Plasmodium vivax malaria is now recognized as a cause of severe morbidity and mortality, resulting in a substantial negative effect on health especially in endemic countries. Accurate and prompt diagnosis and treatment of P. vivax malaria is vital for the control and elimination of the disease.P. vivax (mono and mixed infection) using RDT, site level microscopists and expert microscopists were selected for PCR. Statistical analyses were performed to calculate the proportions, agreement (k), frequencies, and ranges among different diagnostic methods. Fisher’s exact tests and correlation test were used to detect associations and relationship between different variables.A cross-sectional study was conducted from February 2021 to September 2022 at five malaria endemic sites in Ethiopia including Aribaminch, Shewarobit, Metehara, Gambella, and Dubti. A total of 365 samples that were diagnosed positive for P. vivax (mono), P. vivax/Plasmodium falciparum (mixed), P. falciparum (mono) and negative by PCR, respectively. The overall agreement of rapid diagnostic test (RDT), site level microscopy and expert microscopists result with PCR was 90.41% (k: 0.49), 90.96% (k: 0.53), and 80.27% (k: 0.24). The overall prevalence of sexual (gametocyte) stage P. vivax in the study population was 215/361 (59.6%). The majority of these 215 samples  had below 1000 parasites/µl, with only four samples (1.9%) had ≥ 5000 parasites/µl. The gametocyte density was found to be weakly positive but statically significant with asexual parasitaemia .Of the 365 samples, 324 (88.8%), 37(10.1%), 2 (0.5%), and 2 (0.5%) were P. vivax (mono) and P. vivax/P. falciparum (mixed) infections. Therefore, to achieve malaria elimination goals, strengthening routine malaria diagnostic methods by implementing diagnostic tools with a good performance in detecting and accurately identifying malaria species in clinical settings is recommended.Both microscopy and RDT showed moderate agreement with PCR in the detection and identification of  Anopheles mosquitoes. According to World Health Organization (WHO) 2022 report, there were an estimated of 247 million cases and 619,000 deaths from malaria worldwide [Plasmodium vivax is the most geographically widespread parasite and poses a serious threat to public health [Plasmodium falciparum and P. vivax in Ethiopia was 62.8% and 37.2%, respectively [P. falciparum, there has been a rise in the proportion of malaria attributable to P. vivax [Plasmodium vivax is a cause of malaria, associated with a significant public health burden [P. vivax is more challenging than that of P. falciparum, a reflection of key differences in parasite and vector biology [P. vivax is essential for informed decisions on appropriate control strategies to be designed and implemented against this neglected species.Malaria is a life-threatening disease caused by parasites that are transmitted to humans through the bites of infected female c health . In Ethiectively . In co-eh burden . The con biology . Clear uP. vivax infections in blood [Accurate and prompt diagnosis of malaria cases is key to the control and elimination of malaria –14. The in blood . In recein blood , 18. Comin blood . Apart fin blood .Plasmodium vivax distribution has been expanding to the highland, causing occasional malaria epidemics, although the existing deployed interventions seem to have an impact on the prevalence of this parasite [Ethiopia has planned to eliminate malaria from specific geographic areas by 2020 and from the whole country by 2030 –23. Plasparasite .To achieP. vivax infection and P. vivax/P. falciparum mixed infection (referred to as ‘mixed infection’ thereafter) in febrile samples collected from five malaria endemic sites in Ethiopia. These sites represent different transmission intensities and environmental characteristics such as altitude.This study aims to evaluate the performance of routine laboratory diagnostic methods for the diagnosis of pure P. vivax prevalence in the region and from different malaria risk stratification areas. The study sites include Aribaminch, Shewarobit, Metehara, Gambella, and Dubti . Arbaminch hospital is located in the Southern Nation and Nationalities People Regional State in the southern part of the country ; Dubti hospital is located in the Afar Regional State in the northern-east part of the country ; Gambella hospital is located in Gambella Regional State in the western part of the country ; Metehara hospital is located in Oromia Regional State in the eastern part of the country ; and Shewarobit hospital is located in Amhara Regional State in the northern part of the country .A cross-sectional study was conducted from February 2021 to September 2022 at five malaria endemic sites in Ethiopia. The study sites were selected from malaria endemic areas based on their P. vivax (37.2%) from a total positive cases reported in Ethiopia(5). The following formula was used to calculate sample size:Sample size determination was calculated using proportion of P. vivax, Z: z- value at 5% level of significance, ε: margin of error, n: number of minimum sample size. Given that: p = 37.2%, Z = 1.96, and ε = 0.05. So the sample size calculated (n) was 358 and the total sample size with 10% non-response rate (N) was 394 (this is the sample collected at site level). The final sample size after re-examination of blood film slides at the central level was 365.P: proportion of P. vivax or mixed infection using both RDT  and microscopy were included in this study. Blood smears were re-examined blindly by expert microscopists at national level and samples which showed no P. vivax were excluded. A total of 365 samples that were diagnosed positive for P. vivax (mono or mixed infection) based on RDT, site level microscopists and expert microscopists were selected for PCR analysis.A total of 394 participants who were presented with malaria sign/symptom and diagnosed positive for pure The flow chart of the study conducted from February 2021 to September 2022 at five malaria endemic sites in Ethiopia is indicated in Fig. P. vivax and mixed infection using both RDT and microscopy were enrolled in the study. Two millilitre (2 ml) of venous blood sample was collected from volunteer individuals based on the inclusion criteria. The collected sample was used for the preparation of thin and thick blood film , and for the preparation of dried blood spots (DBS). The DBS samples were kept at − 20 °C till shipped to the University of Strasbourg, France, for molecular analysis.Finger prick blood sample was collected from suspected malaria patients who were referred to the laboratory of study sites. A unique identifier was assigned for each study participant to trace them at any time. The sample collected from suspected malaria patients was used for RDT and blood smears preparation. Thick and thin blood smears were stained with 10% working Giemsa solution (pH 7.2) for 10 min. Blood film slides were examined with 100X objective using microscope. Malaria laboratory diagnosis was done using RDT and microscopy in parallel for each suspected malaria patients. Then those who were positive for pure Pf/Pv (05FK80). It is a rapid, qualitative test for the detection of HRP-II (Histidine-rich protein II) specific to P. falciparum and Plasmodium lactate dehydrogenase (pLDH) specific to Plasmodium vivax. Five microlitres (5 µl) of whole blood was used for the test and the result was interpreted at 15 min (up to 30 min).RDT kit currently implemented by the country is SD BIOLINE Malaria Ag P. vivax on the blood film slides with pure P. vivax infection and the asexual stage of both species on the blood film slides with a mixed infection. Whereas the density of gametocyte was estimated only for the gametocyte of P. vivax on both blood film slides with pure P. vivax and mixed species. The parasite density of both the asexual and sexual stages were then classified as low (below 1000 parasites per microlitre of blood), intermediate (1000–4999 parasites per microlitre of blood) and high (≥ 5000 parasites per microlitre of blood) [Microscopic examination of Giemsa-stained thick and thin blood film was used for the detection, identification and quantification of malaria parasites. The first diagnosis using microscopy was done at study site by site level microscopists. Site level microscopists are microscopists who are providing routine laboratory diagnosis service at study site (employees of the study sites). Parasite counts was measured for both sexual and asexual stages on thick film and reported per 200 WBCs or parasite count per microlitre of blood, assuming a total white blood cell count of 8000/µl. The densities of asexual and gametocyte stages in peripheral blood were determined by WHO certified microscopists using microscopy at national level. The quantification of asexual stage parasite density was conducted for f blood) . Blood ff blood) . Blood fGenomic DNA was extracted from DBS collected from study participants using QIAamp DNA Extraction kit  based on manufacture instructions. Each genomic DNA was extracted from one punch (with a diameter of 6 mm) of DBS sample, and DNA was eluted using 100 µL of TE (Tris-EDTA) buffer, then the extracted DNA was kept at − 20 °C till PCR process.Genomic DNA was extracted from DBS collected from study participants using QIAamp DNA Extraction kit  based on manufacture instructions. Each genomic DNA was extracted from one punch (with a diameter of 6 mm) of DBS sample, and DNA was eluted using 100 µL of TE (Tris-EDTA) buffer, then the extracted DNA was kept at − 20 °C till PCR process.The PCR amplifications using Bio-Rad CFX96 Real-Time PCR detection system were carried out following 10 µL of Advanced mix sybgreen (2x) , 4 µL of molecular water, 0.5 µL of each primer  version 25. Descriptive statistics were used to calculate proportions, agreement, frequencies, and ranges. Fisher’s exact tests and correlation test were used to detect associations and relationship between different variables, respectively. The strength of agreement between RDT, site microscopy, and expert microscopists with PCR were determined by Kappa (K) value. Kappa (K) value is classified as: 0.01–0.20 slight agreement, 0.21–0.40 fair agreement, 0.41–0.60 moderate agreement, 0.61–0.80 substantial agreement, and 0.81–1.00 almost perfect agreement , 29. TheFemales accounted for 35.1% 128/365) of the study participants. The mean age of the participants was 24.8 (ranging from 1 to 80 years old) with 137 (37.5%) of the participants age from 15 to 24 years old. Among all participants, 75 (20.5%) were farmer and 212 (58.1%) live in an urban area  were pure P. vivax infection and 17 (5.2%) were mixed infection based on PCR; whereas for the 36 samples diagnosed as mixed infection by RDT, 14 (38.9%) were pure P. vivax infection and 20 (55.6%) were P. vivax/P. falciparum mixed infection based on PCR.For the 329 samples that were diagnosed as pure P. vivax infection by site level microscopists, 310 (94.8%) were pure P. vivax infection, 15 (4.6%) were mixed infection, 1 (0.3%) was pure P. falciparum, and 1 (0.3%) was negative using PCR. Among the 38 mixed infections diagnosed by site level microscopists, 14 (36.8%) were pure P. vivax infection, and 22 (57.9%) were mixed infection using PCR. Of the 68 samples diagnosed as mixed infections by expert microscopists, 49 (72%) were pure P. vivax infection, 18 (26.5%) were mixed infections and 1 (1.5%) was pure P. falciparum infection using PCR.For the 327 samples diagnosed as pure P. vivax, P .vivax/P. falciparum, P. falciparum and negative, respectively using PCR. The overall agreement of RDT, site level microscopy and expert microscopists result was 90.41% (K: 0495), 90.96% (k: 0.534), and 80.27% (k: 0.238), respectively , 37(10.1%), 2 (0.5%) and 2 (0.5%) were P. vivax infection by expert microscopists, 279 (93.9%) were pure P. vivax infection and 18 (6.1%) were mixed infection by RDT and site level microscopists, whereas from 68 samples diagnosed as mixed infection by expert microscopists, 50 (73.5%) and 48 (70.6%) samples were diagnosed as pure P. vivax infection by RDT and site level microscopists, respectively , 10 (15.9%) and 1 (1.6%) were pure P. vivax infection, P. vivax/P. falciparum mixed infection and P. falciparum infection, respectively, based on PCR (Table P. vivax (mono) infection based on PCR.Among all the five sites, Dubti (Afar) health facility had the lowest agreement in diagnostic results between the site level microscopists and PCR (81.43%), whereas the agreement of Shewarobit and Gambella hospitals had the highest agreement of 94.29% and 98.67%, respectively. In Dubti, among the 63 samples diagnosed as pure P. vivax