currently at 4289 entries , 167 positions out of 622 are polymorphic and at least 1383 unique sequences were found . The 449 amino acids (aa) long ectodomain of PfAMA1  has 142 polymorphic positions with a maximum between sequence difference of 42 aa. Many of these variants can be found simultaneously at a single study sitewww.euvaccine.eu), is a first-in-human evaluation done in a staggered multi-centre phase Ia/b clinical trial involving both malaria-naive and malaria-exposed adults and thus allows direct comparison of anti-AMA1 immune responses in both populations20.The phase I fast-track strategy, designed by the European Vaccine Initiative ). Four weeks after the third vaccination IgG level rises ranging between 67 (CAMP Alhydrogel®) and 618-fold (FVO GLA-SE) were observed for the 7 AMA1 variants . Titre rises for CAMP were significantly lower than those for 3D7 . IgG level rises tended to be higher for the 3 DiCo vaccine variants  in the GLA-SE as compared to the Alhydrogel® group , while by contrast rises for the 4 natural variants did not differ for the treatment groups (0.98-fold (0.29 to 3.3), p = 0.98, LMM). The anti-AMA1 IgG levels observed four weeks following the third vaccination ranged between 12.5 and 64.2 µg/mL for the CAMP Alhydrogel® and DiCo2 GLA-SE groups, respectively .IgG levels in malaria-exposed adults to four natural AMA1 variants and the DiCo variants at week 0 and 4 weeks after the third vaccination are shown in Fig. p = 0.0003, LMM). IgG levels to the four natural AMA1 variants increased slightly, but not significantly in the GLA-SE group  .Following vaccination IgG levels to the DiCo vaccine variants increased approximately fourfold in the GLA-SE group  and about 1.5-fold in the Saline group  Fig. . Fold-inly) Fig. . In the Post-vaccination Anti-AMA1 IgG levels in the Saline group ranged between 16.4 and 45.1 µg/mL for CAMP and DiCo2, respectively, whereas in the GLA-SE group levels ranged between 41.1 and 162.4 µg/mL for FVO and DiCo2, respectively. Post-vaccination IgG levels were between 2.5 and 4.1-fold higher in the GLA-SE as compared to the Saline group for CAMP and DiCo3, respectively; significantly higher IgG levels were observed in the GLA-SE as compared to the Saline group for FVO (3.9-fold [1.2 to 12]), DiCo2 (3.6-fold [1.1 to 12]) and DiCo3 (4.1-fold [1.3 to 13]). Supplementary Fig. To confirm that AMA1-DiCo vaccination induces a broad response against AMA1 variants, we compared IgG levels to several AMA1 variants in vaccinated malaria-naive to those in malaria-exposed adults receiving placebo or GLA-SE adjuvanted AMA1-DiCo. Week 30 IgG levels to 7 AMA1 variants in vaccinated naive volunteers were not significantly different from those in placebo-vaccinated Burkinabe as demonstrated with week 30 naive/exposed IgG ratios ranging between 0.54 and 1.88  and a South-American strain (HB3). The number of amino acid differences in the AMA1 molecules of these variants are shown in Supplementary Table p-values < 0.02) as compared to week zero values. No significant differences in GIA levels were observed between the Alhydrogel® and GLA-SE groups . Six months following the third vaccination GIA levels for the HB3 strain decreased significantly below baseline for the Alhydrogel® group −6.3 , whereas GIA levels for the other strains returned to baseline .Unexpectedly, FCR3, 3D7 and HB3 GIA levels at week zero differed from the expected value for malaria-naive subjects (viz. ±20%) for a number of subjects. To compensate for these differences in pre-vaccination GIA levels, the percentage-point difference between the three time points was calculated and statistically evaluated. The left three panels in Fig. p = 0.04, paired t-test), GIA levels to the 3D7 strain remained unchanged over time . The post-vaccination geometric mean IgG1 levels to the DiCo variants were significantly higher (about fourfold) in the GLA-SE group as compared to the Saline group .Pre- and post-vaccination IgG1 levels and fold-increases in malaria-exposed adults are shown in Supplementary Fig. Pre- and post-vaccination IgG3 levels and post-vaccination fold-increases in malaria-naive adults are shown in Supplementary Fig. p < 0.008, paired t-test), whereas no statistically significant differences were observed for DiCo3. No statistically significant differences were observed between the treatment groups for the decrease in IgG avidity  were determined using a NaSCN elution ELISA. IgG AI for the two cohorts are shown in Fig. Competition ELISA was performed to confirm the cross-reactivity of the vaccine-induced responses.50). High IC50 values suggest limited antibody cross-reactivity, whereas low IC50 values suggest a high degree of cross-reactivity. When comparing IC50 values per competitor-coat pair, malaria-naive subjects did not differ from the saline malaria-exposed group and only two differences were found when comparing the malaria-naive subjects with the malaria-exposed GLA-SE group; with a lower IC50 for the FVO-HB3 competitor-coat pair and a higher IC50 for the CAMP-3D7 competitor-coat pair in the malaria-exposed GLA-SE group , homologous competitor-coat pairs , 0.09 (0.04 to 0.21), 0.06 (0.03 to 0.13) and 0.10 (0.06 to 0.18) µg/mL for FVO, 3D7, HB3 and CAMP, respectively. The amounts of heterologous competitor antigens for the FVO coat were significantly higher for 3D7 , and slightly lower  for the HB3 and CAMP competitors. For the 3D7 coat antigen heterologous competitor levels were significantly higher  for the HB3 competitor and slightly higher  for FVO and CAMP, respectively. For the HB3 coat antigen heterologous competitor levels were significantly higher for the FVO and 3D7 competitors  and higher, albeit non-significantly for the CAMP competitor . For the CAMP coat antigen both FVO and 3D7 competitor levels were significantly higher when compared to the homologous competitor  and HB3 competitor levels tended to be slightly higher  than the homologous competitor.Figure oup Fig. . When co20. In malaria-naive volunteers the Alhydrogel® group showed significant increases in the number of IL5 secreting cells following vaccinations, whereas no clear-cut IL5 responses were found in the GLA-SE group. In malaria-naive volunteers the number of IFNγ secreting cells was low (<5) and only few subjects responded following vaccination. Of note is the finding that post vaccination IFNγ responses in the GLA-SE group were low, which is not expected for a TLR4 agonist-containing adjuvant. In the malaria-exposed volunteers, numbers of IL5- and IFNγ-secreting cells increased following vaccination, unexpectedly this increase appeared to be more pronounced in the Saline group. Therefore, the observed increases are more likely due to ongoing malaria transmission than due to AMA1 DiCo vaccination.The numbers of spot-forming cells following overnight stimulation with DiCo variants is shown in Supplementary Fig. Cytokine levels measured in most malaria-naive volunteers were low and, with few exceptions, did not change following vaccination  to generate a vaccine that will induce a broad response to the multitude of strains found in the field, and 2) protecting target groups, e.g., travellers and infants from endemic areas, against severe disease.The results presented here show that the first of the objectives has been achieved, in the sense that the data show that the French volunteers, as a proxy for the ultimate target group, have acquired IgG antibody levels against four natural AMA1 variants, differing between 17 to 29 amino acid residues, that are very similar in magnitude to non-vaccinated malaria-exposed adults. It should be noted, however, that immune responses in malaria-naive infants in endemic countries may differ from those in non-exposed adults.14 and IC50 values for heterologous competitors ranged between six and 88-fold14. The increased breadth of the humoral response is further confirmed by the competition ELISA experiments presented in Fig. 50 values were within a 2.3-fold range. For the HB3 and CAMP AMA1 variants as coating antigens it appears that the broadening separates in two groups where both HB3 and CAMP competitors were within a 2.1-fold range, whereas the FVO and 3D7 competitors were about 10-fold higher. In a previous study with a single variant AMA1-FVO vaccine IC50 values were 0.23 µg/mL (0.21 to 0.26), 1.47 µg/mL (1.08 to 2.02), 4.39 µg/mL (2.93 to 6.57) and 20.15 µg/mL (14.17 to 28.65) for FVO, HB3, CAMP and 3D7, respectively14, spanning an approximate 88-fold range. The ranges observed for heterologous competitors in the current study were decreased as compared to those obtained with a single variant vaccine and are indicative for a broadening of IgG responses.The broadened response following DiCo vaccination is markedly different from the results obtained after vaccination with a single allele PfAMA1 protein that shows significantly lower responses against heterologous PfAMA1 proteins, where up to 50% lower levels have been found50 value differences observed in the competition ELISA patterns. Other exploratory parameters did not reveal any differences between the French and the Burkinabe volunteers, other than a higher level of IgG3 antibodies in the Burkinabe volunteers. The decrease in post-vaccination IgG avidity for DiCo1 and DiCo2 as observed in the malaria-exposed volunteers was also found in the placebo group and, therefore is likely not due to vaccination, but rather due to ongoing malaria transmission.Overall, the DiCo approach, using three different designed AMA1 sequences, here proves to be successful at inducing broadly reactive antibody responses. This is confirmed by the similarity in IgG levels for four natural AMA1 variants in the vaccinated malaria-naive volunteers with placebo-vaccinated malaria-exposed volunteers and the small IC21. The malaria-naive GLA-SE group unexpectedly showed low IFNγ responses, which may be explained by the relatively low dose of GLA (2.5 µg) used in the current study. Other fast-track studies for malaria vaccine candidates have shown that GLA-SE is able to elicit durable Th1-like immune responses22.Cellular responses as determined by ELISpot were modest in the malaria-naive volunteers, and low compared to previously obtained resultsIn the malaria-exposed volunteers, higher numbers of IFN-γ secreting cells were found following vaccination, but as this was also observed for the placebo group, it seems likely that these are the result of ongoing malaria transmission.P. knowlesi non-human primate (NHP) model show that (AMA1-induced) IC50 values close to 3.4 mg/mL total IgG (50% inhibition at an IgG concentration of 3.4 mg/mL) controlled parasitaemia23, while Miura et al.24 and Payne et al.12 show that 100 µg/mL anti-AMA1-specific IgG, resulted in 50% inhibition, illustrating that the observed IgG concentrations (between 16.9 and 31.5 µg/mL) are in line with the observed GIA activities. This implies that vaccine-induced AMA1-specific antibody levels should be three- to fourfold higher to achieve potentially protective GIA levels25. The data presented here also show that neither Alhydrogel® nor GLA-SE are sufficiently potent to achieve these anti-AMA1 IgG levels, underscoring the need for a more potent adjuvant. Alternatively, or additionally, a different vaccination schedule may be used to achieve this goal. That alternative regimens can be successful is illustrated by a previous NHP P. knowlesi study23, in which the PkAMA1 protein was used with a potent adjuvant, combined with an extended vaccination schedule and inter-current infections, eventually resulting in the ability to control Plasmodium knowlesi blood-stage infection. Unfortunately, the development of highly potent, safe adjuvants is not a priority, hampered by the high costs associated with this. Without such adjuvant, potential vaccine candidates like PfAMA1 will not evolve to a stand-alone, efficacious malaria vaccine. Of note, care has to be taken to extrapolate NHP-data directly to the human situation. For example, the CHMI experiment of Payne et al.12 shows no correlation between GIA levels and parasite multiplication rates, while this was observed in the NHP study23, suggesting that the role of AMA1 in invasion may differ between P. falciparum and P. knowlesi.The second objective of the study was to induce potentially protective responses in the target population, in this case exemplified by the French volunteers, where GIA activity is entirely due to anti-AMA1 antibodies. In the GLA-SE adjuvanted group, the highest GIA level induced by vaccination was close to 40%, at an IgG concentration of 10 mg/mL. Calculations in a 12). One explanation may be the fact that malaria-exposed adults have antibodies to many different malaria antigens and that the observed protection is due to this antibody “cocktail”. Cellular responses could provide an alternative explanation, but we do not see a clear fingerprint for the involvement of cellular immunity, as the levels of cytokines are similarly low in both malaria-naive and malaria-exposed volunteers.GIA inhibition levels of the placebo-immunised Burkinabe volunteers were also found to be around 40% Fig. . This sh24.In the Burkinabe volunteers, only small increases were observed for the IgG levels against the natural variants, whereas the increases against the immunogens (PfAMA1 DiCo) were more pronounced. The GIA activity only marginally changed and it raises the question whether the AMA1 DiCo vaccination has had any influence on it. One possible explanation is provided by Miura et al., who show that GIA activity of anti-AMA1 antibodies is counteracted by antibodies against other  antigensIn summary, we have shown proof-of-concept for the “Diversity Covering” approach with the observation that the PfAMA1 DiCo vaccine induces broad range, cross-strain functional AMA1 antibodies in malaria-naive adults to levels similar as in adults in a malaria-endemic area.Whether AMA1-alone vaccination can protect against clinical malaria is still debated.26, a conditional knock-out study shows that 96% of (Plasmodium berghei) AMA1 is dispensable for parasite invasion of hepatocytes27. AMA1-vaccination studies with a highly potent adjuvant in malaria-naive subjects does not give rise to protection12, whereas a field study in malaria-exposed children showed that, although vaccination with AMA1 had no overall protective efficacy (17%), it appeared to have efficacy against parasites bearing a homologous AMA1 variant28. These paradoxical findings may be the result of the stringency of the CHMI, where the clinical end point is set at a much lower parasitaemia level than what is applied for the definition of clinical malaria29. Rhesus macaques are able to control Plasmodium knowlesi malaria infection after multiple PkAMA1 vaccinations , in some cases after having experienced peak parasitaemias close to 2%30. Bearing all data on PfAMA1 in mind, it is evident that PfAMA1-DiCo will not be stand-alone malaria vaccine without a very potent and safe adjuvant, if at all. This does not exclude the possibility of AMA1 being a component of a multi-antigen (multi-stage) malaria vaccine.While deletion of the ama1 gene does not yield viable (blood-stage) parasitesWith the proof of concept for the Diversity Covering approach in people, tackling the polymorphism issue for PfAMA1, further exploration of the combination of PfAMA1 DiCo with a potent adjuvant as a stand-alone vaccine or in combination with other vaccine candidates is warranted.31. An additional reason to drop the Alhydrogel® formulation was that it tends to induce Th2-biassed responses, whereas GLA-SE, containing a TLR4 agonist, is expected to induce Th1-biassed responses. The control group in the Ib phase received Saline.To accelerate early-stage vaccine development, a fast-track strategy was developed by EVI. This involves a staggered multi-centre phase Ia/Ib design for the first-in-human evaluation, which allows proceeding to the phase Ib trial after review of the safety data following the first dose in the French subjects. Both Alhydrogel® and GLA-SE-adjuvanted vaccines were tested in the French volunteers and following confirmation of the GLA-SE vaccine’s safety the Alhydrogel formulation was dropped, because of its low immunogenicity in previous trials20. Participants were healthy males and non-pregnant females aged 20–45 years. The French subjects were recruited from the Paris region. The Burkinabe subjects were recruited from the Saponé Health District, approximately 50 km southwest of Ouagadougou, the capital of Burkina Faso. Malaria transmission is seasonal and is low during the dry season (November–May) and high during the rainy season (June–October). Subjects with symptoms, physical signs or laboratory values suggestive of systemic disorders; positive HIV, HBV and HCV tests were excluded. Additional exclusion criteria for European volunteers were: a history of malaria or travel in malaria-endemic areas within the past six months; positive serology for PfAMA1-DiCo; intention to travel to malaria-endemic countries during the trial. Written informed consent was obtained from each volunteer. The protocol was conducted in accordance with the Declaration of Helsinki and International Committee of Harmonisation Good Clinical Practice Guidelines and approved by the relevant ethics committees and regulatory authorities of France and Burkina Faso.The results reported here apply to the per-protocol subjects as reported by Sirima et al.20. The malaria-naive cohort received either PfAMA1-DiCo-Alhydrogel® (N = 14) or PfAMA1-DiCo-GLA-SE (N = 10), whereas the malaria-exposed cohort received either placebo  or PfAMA1-DiCo-GLA-SE (N = 16). Serum or plasma samples obtained on day 0 and week 30 were available for all analyses, while a week 52 sample was available for evaluation in GIA.The first vaccination was given in January 2014 in France and in July 2014 in Burkina Faso and the final visits were in March and July 2015, respectively. Each vaccine contained a total of 50 µg PfAMA1-DiCo variants (divided 1:1:1 for three DiCo’s) formulated with Alhydrogel® or GLA-SE  in a final volume of 0.5 mL. Vaccines were given intramuscularly in alternating arms on day 0, week 4 and week 2614. IgG levels to the three DiCo variants were reported previously20, and are here complemented with IgG levels to four natural PfAMA1 variants. The number of amino acid position differences for the seven AMA1 variants is shown in Supplementary Table 20.Quantitative enzyme-linked immunosorbent assay (ELISA) was performed in duplicate on serum samples in 96 well half-area flat-bottomed microtitre plates , coated with 1 µg/mL purified PfAMA1 antigens according to published methodsMouse monoclonal anti-human IgG subclass conjugated to horseradish peroxidase was used for IgG1  or IgG3 detection . A standard curve was included on each plate and antibody levels in the samples were calculated using a four-parameter fit. Titres are expressed as µg/mL for total IgG and as arbitrary units for IgG subclasses, where 1 AU/mL yields an OD of 1 over background.14. Briefly, microtitre plates were coated with PfAMA1 variant proteins as described above, following blocking, incubated with a pre-determined level (~2 AU) of IgG for 1 h. Plates were then washed and incubated with a NaSCN concentration range (from 0 to 3.0 M in 0.25 M steps) for 15 min. Plates were washed and developed with goat anti-human IgG alkaline phosphatase conjugate and substrate as previously described14. The avidity index (AI) is expressed as the concentration of NaSCN required for 50% dissociation of bound antibodies (relative to controls without NaSCN).The avidities of the antibodies were determined by sodium isothiocyanate (NaSCN) elution ELISA as previously described18. Dilutions that resulted in 2 AU were calculated for each serum sample and used in the subsequent antigen competition assay. The assay involved co-incubation of different allelic forms of PfAMA1, with test sera in plates coated with naturally occurring PfAMA1 variants, such that competition occurs between the added (competitor) antigens and the coated antigen. For the PfAMA1 variants the competitor antigens were diluted threefold over seven wells from 100 to 0.137 µg/mL with PfAMA1 from the FVO, 3D7, HB3 or CAMP variants. The fraction of IgG remaining bound with competitor added is divided by the IgG levels with no competitor added. The IC50 values  are calculated from the dose-response curves using non-linear regression.Competition ELISA was performed as previously describedP. falciparum strains FCR3, 3D7 and HB3 were cultured in vitro using standard culture techniques in an atmosphere of 5% CO2, 5% O2 and 90% N2. The GIA was performed as previously described, with minor modifications9. Briefly, the effect of purified IgG antibodies on in vitro parasite growth was evaluated at a single IgG (10 mg/mL). An IgG concentration of 10 mg/mL approximates the amount of IgG (7 to 17 mg/mL) found in undiluted human plasma32. Samples were run in triplicate using 96 well half-area flat-bottomed plates  with alanine-synchronised cultures of P. falciparum schizonts at an initial parasitaemia of 0.2–0.4%, a haematocrit of 2.0% and a final volume of 50 µL. After 40 to 42 h, cultures were re-suspended and transferred into 200 µL ice-cold PBS. The cultures were centrifuged, supernatants removed and the plates were frozen. Parasite growth was assessed by measuring parasite lactate dehydrogenase levels with the lactate diaphorase APAD substrate system, and plates were read at 655 nm after 30 min incubation in the dark. Parasite growth inhibition is calculated as follows: 100 × 1 − (OD schizont – OD background)/(OD_IgG – OD background). Parasite cultures without added IgG were used to define maximum parasite growth.Blood samples obtained at weeks zero, 30 and 52 were used for growth inhibition assays (GIA). The antibodies used in GIA were purified from serum or plasma  on protein G columns , exchanged into RPMI 1640 using Amicon Ultra-15 concentrators , filter-sterilised and stored at −20 °C until use. IgG concentrations were determined using a Nanodrop ND-1000 spectrophotometer . 