infection and 37 were mixed infection. Of 324 pure P. vivax samples, 106 (32.7%) were reported from altitude below 500 m and 144 (44.4%) were from altitude of 1000–1500 m. For the 37 mixed infection samples, 36 (97.3%) were from altitude below 500 m and none were from altitude of 1000–1500 m. Pure P. vivax and mixed species identified using PCR were significantly associated with the altitude of study site (p < 0.001). Among the 37 mixed infection, 23 (62.2%) were from rural area, significantly higher than those 324 pure P. vivax, of which 129 (39.8%) were from rural area , 124 (37.7%), and 152 (46.2%) samples had the parasite density below 1000 parasites/µL, between 1000 and 4999 parasites/µL, and ≥ 5000 parasites/µL, respectively. Whereas from 36 samples with P. vivax/P. falciparum mixed infections, the asexual parasite density of 9 (25%), 8 (22.2%), and 19 (52.8%) samples had the parasite density below 1000 parasites/µL, between 1000 and 4999 parasites/µL, and ≥ 5000 parasites/µL, respectively. The asexual stage parasite density has no significant association with the RDT Result (p = 0.138).From a total of 365 samples identified as pure P. vivax and mixed infections by PCR, the mean asexual (the density of both species was quantified in a mixed infection) parasite density was 11,296 parasites/µL . The asexual parasite density of 60 (16.6%) samples were below 1000 parasites/µL, 131 (36.3%) were between 1000 and 4999 parasites/µL, and 170 (47.1%) were ≥ 5000 parasites/µL. For the 60 samples with an asexual stage parasite density below 1000 parasites/µL, 131 samples with an asexual stage parasite density between 1000 and 4999 parasites/µL, and 170 samples with an asexual stage parasite density ≥ 5000 parasites/µL, 4 (6.7%), 11 (8.4%) and 22 (12.9%) were mixed infection, respectively.For the 361 samples identified as pure P. vivax was 215/361 (59.6%). Among these 215 samples with gametocyte, 180 (83.7%) were below 1000 parasites/µL, 31 (14.4%) were between 1000 and 4999 parasites/µL, and 4 (1.9%) were ≥ 5000 parasites/µL. For the 180 samples with a sexual stage parasite density below 1000 parasites/µL and from 31 samples with a sexual stage parasite density between 1000 and 4999 parasites/µL, 16 (8.9%) and 5 (16.1%) were mixed infection, respectively. For the 60 samples with an asexual stage parasite density below 1000 parasites/µL, 6 (10.0%) were from Aribaminch and 19 (31.7%) were from Gambella. For the 131 samples with an asexual stage parasite density between 1000 and 4999 parasites/µL, 36 (27.5%) were from Metehara, which was higher than Dubti 21 (16.0%). For the 180 samples with a sexual stage parasite density less than 1000 parasites/µL, 23.9% were from Shewarobit, which was higher than Gambella (19.4%) and Dubti (13.9%). Both an asexual and sexual stage parasite density were significantly associated with the study site (p < 0.001) , and that of sexual (gametocyte) parasite was 337 parasites/µL . A weak correlation (r) was detected between asexual and gametocyte stages density among the samples   stages of /µL, 31 1.4% were P. vivax and other Plasmodium species is key to the control and elimination of this disease [Accurate and prompt laboratory diagnosis of malaria cases caused by  disease . Previou disease .Plasmodium species, suboptimal storage condition that affects the diagnostic quality of the kits, and/or lack of competency by laboratory personnel in the result interpretation.In this study, the overall agreement of RDT with PCR was 90.41%, which was higher than a study conducted in eastern Sudan with the agreement 81.2%33), and Zambia 84.6% , and Zam and 97.6P. vivax /P. falciparum mixed infection as a mono-infection can lead to inappropriate treatment.Compared to the site level microscopists, the expert microscopists who were certified by WHO showed a lower agreement with PCR. The agreement of site level microscopy with PCR was 90.96%, which was relatively higher than a study conducted in Addis Ababa, Ethiopia with the percent agreement of 71.4% , Adama, P. vivax/P. falciparum mixed infection is higher at low altitude, and that most of the identified P. falciparum infected samples were from sites at low altitude, similar to a prior study conducted in Northeastern Tanzania confirming that P. falciparum prevalence had a negative relationship with altitude [P. vivax in the areas traditionally considered as malaria free is an indication of the expansion of malaria transmission in Ethiopia to higher altitude settings [Our findings showed that altitude . The incsettings , this maP. vivax-infected blood donors [In this study, the correlation between asexual and gametocyte stage density was significant but weakly positive correlation, given that a previous study conducted in Brazil showed a moderate correlation across the range of parasite densities observed in d donors , whereasd donors , Indonesd donors , and wesd donors  reportedP. vivax and mixed infections. It lacks negative samples, which is important to evaluate diagnostic method performance on the identification of true negative. So this made us challenging to evaluate the performance of the methods using different parameters like sensitivity and specificity. The other limitation of this study was conducting parasite density estimation only by microscopists using microscope. It may be a challenge to getting the exact parasite density because of the competency of microscopists.This study was largely limited by its study population. The study population were only malaria patients who were diagnosed with pure P. vivax and P. vivax/P. falciparum mixed infections. Even though microscopy and RDT are used routinely for malaria laboratory diagnosis in Ethiopia, a substantial number of P. vivax mono-infections are misidentified as mixed infections and such an outcome could affect the anti-malarial drug treatment regime. Therefore, to achieve malaria elimination goals, it is recommended to strengthen routine malaria laboratory diagnostic methods by implementing diagnostic tools with a good performance in detecting and accurately identifying malaria species in clinical settings, providing adequate in-service technical training and strengthening the external quality assessment programme.Both microscopy and RDT showed moderate agreement with PCR in the detection and identification of pure