33. Numbers of spot-forming cells for IL5 and IFNγ were calculated as the average number of spots from the three separate DiCo stimuli as previously published20. Supernatants from the overnight stimulations for IL5 and IFNγ ELISpot assays as performed at the trial sites20 obtained at weeks 0, 26, 30 and 52 were pooled per time point and the three separate DiCo stimuli and assayed by a ProcartaPlex 10-plex assay for IFNγ, IL2, IL4, IL9, IL10, IL13, IL17A, IL21, TNFα and TNFβ .The cellular immune response was assessed in vitro by measuring production of the T-cell IL5 and IFNγ cytokines by ELISPOT following overnight stimulation with 10 µg/mL of the three DiCo variants on PBMC samples obtained at Day 0, Week 26, 30 and 52. A negative (unstimulated cells) and a positive control (cells stimulated with PMA-Ionomycin) were included for each subject, as previously described34. Figures were prepared using the ggplot2 package in Microsoft R-open. Antibody levels (IgG and IgG subclasses) were log-transformed to obtain normality and are presented as geometric means with 95% confidence intervals throughout the manuscript. GIA levels and IgG avidity were approximately normally distributed and are presented as arithmetic means with 95% confidence intervals. The statistical significance of between group differences for pre-vaccination IgG levels was initially evaluated using one-way analysis of variance (ANOVA). Significant between group differences were further evaluated by Tukey’s Honest Significant Difference post hoc test, which applies a correction on the p-value for multiple comparisons and provides estimates with 95% confidence intervals (95% CI) for the between group comparisons. The statistical significance of post-vaccination between treatment differences was evaluated using linear mixed models (LMM) with antigen and treatment as fixed effects combined with antigen in subject as random effects.All statistical analyses were performed using Microsoft R-Open version 3.5.0Further information on research design is available in the Supplementary InformationReporting Summary


Plasmodium malariae, which happened in a patient with acute hemorrhagic gastropathy. Case presentation: In April 2019, a 70-year-old Italian man with recurrent spiking fever for four days was diagnosed with a P. malariae infection, as confirmed using microscopy and real-time PCR. The patient had never been abroad, but about two months before, he had received a red blood cell transfusion for anemia. Regarding the donor, we revealed that they were a missionary priest who often went to tropical regions. Plasmodium spp. PCR was also used on donor blood to confirm the causal link. Discussion and Conclusions: The donations of asymptomatic blood donors who are predominantly “semi-immune” with very low parasitic loads are an issue. The main problem is related to transfusion-transmitted malaria. Our case suggests that P. malariae infections in semi-immune asymptomatic donors are a threat to transfusion safety. Currently, microscopy is considered the gold standard for the diagnosis of malaria but has limited sensitivity to detect low levels of parasitemia. Screening using serological tests and molecular tests, combined with the donor’s questionnaire, should be used to reduce the cases of TTM.Transfusion-transmitted malaria (TTM) is a rare occurrence with serious consequences for the recipient. In non-endemic areas, the incidence of transmission of malaria by transfusion is very low. We report a clinical case of transfusion-transmitted malaria due to  Plasmodium spp.; it is responsible for an acute febrile illness with varying severities depending on the species involved and on the subject’s immune status, but also on the process of invasion and multiplication of the parasite in human red blood cells (RBCs) during their complex life cycle. Five species of Plasmodium spp. are currently known to cause malaria in humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. Malaria is found in tropical and subtropical areas where malaria parasites are naturally transmitted by the infective bites of anopheles female mosquitoes during their blood meal. In many temperate areas, such as Western Europe and the United States, economic development and public health measures have succeeded in eliminating malaria. However, most of these areas have anopheles’ mosquitoes that can transmit malaria and the reintroduction of the disease is a constant risk [Malaria is an infectious disease that is caused by intracellular protozoan parasites of the genus ant risk . Just asant risk ,3.Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae are the species that are most frequently detected in TTM [P. malariae associated with transfusion in a patient after having acute hemorrhagic erosive gastropathy.An alternative cause of malaria is transfusion-transmitted malaria (TTM). The incidence of transfusion-transmitted malaria in non-endemic nations due to severe donor choice is very low ,5. Plasmd in TTM . We presE. coli arose, which was treated with ciprofloxacin and subsequently ceftazidime because of the persistence of the febrile state. Physical examination: sensory alertness, fevers up to 38.8 °C accompanied by chills, absence of lymphadenomegaly, and blood pressure (PAO) 90/60. On the first day of hospitalization, the patient underwent microbiological, biochemical–clinical, and instrumental investigations. Regarding the blood: WBC 3390/mmc, Hb 10.5 g/dL, and PLT 131000. Negative abdominal ultrasound. Negative microbiological investigations for toxoplasma, cytomegalovirus (CMV), Epstein–Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), treponema pallidum hemagglutination assay (TPHA), Weil–Felix, and Widal–Wright. Negative urinary culture. An osteomyelobiopsia was performed, which showed hypercellulated bone marrow with reactive type changes. On the third day of hospitalization, the patient has a worsening health condition due to the persistence of fever, thrombocytopenia , anemia (reduction of hemoglobin from 10.5 to 8.2 mg/dL), and acute renal failure  for which dialysis was started. Returning to the donor, we learned that he was a missionary priest who had traveled to endemic regions; however, this was more than 10 years ago. Therefore, he had been subjected to the mandatory donation tests according to the “Provisions relating to the quality and safety requirements of blood and blood components,” resulting in a suitable donation [Plasmodium spp. were sent to the Unit of Microbiology and Virology of the Paolo Giaccone Hospital in Palermo. In the peripheral blood smear, trophozoites and schizonts were identified as P. malariae via their morphologies. Real-time PCR was used as a support for the microscopic examination and was also used on the donor blood to confirm the causal link. After infectious counseling with the Infectious and Tropical Diseases Unit of the Paolo Giaccone Hospital, therapy began with piperaquine tetraphosphate + dihydroartemisinin (Eurartesim) 320/40, which was followed by an improvement of health with a resolution of fever, recovery of liver and kidney function, and a return of the blood count within normal parameters.In April 2019, a 70-year-old male patient of Italian nationality who had never been abroad was observed by the Oncohematology and TMO Unit of the Maddalena Clinic for about two months after having a recurrent spiking fever for four days. Regarding the pathological history: erosive gastropathy for which the patient received a transfusion due to anemization (Hb 6.5 mg/dL) in January. Fever started after the transfusion, and a urinary tract infection due to Plasmodium spp. infection that is caused by a whole blood or blood component transfusion from a malaria-infected donor to a recipient. The first case of malaria as an accidental consequence of blood transfusion was described in 1911 by Woolsey [Plasmodium species were detected in 100 TTM case reports with different frequencies: 45% P. falciparum, 30% P. malariae, 16% Plasmodium vivax, 4% P. ovale, 2% P. knowlesi, and 1% mixed infection P. falciparum/P.malariae [Plasmodium spp. are able to survive in stored blood, even if frozen, and retain their viability for at least 1 week, possibly well over 10 days depending on the conditions of storage [P. falciparum, 3 years with P. vivax, and decades with P. malariae. [P. malariae due to a blood transfusion that was received from an asymptomatic donor who had been in endemic regions a decade earlier. Infection was confirmed in the donor using molecular biology. This suggests that all blood donors who were in endemic areas should be screened for anti-malarial antibodies, even if they were there a long time beforehand. To reduce cases of transfusion-transmitted malaria, diverse surveillance strategies can be used, including pre-donation questionnaires and/or laboratory screening [Plasmodium spp. antibodies and, in case of a positive result, to be excluded as blood donors for 3 years [Transfusion-transmitted malaria (TTM) is an accidental  Woolsey . In Ital Woolsey . Plasmodmalariae . These amalariae , and expmalariae . All Pla storage . Furtheralariae. ,14,15,16creening . The usecreening  but the  3 years . The lim 3 years ,21. An i 3 years ,23.The WHO recommends that nucleic acid amplification tests should be considered for epidemiological research and survey mapping of sub-microscopic infections. Indeed, the polymerase chain reaction (PCR) is the most sensitive method available, detecting parasitemia from 2–5 parasites/μL, unlike microscopy and RDTs, which have a sensitivity of 50–500 parasites/μL. and ~100 parasites/μL respectively. However, PCR is an expensive and complex method ,25.Transfusion-trasmitted malaria is a rare but serious possibility. The optimal strategy to reduce the risk of transfusion-transmitted malaria in non-endemic countries without unnecessary exclusion of blood donations is still debated and semi-immune individuals represent the biggest challenge for TTM screening, as they might become asymptomatic carriers with a very low parasite density, which is difficult to detect with current direct diagnostic methods. Currently, microscopy represents the gold standard analysis method for the diagnosis of malaria despite the limited sensitivity when detecting asymptomatic infections with low parasitemia. Molecular tests and serological tests, combined with a donor’s questionnaire, should be used to prevent transfusion-transmitted malaria in non-endemic areas.