Plasmodium knowlesi (Pk) causes zoonotic malaria and is known as the “fifth human malaria parasite”. Pk malaria is an emerging threat because infections are increasing and can be fatal. While most infections are in Southeast Asia (SEA), especially Malaysia, travelers frequently visit this region and can present with Pk malaria around the world. So, clinicians need to know (1) patients who present with fever after recent travel to SEA might be infected with Pk and (2) Pk is often misdiagnosed as P. malariae . Here we review the history, pathophysiology, clinical features, diagnosis, and treatment of Pk malaria. Severe disease is most common in adults. Signs and symptoms can include fever, abdominal pain, jaundice, acute kidney injury, acute respiratory distress syndrome, hyponatremia, hyperparasitemia, and thrombocytopenia. Dengue is one of the diseases to be considered in the differential. Regarding pathophysiologic mechanisms, when Pk parasites invade mature red blood cells  and reticulocytes, changes in the red blood cell (RBC) surface can result in life-threatening cytoadherence, sequestration, and reduced RBC deformability. Since molecular mechanisms involving the erythrocytic stage are responsible for onset of severe disease and lethal outcomes, it is biologically plausible that manual exchange transfusion (ET) or automated RBC exchange (RBCX) could be highly beneficial by replacing “sticky” parasitized RBCs with uninfected, deformable, healthy donor RBCs. Here we suggest use of special Pk-resistant donor RBCs to optimize adjunctive manual ET/RBCX for malaria. “Therapeutically-rational exchange transfusion” (T-REX) is proposed in which Pk-resistant RBCs are transfused (instead of disease-promoting RBCs). Because expression of the Duffy antigen on the surface of human RBCs is essential for parasite invasion, T-REX of Duffy-negative RBCs—also known as Fy(a-b-) RBCs—could replace the majority of the patient’s circulating normocytes with Pk invasion-resistant RBCs (in a single procedure lasting about 2 h). When sequestered or non-sequestered iRBCs rupture—in a 24 h Pk asexual life cycle—the released merozoites cannot invade Fy(a-b-) RBCs. When Fy(a-b-) RBC units are scarce , clinicians can consider the risks and benefits of transfusing plausibly Pk-resistant RBCs, such as glucose-6-phosphate dehydrogenase deficient (G6PDd) RBCs and Southeast Asian ovalocytes (SAO). Patients typically require a very short recovery time (<1 h) after the procedure. Fy(a-b-) RBCs should have a normal lifespan, while SAO and G6PDd RBCs may have mildly reduced half-lives. Because SAO and G6PDd RBCs come from screened blood donors who are healthy and not anemic, these RBCs have a low-risk for hemolysis and do not need to be removed after the patient recovers from malaria. T-REX could be especially useful if (1) antimalarial medications are not readily available, (2) patients are likely to progress to severe disease, or (3) drug-resistant strains emerge. In conclusion, T-REX is a proposed optimization of manual ET/RBCX that has not yet been utilized but can be considered by physicians to treat Pk malaria patients. Plasmodium knowlesi (Pk) was first observed by Giuseppe Franchini when examining the blood of Macaca fascicularis  . M,9. MPk pia (SEA) A, and PeSEA)   ,18Plasmoutdoors) ,20,21,22utdoors) ,24. (Notor of Pf .) The do Sarawak . Experimbacensis , but susbacensis . Pk malaria  iRBCs, which are highly virulent in rhesus macaques, can be found in rhesus macaques with functional spleens. When SICA[+] iRBCs are passaged in splenectomized rhesus macaques, they become less virulent SICA[-] iRBCs. If SICA[-] iRBCs are passaged in rhesus macaques with function spleens, either a milder disease is observed or SICA[+] iRBCs form and lead to more severe disease. The exact mechanism by which the spleen regulates y PfEMP1 . In contaginate) . An autoothelium . In contm  hypoxanthine uptake assay [50 of chloroquine for Pf strain 3D7, which was 6 nM at 48 h. Thus, the authors concluded that this Pk strain was chloroquine-sensitive. Chloroquine can disrupt Plasmodium parasites from degrading hemoglobin to hemozoin, leading to a buildup of heme, which is toxic for Plasmodium [Pk is chloroquine-susceptible, it may suggest that it needs hemoglobin as a nutrient source. Interestingly, some Plasmodium species can replicate in reticulocytes without hemozoin formation, giving them chloroquine resistance [A study from 2016 found that the half-maximal inhibitory concentration  was similar for artemether, artesunate, artemisinin, and dihydroartemisinin [Pk malaria patients to continuously analyze blood smears every 12–24 h to confirm that antimalarial treatment is causing a decline in parasitemia and to monitor the patient’s clinical status [Some articles explicitly state that there is no evidence of sistance . For exafe cycle . The halemisinin . The CDCl status ,112. Pk malaria is IV artesunate and oral doxycycline [Artemisia annua plants [In Malaysia, the recommended first-line treatment of severe ycycline . The CDCycycline ,112. Arta plants . Tu Youya plants . If it ca plants ,112,149.a plants . More than 4 h after the third dose of IV artesunate, parasitemia needs to be measured . , then the apheresis circuitry can be primed with RBC units . AlternaIn this procedure, the patient’s blood is anticoagulated and flows through tubing to the apheresis machine, where it can be fractionated into plasma, platelet-rich plasma, leukocytes, and RBCs by a centrifuge. The centrifugal force separates blood according to specific gravity, with RBCs having the highest specific gravity ,153. TheManual ET removes the patient’s whole blood and replaces it with donor RBCs reconstituted in albumin solution or plasma. Manual ET can be used if apheresis equipment is not available or there are concerns about the extracorporeal blood volume in the machine representing a high percentage of the patient’s blood volume. Manual ET can be performed using several techniques. For example, a large-bore catheter can be inserted into each vein of the antecubital fossa . One traPk malaria. In addition, this procedure is rarely used in Africa, possibly because RBCX requires an apheresis machine, which is expensive. The benefits of manual ET may include the removal of (1) proinflammatory cytokines in plasma, such as tumor necrosis factor