The Solomon Islands has made significant progress in the control of malaria through vector control, access and use of improved diagnostics and therapeutic drugs. As transmission is reduced there is a need to understand variations in transmission risk at the provincial and village levels to stratify control methods.Plasmodium species parasites in blood samples was detected using PCR.A cross-sectional survey of malaria in humans was conducted in the Solomon Islands during April 2018. Nineteen villages across 4 provinces were included. The presence of Plasmodium falciparum was 1.2 % (n = 23) and for Plasmodium vivax was 1.5 % (n = 28). 22 % (n = 5/23) of P. falciparum DNA positive participants were febrile and 17 % of P. vivax DNA positive participants (n = 5/28). The prevalence of both P. falciparum and P. vivax was extremely spatially heterogeneous. For P. falciparum, in particular, only 2 small foci of transmission were identified among 19 villages. Plasmodium falciparum infections were uniformly distributed across age groups. Insecticide-treated bed net use the night prior to the survey was reported by 63 % of participants and significantly differed by province.Blood samples were analysed from 1,914 participants. The prevalence of DNA of P. vivax. Village-level stratification of targeted interventions based on passive and active case detection data could support the progress towards a more cost-effective and successful elimination programme.Malaria transmission across the Solomon Islands has become increasingly fragmented, affecting fewer villages and provinces. The majority of infections were afebrile suggesting the need for strong active case detection with radical cure with primaquine for The online version contains supplementary material available at 10.1186/s12936-021-03779-y. Since the beginning of the millennium, substantial progress was made to reduce the global incidence of human malaria, with 12 new countries being certified as malaria-free since 2007 . As malaPlasmodium falciparum replaced by Plasmodium vivax as the dominant malaria parasite in the Solomon Islands [The Solomon Islands has made significant progress to control malaria through vector control, access and use of improved diagnostics and therapeutic drugs. Vector control has been the principal preventative intervention to reduce malaria transmission over the past two decades with long-lasting insecticidal nets (LLINs) and IRS, both globally and in the Solomon Islands. These strategies have seen  Islands .Here the annual parasite incidence (API) was reduced from over 200 per thousand in 2003 to just below 30 per thousand in 2014. However, since 2015, the API has increased reaching 107 per thousand in 2019. This resurgence has delayed the goal of malaria elimination with the priority of the 2021–2025 Strategic Plan now seeking to reach zero cases by 2034 . In 2019The cross-sectional study was conducted in the Solomon Islands  in April 2018. The Solomon Islands is hot and wet with an annual rainfall of 2,005 mm . Rain falls year round with a peak during January to March. The mean daily coastal temperature ranges between 24 and 30 °C with a mean of 26 °C.P. falciparum cases based on passive case detection at health facilities. Inclusion criteria required villages to have a minimum resident population of 200, and to be accessible by sea or road. The sites encompassed 19 suburbs and villages  name, age, gender, household number, (2) fever history, (3) malaria diagnosis and treatment history and (4) access/use of mosquito protection measures. The typanic temperature of participants was measured (Welch Allyn Braun ThermoScan Pro 6000) and any febrile individuals (temperatures > 38 °C) were immediately referred to the nearest health facility.Each participant provided a ≤ 10 ml blood sample by venepuncture using vacutainers, drawn by a nurse trained and employed by the Ministry of Health and Medical Services. Five µl of selected blood samples was immediately tested for malaria using the AccessBio CareStart rapid diagnostic test (RDT) (G0161) according to the manufacturer’s instructions. Concurrently, 3 × 50 µl blood spots were placed onto cellulose chromatography papers  and dried under ambient conditions. Dried blood spots were stored in individual snap-lock bags and sealed in an airtight container with silica gel.g for 10 min. Serum and clots were initially stored at 4 °C, then frozen at − 20 °C within 4 days, until shipped internationally on dry ice and subsequently stored at − 80 °C until analysed. A unique code was assigned to each participant and their associated samples.Serum was separated by centrifugation at 1,500Plasmodium 18 S gene [18 S Plasmodium followed by a multiplex reaction targeting P. falciparum, P. vivax, Plasmodium malariae and Plasmodium ovale.Dried blood spot (DBS) samples were pooled (4 samples/pool) for extraction using the Chelex/saponin method [adapted from 8 S gene , 11. Pos8 S gene , 11. Whe8 S gene  targetinchisq.test).The relationship of province on use of insecticide-treated nets (ITNs), other vector control measures, domestic and international travel history and malaria positivity were compared using a chi-squared contingency table (MASS) with a binomial distribution, a binary dependent variable . Step-forward multi-model inference (MMI), based on ranking the value of the Akaike’s Information Criterion (AIC), was used to select the explanatory variables that best described malaria PCR positivity. The relative strength of evidence for each model within the set of alternatives was assessed using Akaike weights (wAIC) where the wAIC for each model is interpreted as the probability for the most likely model, with support varying from 0 (no support) to 1  [Χ2 distribution [The relationship between malaria PCR positive explanatory variables was analysed using a generalized linear model  –15. The ribution , 17. TheA total of 1,977 individuals  participated in the study. Participants had a median age of 30 years, with 62 % being female , with higher mosquito coil use in Honiara and Guadalcanal , and 16 % of participant houses in Western Malaita having window-screens ) the night prior to the survey was reported by 63 % of participants and significantly differed by province : 46 participants were malaria positive with 17 individuals positive for P. falciparum, 22 with P. vivax, 6 with both P. falciparum and P. vivax, and a single individual with P. ovale : 2 were positive for P. falciparum, 2 were positive for P. vivax and 3 were PCR positive with both P. falciparum and P. vivax; Table P. falciparum was 22 % (n = 5/23) and for P. vivax was 17 % (n = 5/28).Blood samples from 1,914 participants were analysed by PCR for P. falciparum, the base GLM model was most substantially improved by adding village (100 % wAIC support). Sequentially the model was improved by adding temperature  and the remaining 22 positive participants were from only 2 villages in Malaita. Overall, 69 % of the P. falciparum positives were from a single village, where the prevalence was 15.5 % . Sequentially the model was improved by adding temperature  and gender (51 % wAIC support). These explanatory variables of village, temperature, bed net use and gender were significant (log-likelihood ratio test), and were included in the most parsimonious model , Honiara (n = 4) and Malaita (n = 20), across 7 villages. Overall 63 % of the P. vivax positive individuals were found in 2 villages, where the prevalence was 8.0 and 9.7 % , and provides evidence supporting the implementation of the strategic plan to target these foci effectively.The current WHO recommendations for elimination and post-elimination settings calls for stratified targeting of control efforts by transmission intensity zones , 38. In An. farauti, the dominant malaria vector, occurs early in the evening and pre-dominantly in the peri-domestic areas of house verandas and kitchens [The strategic plan outlines the strategies for vector control, case management and surveillance and response. For vector control, the aim is to achieve universal coverage with LLINs, and to use IRS to rapidly reduce incidence in high transmission and outbreak areas. Previous work demonstrated that the risk of being bitten by kitchens . Hence, kitchens . For caskitchens  and datakitchens .P. vivax. Village-level stratification of targeted interventions could support the progress towards a cost-effective and successful elimination programme.Pockets of malaria were detected in highly localized foci in the Solomon Islands. This highlights the need for stratified malaria control with increased vector control in high transmission areas and case-based surveillance using RACD in low transmission areas with anti-malarial treatment including radical cure for Additional file 1: Table S1. The number and percentage of participants that were positive for either P. falciparum or P. vivax, summarized by the various explanatory variables. Figure S1. Age trends of Plasmodium species infections detected by PCR.