alpha; (2) iRBCs; (3) parasites; and (4) parasite toxins  [Reports from both developed and developing nations attribute manual ET/RBCX to improved outcomes for malaria patients ,164,165.nositol) ,166,167.nositol) . Unfortunately, manual ET/RBCX for malaria is rarely carefully reported or evaluated. Descriptions of the specific donor RBCs delivered to malaria patients  are almost always incomplete in case-reports and case-series ,158,161.However, when automated RBCX was used to treat a 13-year-old and two adult patients with CM, sickle cell trait RBC units were deliberately excluded . . Of major clinical importance, exposure to RBC alloantigens following pregnancy or transfusion can increase the likelihood of RBC alloimmunization against other alloantigens, which increases the probability of transfusion complications and reduces the availability of compatible RBCs for future transfusion [T-REX refers to replacing a patient’s iRBCs with special invasion- or disease-resistant donor RBCs instead of nondescript standard-issue RBCs  that may contain nsfusion ,189,190.nsfusion ,194,195.nsfusion ,201,202,nsfusion ,204,205,Pf malaria [Pf malaria because blood-group-O patients have better clinical outcomes , in part due to reduced rosetting (the binding of iRBCs to uRBCs) [Plasmodium strains [Previously, several T-REX options have been proposed as candidate non-drug adjuncts for  malaria ,212,213  malaria . For exao uRBCs) . It seem strains . Pk-resistant RBC variants currently available in blood banks. Blood donors with these RBC variants are eligible to donate RBCs, based on their responses to screening questions, whether they have adequate hemoglobin, testing negative for infectious disease, and other data. Because these variant RBCs are not thought to be harmful, they are not routinely tested for and are part of the blood bank’s inventory.Here we propose use of FY*A allele leads to Fya expression, while the FY*B allele, caused by c.125G>A (coding DNA mutation at nucleotide 125 from guanine to adenine), leads to the expression of Fyb. Fy(a-b-) is the most common phenotype in Blacks and is usually caused by the mutation c.-67T>C in the erythroid promoter GATA-1-binding motif of FY*B. This allele is known as ESFY*B, in which “ES” stands for “erythrocyte silent” [b expression on RBCs only, but not other tissues. In contrast, Fy(a-b-) phenotype is extremely rare among White and Asian populations. Duffy is a seven transmembrane domain glycoprotein that binds chemokines in the C-X-C and C-C classes  ,217. The silent” . This muPk parasites need to bind to the Duffy antigen [Pk invasion-resistant Duffy-negative RBCs, also known as Fy(a-b-), could be delivered via manual ET/RBCX to prevent or treat severe malaria. However, three studies found that 0% of Malaysians had the Fy(a-b-) phenotype [a and Fyb expression in Pk malaria patients versus controls [ antigen ,218,219  antigen ,220,221.henotype ,223. In controls ,225. ThuPk malaria [Pk infection [P knowlesi in a human was reported in 1965 in a man who returned to the USA after visiting peninsular Malaysia” [Pk cases have been reported in Africa, there is concern about Pk malaria patients presenting for care on the continent and the potential for misdiagnosis [It is important to consider that Malaysia has approximately 25 million visitors annually . A 2014  malaria . So, clinfection . Interesalaysia” . The Fy [The Fy(a-b-) phenotype can be prevalent outside of Africa. Studies have found the Fy(a-b-) phenotype in 78.32% of southwestern Saudi Arabian blood donors ; 61% of Nicobar) ,234,235.Pk [Pk was also found in Anopheles mosquitoes from these islands, which also harbor M. fascicularis [a and anti-Fyb blood typing antibodies, Fy(a-b-) RBC units could be identified and used for anti-Pk T-REX. One study found that 82.76% of the Jarawas tribe of the Andaman and Nicobar Islands were Fy(a-b-) . AnotherPk . In addiicularis . By phenG6PD is an enzyme in the pentose phosphate pathway (PPP), which leads to reduction of nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH ,238. ThiPk T-REX of G6PDd RBCs because they may reduce the risk of severe Pk malaria. A case–control study examined Pk malaria patients that presented to two primary referral hospitals in Malaysia from 2012 to 2015 [Pk malaria mono-infections (confirmed by PCR) and 683 controls. G6PDd was present in 2 (0.9%) of 227 Pk malaria patients who were tested and 43 (6.5%) of 663 controls that were evaluated. The adjusted odds ratio was 0.20, and the p value was 0.045. To further support its potential benefit, there is evidence that the G6PDd phenotype was selected by human evolution because it reduces the risk of severe malaria and death [If Fy(a-b-) RBC units are scarce in given region , healthcare providers may consider the anti- to 2015 . The stund death ,240,241.Pk malaria would strengthen the case for the T-REX of G6PDd RBCs. One study looked at baseline features of malaria patients [< 12 years of age infected with Pk, Pf, and Pv, respectively. In addition, G6PDd was found in 1.1%, 4.3%, and 2.1% of adults infected with Pk, Pf, and Pv, respectively. Interestingly, G6PDd was least common in Pk patients of both age groups. It is worth noting that primaquine can trigger hemolysis in G6PDd individuals. However, this medication is not typically used to treat Pk malaria [Pk malaria patients  may be treated with primaquine to prevent relapse due to hypnozoites in the liver [Additional studies showing improved outcomes of G6PDd individuals with patients . G6PDd w malaria ,242,243.he liver ,243. The WHO recently reclassified G6PD variants into four classes. Persons in Class A, B, C, and U have median G6PD activity <20%, <45%, 60–150%, and 45–60%, respectively . Class CPk malaria T-REX.Because most G6PDd individuals do not have chronic anemia, they have a hemoglobin level that is sufficient to allow for blood donation. Thus, persons with G6PDd donate blood in many countries. For example, about 7.6% of Thai blood donors, more than 16% of Iranian blood donors, and 25.5% of Nigeria blood donors were G6PDd ,247,248.The FST test starts by incubating blood with NADP and glucose-6-phosphate . G6PD inThe safety of transfusing G6PDd RBCs needs to be considered. The WHO states that G6PDd blood donors should be allowed to donate if do not have a history of hemolysis . HoweverThe safety of transfused G6PDd RBCs has been studied in SEA. In 2022, there was a randomized controlled clinical trial of G6PDd versus non-G6PDd RBC units transfused into patients with hypoproliferative anemia in Thailand . The tra- anion in the opposite direction of an HCO3− ion across the RBC membrane [Pv and CM caused by Pf [Southeast Asian Ovalocytosis (SAO) is caused by a 9 amino acid deletion in the band 3 protein (AE1), which is an anion exchange protein on RBCs ,265. Banmembrane . Heterozmembrane . Thus, omembrane . SAO wased by Pf ,264,269.Pk invasion of RBCs from persons with SAO, also known as Melanesian elliptocytosis [Pk ring forms per 100 RBCs for the controls and 0.4 and 0.5 for two SAO samples. A similar trend was noted in the second experiment. Importantly, all SAO RBCs and control RBCs expressed the Duffy antigen. The study also showed that SAO RBCs had fewer Pk merozoites attached to them, compared to the controls. However, only five samples were tested, and the findings have not been confirmed by others. One in vitro study showed that the ocytosis , was “maocytosis . In the Pk malaria would strengthen the case for T-REX of SAO RBCs. Pk-resistant.The prevalence of SAO can be substantial in parts of Malaysia, the Philippines, Indonesia, Thailand, and Papua New Guinea . The vasPk T-REX, healthcare professionals will need to ask blood bank staff if they can provide Fy(a-b-), G6PDd, and/or SAO RBC units. Using tubing segments from RBC units, Fy(a-b-), G6PDd, and SAO RBCs can be identified using standard blood-typing reagents, the rapid FST test, and based on their unique microscopic appearance, respectively. The availability of these units depends on the blood donor population demographics and the geographic location of the blood bank. Importantly, the identification of these Pk-resistant RBC units can be concomitant as the patient awaits and then obtains the vascular access needed for the procedure. To perform anti-Pk-resistant RBC units can be obtained, it is advisable to start with transfusion of standard-issue RBCs at the beginning of the procedure, followed by Pk-resistant RBCs at the end. This sequence will minimize the removal of the Pk-resistant RBCs during the procedure. The RBC units should be transfused in a specific order. Manual ET/RBCX slowly and continuously removes RBCs from the patient, while RBC units are slowly and continuously transfused. So, the first transfused RBC unit will have a higher percentage of RBCs removed than the last unit transfused. If only a limited number of Pk’s asexual replication, the therapeutic benefit of T-REX and antimalarials, and patient deaths. If T-REX of Fy(a-b-) RBCs is performed, then the circulating uRBCs and iRBCs will be removed during the ~2 h procedure. Because sequestered iRBCs adhere to the endothelium, they will not be removed by T-REX. However, over the next 24 h , the schizonts will rupture, releasing merozoites and reducing sequestration. The merozoites will not be able to invade Fy(a-b-) RBCs. Thus, the benefits of T-REX would be realized during the procedure, within 24 h , and during subsequent asexual life cycles. This is important because a case-series about Pk malaria fatalities found that the median time from admission to death was 41 h [It is important to consider the timing of Pf malaria [Pf parasites inside of RBCs being killed by drugs, these iRBCs can still cytoadhere for hours because PfEMP-1, a key molecule this process, remains on the surface of iRBCs [The timing of the benefit of antimalarials is complex. This topic came up in the South East Asian Quinine Artesunate Malaria Trial (SEQUAMAT) and African Quinine Artesunate Malaria Trial (AQUAMAT), in which clinical outcomes from quinine and artesunate treatment were compared in patients with severe  malaria ,273. Bot malaria ,273. Thi malaria . A studyof iRBCs . So, parof iRBCs . A randoof iRBCs . Still, After the patient’s malaria resolves, there is no need to perform RBCX to remove the transfused Fy(a-b-), G6PDd, or SAO RBCs. This is because these RBCs were donated by healthy non-anemic persons who met blood donor eligibility requirements. However, the duration of survival of Fy(a-b-), G6PDd, and SAO RBCs is worth discussing. Healthy human RBCs can circulate for about 120 days . Fy RBC units. The patient’s clinical status and laboratory values  before and after T-REX can be reported. For a more complete list of procedure parameters to report, view reference [If healthcare professionals publish anti-eference .Pf malaria, which likely explains its high prevalence in Pf malaria “hot-zones” [Pk malaria patients [Pk, Pf, and Pv malaria patients admitted to a Malaysian hospital. However, the prevalence of blood group O in non-malaria patients was not measured. So, it is not known if blood group O provides some benefit to Pk malaria patients. Studies assessing the impact of ABO blood group on Pk malaria would be beneficial. If blood group O protects against severe Pk malaria, it could be a feasible anti-Pk malaria T-REX option. This is because (1) blood group O is a “universal” blood type, (2) it is found in about 30% or more of the global population, and (3) no additional testing is required. Blood group O has been shown to reduce the risk of severe t-zones” ,209,281.patients . This stPk T-REX is a theoretical optimization of manual ET/RBCX, in which Pk-iRBCs are replaced with Pk-resistant RBCs. Fy(a-b-) and SAO RBCs are likely to resist Pk invasion, while G6PDd RBCs are likely to reduce the risk of severe Pk malaria. Anti-Pk T-REX may be beneficial if IV artesunate or other antimalarial medications are not readily available, if patients are likely to progress to severe disease, or if parasite drug-resistance develops. Anti-