Plasmodium antigens, and Plasmodium DNA, respectively. Bone and tooth samples from ten individuals who displayed skeletal lesions associated with anaemia, from a site in southern Egypt (third to sixth centuries AD), were selected. Plasmodium antigens were detected in five of the ten bone samples, and traces of Plasmodium aDNA were detected in six of the twenty bone and tooth samples. There was relatively good synchronicity between the biomolecular findings, despite not being able to authenticate the results. This study highlights the complexity and limitations in the conclusive identification of the Plasmodium parasite in ancient human skeletons. Limitations regarding antigen and aDNA preservation and the importance of sample selection are at the forefront of the search for malaria in the past. We confirm that, currently, palaeopathological changes such as cribra orbitalia are not enough to be certain of the presence of malaria. While biomolecular methods are likely the best chance for conclusive identification, we were unable to obtain results which correspond to the current authentication criteria of biomolecules. This study represents an important contribution in the refinement of biomolecular techniques used; also, it raises new insight regarding the consistency of combining several approaches in the identification of malaria in past populations.Although malaria is one of the oldest and most widely distributed diseases affecting humans, identifying and characterizing its presence in ancient human remains continue to challenge researchers. We attempted to establish a reliable approach to detecting malaria in human skeletons using multiple avenues of analysis: macroscopic observations, rapid diagnostic tests, and shotgun-capture sequencing techniques, to identify pathological changes, The online version contains supplementary material available at 10.1007/s12520-021-01350-z. Discussions of disease in the past must take a multi-disciplinary approach, relying on archaeological contextual information, material culture studies, and, most importantly, analyses of human remains . The parasite is transmitted to humans through the bite of infected female Anopheles mosquitoes. In 2018, an estimated 228 million cases of malaria worldwide were reported . In P. falciparum infections, the later developmental stages (trophozoites and schizonts) are sequestered in the lumen of small vessels via their adhesion to microvascular endothelial cells, potentially leading to severe disease , malaria is a life-threatening disease in humans caused by the protozoan parasite of the genus rted WHO . The disrted WHO . PlasmodP. vivax was present and recorded in the Eastern Mediterranean in ancient texts by the fifth century BC  and which can be localised in the skeleton  rapPlasmodium DNA, using a similar approach as described by Marciniak et al.  are lesions associated with anaemia and therefore possibly malaria  Anaemia has a twofold relationship with malaria; it is not only closely associated with the Plasmodium parasites, which cause ‘malarial anaemia’ or haemolytic anaemia, but it is also believed to confer a degree of acquired immunity or resistance through inherited blood disorders, such as thalassemia and sickle-cell anaemia  method, in a sample from Lugnano, Italy   Table ; Fig. 2.O) Table  and was O) Table . The sitO) Table , 1980. CO) Table : 12. CemThe individuals from the graves are recorded here with the cemetery identifier, the grave number, and if there was more than one individual in the grave, then the individual number is also provided (Cem/Gr/Ind). Each individual has two EURAC laboratory identifiers — a number for the tooth sample and a number for the bone sample Fig. .Fig. 2ToThe ten individuals from Sayala were all re-examined macroscopically at the Natural History Museum, Vienna. Standard methods of assessment were applied to assess for age, sex, and any pathological changes  malaria card test Pan/Pf, DiaSys  for the detection of the HRP2 antigen of P. falciparum and pLDH for a pan-malarial antigen. For further details regarding the methods employed here, see Loufouma Mbouaka et al.  The tooth was transversally split with a clean and disinfected Dremel diamond blade and the inner part, composed of pulp and dentine, was collected with a driller and weighed, and (2) a hole was created with a single-use drill bit in the bone at a point where the dense medullary bone was expected, and the interior surface of the cortical bone was collected and weighed. Approximately 60 mg of tooth powder and 230 mg of bone powder were taken for each respective sample and subjected to a silica-based DNA extraction . Pre-processed shotgun reads were first aligned to the human full genome . The minimum mapping and base quality were both 30. The resulting bam files were used to check for characteristic aDNA nucleotide misincorporation frequency patterns using mapDamage2 v.2.0.9  and Plasmodium vivax  and against the mitochondrial genomes of both organisms  using BWA , 2230 (CIII/8/2), 2239 (CII/139), 2240 (CII/139)) were further subjected to a capture-enrichment assay targeting Plasmodium mitochondrial genomes. The capture in-solution bait assay was designed according to Marciniak et al.  and excluding parts of the COXIII gene  and positions within the intergenic and rRNA regions  that previously displayed high rates of unspecific bindings . Thereby, retrieved sequence reads were subjected to the same pre-processing and analysis pipelines as described before for the shotgun sequence reads. Mitochondrial capture results were compared to the captured sequence data of two Plasmodium positive samples (LG20 and LV13) from Imperial period Italy published by Marciniak and colleagues   platform. Paired Illumina reads were quality-checked and processed  using the SeqPrep tool v1.2  Fig. . It is dls Lewis : 193–195Plasmodium antigens, as hoped. To summarise, 40% (n=4) of the samples presented positive results for Plasmodium falciparum and/or Plasmodium vivax, and/or the pan-malaria, with a further sample testing positive for the pan malaria antigen only.The immunological assessment was performed only with the bone samples from the ten individuals, since there was not enough material remaining after the DNA analysis to perform immunoassays with tooth samples . In addition, the capture assay of two samples  that contained no Plasmodium DNA in the shotgun datasets were included in as negative control, and for the subsequent data analysis, two individuals from the study of Marciniak et al. . The four individuals who tested positive for the presence of malaria did not display any consistent pathological lesions which set them apart from the individuals who were negative for malaria. The following section will provide a discussion of the results and some of the implications of this study for the detection of malaria in ancient human remains.Both the immunological assay and the aDNA results reveal consistent indications of the presence of ls Table . While sPlasmodium parasite DNA, or evidence of haemolytic anaemia resulting in porosity in the bones, it is particularly useful that the results show some correlation. This section will present our discussion on the results obtained with each method, with particular focus on the limits of each, including biomolecule survival, test specificity and sensitivity, and sample selection. We conclude that at this stage of research into malaria in the past, it is important to apply multiple techniques to establish the presence of malaria in ancient human remains and ensure authentication of the results for conclusive statements to be made.This research has provided the first comparative study between two widely used biomolecular techniques and skeletal morphological changes in the investigation and identification of malaria in a past population. Previous research on the identification of malaria in archaeologically derived human remains has used only one approach: immunoassay ; however, asymptomatic individuals pose a conundrum for our analysis, as typically they carry a lower parasite density, yet may be more likely to suffer from anaemia , and thus, the skeletal response would reflect the anaemia, while the biomolecular results may not support a malaria diagnosis in archaeological populations in endemic areas  (see Brickley n = 14) displayed some level of porosity, while only 14.5% of adults with intact orbits (n = 227) exhibited porosity (n = 33), with females displaying more severe cases and a higher prevalence   and Plasmodium falciparum histidine–rich protein-2 (pfHRP2) which is specific to the P. falciparum species and parasite lactate dehydrogenase (pLDH) which is an example of a pan-/species-specific malaria antigen), and subsequently, the ability of the commercially available rapid diagnostic tests (RDTs) to detect the fragmented or small quantities of these antigens which are likely present in ancient remains. Research seems to suggest that the antigens that the RDTs detect can remain measurable in the body from 5 days to up to 2 months after treatment and can still be detected after death caused by malaria   which could increase risk of contamination from soil; this does not seem to have a bearing on our results . Therefore, if we consider that Strouhal observed that 68.6% of the infants and children with intact orbits and 14.5% of adults with intact orbits display cribra orbitalia, it is possible that malaria was more frequent than our biomolecular analyses have revealed. Certainly, it seems that this population faced nutritional or haemopoietic stresses which is consistent with other Nile Valley populations around this time  individuals examined presented a positive result of either both or one biomolecular test, and of these, five of seven (71%) of the genetically female skeletons tested were positive, while only one of three (33%) of the male skeletons are. The greater number of females in the sample group reflects a blind aspect of the study, as most of the individuals could not be osteologically assessed for sex, and it was only genetically identified later; this implies that more females than males displayed osteological markers of possible anaemia . Clinical research indicates that children and pregnant women are at the greatest risk of acquiring malarial anaemia  represent both sexes, albeit disproportionately female, and predominately are over the age of 12 years-at-death. Sayala was a large population situated on the banks of the Nile, and there are still