Plasmodium and is transmitted by the bite of an infected Anopheles mosquito. The disease is commonly characterized by fever, edema, thrombocytopenia, hypoglycemia, anemia, and myalgias; however, the infection’s cutaneous presentations are not commonly emphasized and tend to be overlooked.Malaria is a vector-borne tropical infection caused by protozoa of the genus A literature search was conducted that focused on the various skin pathologies that malaria patients have been noted to present with using case reports and currently available literature. We describe the various skin manifestations associated with malaria, such as purpura fulminans, febrile urticaria, cutaneous leishmaniasis co-infections, urticaria infectiosum, vivax-induced severe thrombocytopenia petechiae, acral skin necrosis, and reticulated erythema, and how each of these skin manifestations may provide insight into the patient’s prognosis.Documentation and vigilance regarding these cutaneous manifestations must be emphasized as they may lead to better patient outcomes and a stronger understanding of the patient’s underlying malaria. Plasmodium, is transmitted by the bite of an infected Anopheles mosquito. Following invasion and growth within erythrocytes once inside the human body, the Plasmodium parasite causes the disease's periodic fever episodes and systemic symptoms. Malaria is a significant global concern since it affects 10,000 to 30,000 of the 125 million visitors to endemic regions yearly, with a 1% mortality rate [Malaria, a blood-borne tropical infection caused by protozoa of the genus ObjectivesOur narrative review details malaria’s relationship with its various skin pathologies, some less common than others, and strives to illuminate the prognostic value of the infection’s cutaneous manifestations.MethodsA literature search was performed using PubMed, MEDLINE, Scopus, and Google Scholar. Through the use of case reports, narrative reviews, and systematic reviews, our investigation focused on the various skin pathologies that patients afflicted with malaria have presented with. Articles were selected and narratively reviewed by two reviewers. A third reviewer resolved any incongruity in the initial article selection process. A total of 11 cases were ultimately selected to be included in this analysis.Seven malarial manifestations were assessed using a severity index (SI) score based on four metrics, namely “cutaneous findings,” “functional outcomes,” “degree of sepsis,” and “time to resolution.” The SI was calculated using an evaluation matrix, where each metric received a weighting out of three based on the severity associated with malaria. Specifications of weighting for each metric are given in Table Purpura fulminansPlasmodium falciparum infection is noted to be associated with low levels of protein C, protein S, tissue plasminogen activator, and antithrombin with an increased level of plasminogen activator inhibitor-1 [Purpura fulminans (PF) is a condition characterized by disseminated intravascular coagulation (DIC) that commonly occurs in patients with an acquired deficiency in anticoagulants, often due to a septic infection . Historiibitor-1 ,5.3 , and had a decreased platelet count . Additionally, he had moderately elevated liver enzymes, an increased prothrombin time (25.9 seconds), and a blood smear positive for P. falciparum antigen [Chaudhary et al. reported a case of a 22-year-old man with rigor, fever, and chills for seven days who presented with bluish-black discoloration of both feet and legs to a hospital in South East Asia . The pat antigen . The patP. falciparum. He received intravenous fluid, norepinephrine, low-dose hydrocortisone, intravenous quinine, vitamin K supplementation, and ceftriaxone, and recovered gradually. However, the purpura progressed to skin necrosis of the feet, and both were amputated two months later.Another report describes a case of a 51-year-old Caucasian male with a past medical history of post-traumatic splenectomy who had just returned from a vacation in Madagascar . He had P. falciparum malaria. PF can be fatal, often requiring eventual amputations; thus, early recognition is paramount.PF is characterized by symmetrical distal ischemic damage and hemorrhagic infarction of two or more locations without obstructing large vessels . The afoPF received an SI-weighted score of 9 for “cutaneous finding,” 6 for “degree of sepsis,” 9 for “time to resolution,” and 3 for “functional outcome,” giving the cutaneous manifestation a total SI score of 27.Febrile urticariaP. falciparum and Plasmodium vivax [A classic presentation of febrile urticaria in the pediatric population is the appearance of wheals associated with edema of underlying subcutaneous tissues. This skin manifestation is believed to result from the malarial subtypes um vivax ,10. It ium vivax . Scientium vivax . In thesP. vivax. She was successfully treated within three days without complications with chloroquine and antihistamines. The patient additionally received a 14-day course of primaquine for radical cure. A second case study by Sharma et al. described a family where three members, on separate occasions, presented with urticarial lesions that occurred concomitantly with a fever secondary to P. falciparum infection, suggestive of a potential genetic predisposition nature of febrile urticaria [Zaki and Shanbag described a case involving a nine-year-old Indian girl with a high-grade fever and bilateral upper and lower limb pruritic urticarial rashes . Peripherticaria . It was Febrile urticaria received an SI-weighted score of 3 for “cutaneous finding,” 2 for “degree of sepsis,” 3 for “time to resolution,” and 1 for “functional outcome,” giving the cutaneous manifestation a total SI score of 9.Malaria and cutaneous leishmaniasis coinfectionMalaria and cutaneous leishmaniasis (CL) are two of the world’s most prominent vector-borne parasitic diseases affecting developing nations ,13. Mala9/L, white blood cell count was 2.90 x 109/L, and platelet count was 63 x 109/L. An intracellular Leishmania-Donovan body and schizonts of P. vivax were observed on peripheral blood smear, as well as a positive RK 39 immunochromatographic and malaria rapid diagnostic test, further corroborating the diagnoses [One case report details a rare case of a child, age 5, who exhibited black necrotic lesions, skin darkening, and fever in Nepal . The youiagnoses . A totaliagnoses .P. falciparum, 5 (11%) were positive for P. vivax, and a total of 16 (4.2%) individuals were found to be coinfected with VL and malaria. In the interest of further exploring potential etiologies of this joint infection, it was estimated that 188 (46.9%) study participants traveled in the past, with 10 (5.6%) of these individuals presenting with a combined VL-malaria infection.A cross-sectional study conducted in Ethiopia assessed the prevalence of coinfection of visceral leishmaniasis (VL) with malaria . A totalCL coinfection received an