questions regarding the nature of the community and the social structure associated with it, which, once answered, may help to understand the prevalence of malaria within this community. Our results, across the three methods of analysis, indicate that there is consistency across biomolecular methods, but further refinement is still needed to be able to authenticate the results for precise identification of malaria in the past. It is only when this is possible that prevalence and distribution of the disease and the different species of malaria can be discussed, alongside the impact such an endemic disease would have had on the community.Plasmodium DNA, suggest that there is great promise in applying multiple analyses to an archaeologically derived skeletal collection. At the moment, the limitations predominantly relate to the thresholds imposed by the specificity and sensitivity of the tests applied; thus, with continual improvements to the technology, both these methods could have a great impact on our understanding of disease prevalence and its effect on populations in the past. The methods of DNA extraction and analysis are improving with great speed, and specific DNA fragments are consistently identified with greater precision from very ancient remains. Still, however, there is a limit to analysing ancient DNA when the material is highly degraded. Likewise, although the use of RDTs in identifying malaria in the past has had a relatively long history, there still remain issues with specificity and identification of the binding antibodies. Thus, while a false positive is not likely in aDNA analyses, it is certainly still possible with the RDTs, and conversely, a negative result in either analysis is not a conclusive negative. Importantly, proteins tend to be more stable than DNA in ancient human remains. Therefore, focus of future analyses should be put on the identification of specific Plasmodium proteins to avoid false positive diagnostics. Overall, there was no evidence from the palaeopathological analysis of the skeletal material of distinctive consistent osseous changes between individuals who subsequently showed positive genetic and immunological results for the presence of Plasmodium and those who did not.The results of this study, in which four of the ten individuals sampled provided matching positive results for both malarial antigens and indications of We suggest, as a result of our investigation, that bones, with good preservation of the medullary bone, are likely an effective sample source in the search for malaria and that collaboration between multiple strands of biomolecular testing contributes to the confirmation and success of the results. Irrefutable evidence of the presence of malaria through macroscopic observations is the goal of palaeopathologists to avoid destructive analyses; however, this will be most effectively realised through rigorous comparison amongst methods on a skeletal collection which is well-preserved with documented malaria cases. As we have noted above, in terms of biomolecular analyses, the survivability of proteins encourages further exploration into immunological assays in particular. It is only when the results are consistent and replicable that malaria will be identified with certainty in ancient remains, and the evolutionary history of this significant disease can be explored thoroughly as researchers seek to create a vaccine. Our research provides new insight and shows the complexity of investigating diseases in the past through multidisciplinary approaches; it also demonstrates the benefit of combining different techniques in the refinement of the process of identification of malaria in ancient human remains.ESM 1(DOCX 14 kb)ESM 2(DOCX 17 kb)ESM 3(XLSX 30 kb)ESM 4(JPG 5321 kb)ESM 5(JPG 4632 kb)ESM 6(JPG 5698 kb)ESM 7(JPG 5139 kb)ESM 8(JPG 1334 kb)ESM 9(JPG 1363 kb)ESM 10(JPG 1349 kb)ESM 11(JPG 1028 kb)ESM 12(JPG 1341 kb)ESM 13(JPG 4192 kb)ESM 14(JPG 174 kb)ESM 15(DOCX 270 kb)ESM 16(DOCX 120 kb)ESM 17(DOCX 823 kb)ESM 18(DOCX 166 kb)


Khuzdar, the largest city of Southern Balochistan, is endemic for malaria with API of 6. The study was aimed at comparing the clinico-lab profile of severe and uncomplicated malaria in the region and to determine any association with age. No such study is reported in the region so far.A prospective clinical observational study was conducted in Combined Military Hospital, Khuzdar between 2018 and 2020. A total of 210 Malaria patients, irrespective of age and gender were included. Cases were categorized into severe and uncomplicated according to WHO criteria. The clinical parameters and lab profile of severe and uncomplicated cases were compared and data was analyzed using SPSS 23.0. Categorical variables were analyzed for association of clinical features with severe malaria using Fisher exact test. Continuous variables were compared between uncomplicated and severe malaria using Mann-Whitney U –test. Statistical significance of lab parameters with type of malaria was derived using Kruskal Wallis.Uncomplicated and severe cases were 191 (91%) and 19 (9%) respectively. Severe malaria was significantly associated with jaundice, bleeding from gums, epistaxis, pallor, vomiting, respiratory distress, thrombocytopenia, low Hb, raised serum bilirubin and raised PT . In children, frequency of multiple complications was significantly higher than adult patients. .Overall case fatality of severe malaria was 5.2% (1/19). However, case fatality rate was 100% in cerebral malaria.Certain clinical and lab parameters can be used to predict prognosis and thus avoid adverse outcome in malaria patients. P.falciparum, the region contributed to 53% of P.vivax burden in 2018. According to WHO, Malaria Incidence rate in Pakistan in 2018 was 1-10(cases per 1000 population at risk).2Malaria remains one of the most prevalent parasitic diseases across the world, claiming about half a million deaths globally with majority occurring in Africa followed by Southeast Asia and South America.P.vivax accounted for 84% of all malaria cases. Lack of access to healthcare facilities/low blood examination rates may be the reason of low reported cases in Balochistan.4Pakistan is among seven countries of WHO Eastern Mediterranean Region sharing 98% of total regional malaria burden. In 2018, a total of 374,513 confirmed malaria cases were reported from all health facilities to Federal Directorate with 16.4%  cases from Balochistan. P.vivax was considered to cause uncomplicated malaria previously, systematic review of clinical studies of severe malaria has shown increase in severe and even fatal cases of vivax over past 15 years.11Clinical presentation of malaria varies from asymptomatic infection to benign/uncomplicated disease and even severe/fatal cases depending on host and environmental factors.Most of the studies regarding clinico lab profile of malaria are restricted to areas of high endemicity like AfricaThe study was conducted in the department of pathology at Combined Military Hospital Khuzdar.This was a prospective clinical observational study. Non probability consecutive sampling technique was used. The study was conducted over a period of two years from October 2018 to September 2020. Approval was obtained from the hospital ethics committee for conducting the study (File No.02/ERC/CMH Khz) Dated 29 October 2020.P.vivax, P.falciparum and Mixed  malaria infection. Patients with age less than 12 years were classified as paediatric population. Cases of relapsed malaria, those labeled as Clinical Malaria or Cases with any other coinfection were excluded from the study.Subjects of the study included all patients irrespective of age and gender, admitted with the diagnosis of Data were collected in a tabulated form in a proforma after taking informed consent and consisted of demographic profile, detailed history, and general and systemic examination of patients. Patients were classified as uncomplicated or severe malaria cases according to WHO clinical and laboratory criteria. Severe falciparum malaria was defined as one or more of the clinical or laboratory parameters, occurring in absence of an identified alternative cause, and in the presence of P.falciparum asexual parasitemia. The criteria for severe vivax malaria were the same as for severe falciparum with no parasitaemia density thresholdsR Line Malaria Pf/Pv Rapid test Cassette (Whole Blood) was used. Blood samples of patients with clinical suspicion of malaria were evaluated for malarial parasite through combined peripheral film examination and RDT. The RDT is a lateral flow chromatographic immunoassay for the simultaneous detection and differentiation of P.falciparum and P.vivax antigens in whole blood. The membrane is pre-coated with anti HRP –II antibodies. During testing, the whole blood reacts with pre coated dye conjugate on test cassette, the mixture then migrates upward by capillary action, reacts with anti HRP-II on P.falciparum test line region or with anti P.LDH on P.vivax line region depending on whether the specimen contains HRP-II or Plasmodium-specific P.vivax LDH or both yielding colored lines in the specific regions.Diagnosis of malaria was made on microscopy through peripheral smear examination of conventional thick and thin blood films, stained with Giemsa stain, and rapid diagnostic tests (RDTs). The TuberComplete blood cell counts were performed on automated haematology analyzer . Sera for biochemical parameters were run on semi-automated chemistry analyzer (Merck Microlab 300 LX). Prothrombin Time (PT) was performed manually using Helena Bioscience Europe Thromboplastin L KIT. Patients with other coinfections like typhoid and viral hepatitis were excluded from the study after appropriate testing.P ≤ 0.05, with confidence interval of 95%, was considered as statistically significant.It was performed using Statistical Package for the Social Sciences (SPSS) for Windows, Version 23.0. Chicago, SPSS Inc. Categorical variables were analyzed for association of clinical features with severe malaria using Fisher exact test. Continuous variables were compared between uncomplicated and severe malaria using Mann-Whitney U –test. Statistical significance of lab parameters with type of malaria was derived using Kruskal Wallis test. A total of 210 admitted patients positive for vivax, falciparum and mixed  malaria on peripheral smear examination and/or antigen-based RDT were included in this study. Descriptive statistics and frequencies of various malaria species in study population and clinical features, are shown in Patients with age<12 years were classified as children. Out of 210 cases, eight (3.8%) were children . All children presented with complications and were classified as having severe malaria.Frequencies of clinical features were significantly higher among severe cases of malaria. .Descriptive statistics of lab parameters assessed for patients and correlation with parasite species are shown in All lab parameters, except TLC and creatinine showed significant difference with the clinical severity. Based on these findings, the study suggests that Hb, platelet count, T-Bilirubin and PT can be used as prognostic markers for malaria. .) were classified as severe malaria. Fifteen patients (78.9%) had a single complication including one patient of cerebral malaria, while only four (21.05%) patients had multiple complications.A total of nineteen (9%) patients  Chi square Test=8.872, p-value 0.002.P.vivax contributing to severe malaria burden.To our knowledge, this is the first study carried out in Khuzdar on the clinical and laboratory profile of severe and uncomplicated malaria. We found that certain clinical and laboratory parameters are significantly associated with severity of the disease and can be used as prognostic markers. As compared to past, there is now sufficient evidence available in literature supporting the evidence of P.vivax as the leading cause of malaria (63.8%) followed by P.falciparum (29.5%) and mixed (6.7%). This was in accordance with Pakistan Annual Malaria report 2019P.vivax.P.falciparum as the more prevalent species in Barkhan and Kohlu, bordering areas of East Balochistan.6We report malaria 6.8% folloAll patients presented with fever. However, symptoms of vomiting and signs of pallor and icterus were more frequently seen in patients of severe malaria. These were the only warning signs of malaria observed in our study population. Mathews SM from Delhi reported abdominal pain, headache, altered consciousness and cough with breathlessness as potential risk factors for development of severe malaria.6P.vivax contributed to 47.3% of severe malaria cases, 42% severe vivax cases were reported from a tertiary hospital in Delhi.6The high prevalence of uncomplicated cases as seen in our study, which may be due to better knowledge, attitude and practices in the study population, early access to healthcare facilities, prompt diagnosis and effective management of malaria cases, was in agreement with similar studies carried out in Khuzdar between 2003 to 2004Fever, jaundice and pallor were the most frequent complications in our study which was similar to a study done at Peshawar, where fever (100%) and pallor (50%) were also the most common presenting symptoms of children with severe malaria.69/l) was seen in 50.9% of all cases of malaria and severe thrombocytopenia (<50 x 109/l) was seen in seven cases only. Six out of these seven cases developed severe malaria while one case had uncomplicated malaria. These findings correlated with the results of study carried out in Columbia9/l. Only two patients inducted in our study, out of a total of seven with severe thrombocytopenia, presented with spontaneous bleeding. Both anaemia and thrombocytopenia could be useful lab parameters to predict severity of malaria. There are multiple factors responsible for etiology of both these complications, including immune mechanismsHemoglobin levels, platelet count, total bilirubin and prothrombin time were significantly different among uncomplicated and severe malaria cases. However, we could not find any significant association between type of malaria species and lab profile. Contrary to this, Zubairi et al. reported bilirubin, Hb and platelet count to be significantly associated with falciparum malaria.Children comprised of 75% of study population presenting with multiple complications. Similar findings were reported in a study carried out in Karachi, which reported multiple organ dysfunction in 70% of children with severe malaria.6The case fatality rate was 5.2% (1/19) in severe malaria cases in our study. The same, labeled as incidence of mortality, was reported to be 3.17% 2/63) in the study carried out in Delhi.3 in the The study was conducted in Khuzdar and included admitted malaria patients only. Therefore, the results of study are not representative of the entire Balochistan population.The study showed a high prevalence of uncomplicated malaria cases which may be associated with early diagnosis and effective management. However, paediatric population particularly remains at risk for developing severe malaria and multi organ involvement.Moreover, the study also signifies the importance of certain clinical and lab parameters which could be used as prognostic markers for disease progression and such cases could be vigilantly monitored to avoid adverse outcomes.MK: conceived, designed and editing of manuscript. She is also responsible for the accuracy or integrity of the work.HN: did Data collection and manuscript writing.NM: did review and final approval of manuscript.


Plasmodium malariae occurred in Sonsogon Paliu village in the remote area of Ulu Bengkoka sub-district of Kota Marudu, Northern Sabah, Malaysian Borneo from July through August 2019. This was the first outbreak of malaria in this village since 2014. On 11th July 2019 the Kota Kinabalu Public Health Laboratory notified the Kota Marudu District Health Office of a Polymerase Chain Reaction (PCR) positive case of P. malariae. This index case was a male from Sulawesi, Indonesia working for a logging company operating in Sonsogon Paliu. During the resulting outbreak, a total of 14 symptomatic cases were detected. All of these cases were positive by thick and thin blood smear examination, and also by PCR. During the outbreak, a mass blood survey screening was performed by light-microscopy and PCR. A total of 94 asymptomatic villagers 31 (33.0%) were PCR positive but thick and thin blood smear negative for P. malariae. Both symptomatic and asymptomatic cases received treatment at the district hospital. When symptomatic and asymptomatic cases were considered together, males (29/45. 64.5%) were infected more than females , the male:female ratio being 1.8:1. Adults were the predominant age group infected  followed by adolescents  and children under five years of age . This report illustrates that symptomatic and submicroscopic cases pose a challenge during P. malariae outbreaks and that PCR is a valuable tool for their identification. The rapid identification and control of imported malaria is crucial for the continued control of malaria in Malaysia.An outbreak of  Plasmodium knowlesi parasites and outbreaks caused by imported parasites. One such outbreak occurred in 2019 in a village in northern Sabah. This outbreak was unusual, as it was found to be caused by P. malariae, an often-benign human malaria parasite rarely seen in Malaysia. We investigated this outbreak and found that the index case could be traced to an Indonesian migrant worker employed in the logging industry. An additional 13 people in the village developed malaria symptoms and were subsequently found to be positive for P. malariae. A further 94 inhabitants of the village without symptoms were then tested by a sensitive PCR technique, and 31 of them were also found to be positive for the parasite. These results illustrate the threat posed by asymptomatic carriage of malaria parasites, and the importance of PCR testing for outbreaks of residual malaria in elimination settings.In Malaysia, malaria caused by the human-only malaria parasites has been all but eliminated, and the major obstacles to a malaria-free status are now emerging malaria caused by zoonotic transmission of  Plasmodium knowlesi [Plasmodium falciparum and eight cases of Plasmodium vivax recorded in Sabah 2017 [P. knowlesi were reported [Malaysia has been identified by the World Health Organization (WHO) as one of 21 countries with the potential to eliminate human malaria by 2020 [knowlesi . Howeverreported .Plasmodium malariae infection is often asymptomatic, rarely leads to severe clinical illness or death, and is typically associated with a low-grade chronic infection that can persist for extended periods of time, perhaps even decades [P. malariae is rather patchy, it has been observed in all major malaria-endemic regions of the world, throughout tropical Africa, Southeast Asia, and Central and South America [P. falciparum and P. vivax globally, it can cause long-term chronic infections and possesses the ability to recrudesce due to the persistence of subclinical parasitemia [P. malariae remains relatively little studied, both at the biological and epidemiological levels [P. malariae in Sonsogon Paliu village, Kota Marudu district, Sabah during July and August 2019. Our findings highlight the challenges posed by imported malaria for the achievement of malaria elimination. decades ,8. The d decades ,10. Alth America –13. Althasitemia . Of the l levels ,12. HereThis study was approved by the Medical Ethics and Research Committee, Ministry of Health Malaysia (NMRR-20-1523-55702). The study protocol was registered under the National Medical Research Registry. Written informed consent was obtained from participants and in the case of children informed consent was obtained from guardians.th of July 2019, the Kota Kinabalu Public Health Laboratory (KKPHL) notified the Kota Marudu District Health Office of a P. malariae case in a village in their administrative region. KKPHL is the reference laboratory of Sabah state for malaria and other infectious diseases. Venous blood was sent to this laboratory for confirmatory diagnosis of malaria. All cases were diagnosed using thin and thick blood smear microscopy and PCR. An outbreak investigation was carried out that involved the formation of three teams for population screening, outbreak control, and entomological investigations.On the 11via venipuncture using needles and syringes. Samples were stored in sampling tubes, and transported to the laboratory in a cold-box. Thick and thin smear microscopy was carried out within 24 hours of blood collection. Staining and microscopy were conducted according to standard operating procedures provided by the Public Health Laboratory. All slides were examined by a trained microscopist. Slides were examined at 100 × magnification and the number of parasites per 200 leukocytes determined. Slides were considered negative when no parasites were observed after counting >100 microscopic fields.Population screening for malaria parasites was performed by Mass Blood Survey (MBS) using thin and thick blood smear microscopy and PCR. The target population for this study were all villagers who resided in the study village. Villagers’ blood was withdrawn from the cubital fossa An epidemic curve was plotted using the time of first reported symptoms as a start point. Using a structured questionnaire , face-toThe entomology team analyzed the effectiveness of vector control measures. These measures include indoor residual spraying (IRS) and the distribution of Long-Lasting Insecticide Nets (LLIN). The analysis was based on the effectiveness of IRS and LLIN and to ensure that the activities followed standard procedures. Entomological surveys were conducted for three nights from 29 July to 31 July 2020. Both indoor and outdoor landing catches were carried out with two men sitting indoors and six outdoors from 6.00 pm until 12.00 am. All staff were given doxycycline prior to the survey. Adult mosquitoes were collected using small vial tubes . All moshttps://www.naturalearthdata.com) administrative area data was used for administrative boundaries. Road data was obtained from OpenStreetMap Data Extracts (https://download.geofabrik.de). For the location information of each village included in the district, records from the Kota Marudu Health Office were used. The coordinates of houses of all cases were determined via GPS.A map for outbreak cases was created using R, and its graph drawing packages . Natural Earth  and insecticide repellent spray to the community. As a continuation of vector control activities each malaria case received a personal repellent following discharge from hospital.Malaria control measures were last implemented in the village between 17The outbreak control team identified the index case as an Indonesian male from Sulawesi who had been working for a logging company in Sonsogon Paliu from the end of March 2019. He had no history of travel to other countries except Sulawesi, from where he had travelled at the end of February 2019.st April 2019, and an outbreak was declared on the 11th of July 2019 when the district health office received a P. malariae positive PCR result. The first case-patient became symptomatic on the 31st of May, and on the 12th of June the peak was noted followed by a decrease with the last case-patient becoming symptomatic on the 25th of July  of 12.96%. Among these, nine (64.3%) were male and five (35.7%) were females . Except for three cases the houses of all the symptomatic cases were very close to each other . Among them, 14 cases of symptomatic ch other . The firP. malariae positive patients were 16.5 years (range 3–30 years). During the outbreak, the highest number of patients  belonged to the 5–18-year age group followed by those above 18 years  and less than five years . Thirty-one individuals were identified as asymptomatic P. malariae parasite carriers by MBS. Among these, 20 (64.5%) were males and 11 (35.5%) were females (ratio 1.8:1). The median age of these patients was 21 years (range 2–86 years). The highest number of patients  were over 18 years of age, followed by 5–18 year-olds  and those below five years . When symptomatic and asymptomatic cases were considered together, males (29/45. 64.5%) were more likely to be infected than females . Age distribution showed that adults were most at risk  followed by adolescents  and children under five years of age .The median age of symptomatic There were no deaths during this outbreak. All cases were treated orally with Riamet (artemether plus lumefantrine) at Kota Marudu District Hospital. There were no recorded adverse effects associated with drug treatment.Anopheles balabacensis, two were identified as Aedes albopictus and one sample was An. barbirostris. Further dissection of mosquito ovaries revealed that all the mosquitoes were nulliparous. No oocysts or sporozoites were detected in the midguts and salivary glands respectively. All mosquitoes were collected from outdoor biting collection, and none were found indoors. Anopheles barbirostris larvae were detected in two ponds within 50 meters from the residence of one P. malariae positive case. Our survey also showed that the usage rate of ITNs and insecticide repellent spray by village households was 100%.During 136 person-collecting hours, a total of seven mosquitoes were caught, four were identified as P. vivax and three cases of P. falciparum. From April 2014 onwards, no cases of malaria caused by human-only malaria parasite species were reported, however, there were 20 cases of P. knowlesi infection during this period. Therefore, when the index case was first notified it was suspected to be a case of P. knowlesi as this is the dominant species in Sabah [Plasmodium malariae accounts for only 0.6–0.8% of Plasmodium infections in Sabah [P. malariae is endemic in Sulawesi, Indonesia [According to data retrieved from the Kota Marudu District Health Office, the last cases of infection by human-only malaria parasites in Sonsogon Paliu village occurred in 2014. These were a case of in Sabah . Plasmodndonesia ,16, wherst, 2019, and it took two months for the first symptomatic case to appear. The spread peaked after 11 days, and cases slowly came to an end 55 days from the first appearance of the first case-patient. It is possible that this slow spread was linked to the fact that vectors were not abundant during this time. Larviciding was performed to control the development of mosquitoes which breed in this water despite the fact that no An. balabacensis larvae were found in ponds or other standing water. Anopheles barbirostris larvae were found in stagnant water flowing from a river near one of the positive case residencies. However, there is no report implicating A. barbirostris as a malaria vector in Malaysia. The infectiousness of P. malariae for Anopheles mosquitoes in malaria-endemic areas remains unclear and merits further investigation [The shape of the epidemic curve suggested a common source outbreak. The first case–patient developed symptoms on May 31tigation .All families surveyed in the village during this investigation reported the use of ITNs. This may be one of the reasons that few children under five were infected during the outbreak. Although there were some differences in age distribution between symptomatic and asymptomatic cases, adults were most at risk followed by 5–18 year-olds. As there is no electricity in the village, families spend their time outside engaged in activities such as storytelling, eating together, playing games and web-surfing, and only go inside the house to sleep. This tendency to be outside during peak mosquito-biting time might contribute to the risk of infection.P. malariae [P. malariae infection in children exclusively [P. malariae in Africa and Asia may be due to multiple factors including differences in host and parasite genetics, cross-immunity between malaria parasite species, and host behavioral differences.A similar study from Indonesia also found that older people were more likely to be infected with malariae  than youlusively ,18. ThisP. malariae infection, further reinforcing the assumption that outdoor biting and proximity to forested areas contributes to infection risk. We assume that such individuals are infected whilst at work in the forest, but it is also possible that they are infected when returning from work late in the evening. This warrants the distribution of personal anti-mosquito repellents to villagers, as their activities such as farming, hunting and logging mainly occur outdoors. Insect repellents are an additional self-protection tool for avoidance of mosquito bites. These could complement other preventive measures such as IRS and the distribution of LLINs. The distributed repellent has a long lasting effect for up to eight hours.We observed that engagement in the logging industry is associated with increased P. malariae, which is in agreement with the present study. Men are more likely than women to spend time outside houses during the evening, and will occasionally sleep outdoors, both of which increase the risk of infection.Logging can drastically alter the forest landscape. Environmental modification through deforestation is an important driver of malaria transmission in Sabah –4, and tP. malariae is more common than previously thought, and asymptomatic carriage of this parasite might be commonplace. This is due mainly to the increased sensitivity of malaria parasite detection methods, including PCR and LAMP technologies [P. malariae because parasitaemia is typically below the threshold of detection [P. malariae can persist for extremely long periods, asymptomatically [Notably, we found that over 60% of the infections identified during this outbreak were asymptomatic. Asymptomatic malaria poses a challenge to malaria control and elimination programs, as it makes identification of parasite carriers difficult . It is bnologies . Surveysetection ,10. Seveatically ,8,10.P. malariae [P. malariae may be intrinsically less susceptible to artemisinin than P. falciparum [Despite the high attack rate in the present report, there were no cases of anemia or nephritis, which are the most serious clinical manifestation of malariae –9. Altholciparum ,12, all Adequate-quality housing reduces the opportunities for entry of vectors and can radically reduce the human biting rate of malaria-transmitting mosquitoes . We founP. malariae in the village of Sonsogon Paliu in Malaysian Borneo. The index case was an immigrant worker from Sulawesi, Indonesia who was working for a logging company in the area. We identified a cluster of sub-microscopic, asymptomatic infections, and analyzed the risk factors associated with parasite carriage. We suggest that district health offices should collaborate and co-ordinate with the administration section of commercial logging companies in this region in order to consolidate the effective implementation of anti-malaria interventions among workers. Methods for interrupting the exposure of workers to malaria vectors inside the forest need to be formulated in order to prevent further outbreaks.In conclusion, we describe an outbreak of S1 Table(DOCX)Click here for additional data file.S1 FigPhotographer: Nurul Athirah Naserrudin.(DOCX)Click here for additional data file.S2 FigPhotographer: Nurul Athirah Naserrudin.(DOCX)Click here for additional data file.S3 FigPhotographer: Nurul Athirah Naserrudin.(DOCX)Click here for additional data file.