SI-weighted score of 3 for “cutaneous finding,” 4 for “degree of sepsis,” 6 for “time to resolution,” and 1 for “functional outcome,” giving the cutaneous manifestation a total SI score of 14.Plasmodium vivax malaria presenting as urticaria infectiosumAlthough rare, cutaneous malaria involvement can manifest as severe urticaria and angioedema . The mosP. vivax malaria trophozoites, contrary to the previous negative initial peripheral smear. After starting the patient on oral chloroquine, his health dramatically improved within three days, resulting in his fever and the urticarial rash resolving.Kapse reports a case of vivax malaria that presented clinically as urticaria and angioedema . A sevenFurthermore, 10 individuals who presented with urticaria as a manifestation of malaria, either with or without fever, were described by Godse and Zawar . All theUrticaria infectiosum received an SI-weighted score of 3 for “cutaneous finding,” 2 for “degree of sepsis,” 3 for “time to resolution,” and 1 for “functional outcome,” giving the cutaneous manifestation a total SI score of 9.Plasmodium vivax malaria with severe thrombocytopenia petechiae and varied cutaneous manifestations3, and she was hypotensive with signs of hypovolemic shock. Her hemoglobin was low at 7.2 g/dL, she displayed mild hepatosplenomegaly, and her antigen and PCR testing were positive for P. vivax infection. Coagulation assays were in the normal range, including prothrombin time, activated partial thromboplastin time, fibrinogen count, and D-dimer levels. Platelet transfusion was attempted but failed to raise the patient's platelet count. Subsequent malarial treatment with parenteral artesunate, oral mefloquine, and primaquine was initiated, leading to fever resolution and rapid clearance of the malarial parasite. Intravenous immunoglobulin (IVIG) was begun in an attempt to address thrombocytopenia and bleeding. Platelets 10 days post-IVIG infusion was 74,000 and returned to an average count at six weeks. Given the rapid and successful response to IVIG, it is speculated that epitope mimicry between the P. vivax parasite and platelets might have been the driving mechanism behind the patient’s thrombocytopenia and bleeding [Malaria is known to cause mild thrombocytopenia during its course of infection. In some cases, however, the extent of platelet drop can be rather severe. In one such instance, a four-year-old girl from India experienced severe thrombocytopenia, which manifested as cutaneous and mucosal bleeding . The casbleeding .Plasmodiumvivax malaria with severe thrombocytopenia petechiae received an SI-weighted score of 6 for “cutaneous finding,” 6 for “degree of sepsis,” 6 for “time to resolution,” and 1 for “functional outcome,” giving the cutaneous manifestation a total SI score of 19.Acral skin necrosis in patients with malariaP. falciparum malaria [Superficial skin necrosis of the extremities may arise from local trauma or microvascular obstruction. This condition is abnormal in younger patients and has been associated with atrial fibrillation, myocardial infarction, aneurysm, endocarditis, cryoglobulinemia, and vasculitis . Dos San malaria . The patPlasmodium malarial infection is not well documented. In the case presented by Dos Santos et al., the etiology of these lesions may be hypothesized to be due to the increased adherence of Plasmodium-parasitized erythrocytes to endothelial cells in the microvascularization of the lower limbs [The association of acral necrosis with er limbs .Acral skin necrosis received an SI-weighted score of 6 for “cutaneous finding,” 4 for “degree of sepsis,” 3 for “time to resolution,” and 1 for “functional outcome,” giving the cutaneous manifestation a total SI score of 14.Reticulated erythema in malariaP. falciparum trophozoites. The patient was treated with antimalarial drugs α-β arteether, quinine, supportive platelet concentrate, and intravenous fluids. The patient's rash responded well on day 3 of treatment and was resolved by day 9 [P. falciparum malaria, physicians treated him with α-β arteether, supportive platelet concentrates, and intravenous fluids. His rash improved by day 7.Reticulated erythema is a clinical description that describes a skin rash with a "net-like," "chicken wire," or "sieve-like" pattern. The rash is associated with systemic, infectious, acquired, and congenital dermatoses . Two casby day 9 . Vaishnaby day 9 . The patP. falciparum, resulting in her treatment with chloroquine and antihistamine. The patient's rash responded appropriately to the medications and resolved without further complications. It is proposed that erythema and urticaria occur due to the release of histamine, leukotrienes, and platelet-activating factor. Notably, the associated petechiae seen in the first two causes are suspected to be due to an immune complex resulting in vessel damage [The aforementioned case report from Morocco involved a 20-year-old female admitted with an acute febrile rash . Her phyl damage .Reticulated erythema received an SI-weighted score of 3 for “cutaneous finding,” 4 for “degree of sepsis,” 3 for “time to resolution,” and 1 for “functional outcome,” giving the cutaneous manifestation a total SI score of 19.The comprehensive summary of the cutaneous manifestations, malaria types, interventions, outcomes, and major clinical findings of each of the cases presented in this review are given in Table Malarial severity analysisThe malarial severity analysis revealed SI scores ranging from 9 to 27, with a maximum score of 27 obtainable from the matrix. The SI scores for the cutaneous manifestations of malaria (including PF), thrombocytopenia petechiae, acral skin necrosis, CL coinfection, reticulated erythema, urticaria infectiosum, and febrile urticaria scored 27, 19, 14, 14, 11, 9, and 9, respectively, as shown in Table Malaria's cutaneous manifestations indicate the degree of malaria infection and provide insight into an individual's prognosis. By using a total SI to assess cutaneous findings, functional outcomes, degree of sepsis, and time to resolution, we can better analyze a patient’s cutaneous presentation of malaria. PF yielded the highest total SI weight at 27, followed by P. vivax malaria with severe thrombocytopenia petechiae (19), acral skin necrosis (14), CL coinfection (14), reticulated erythema (11), febrile urticaria (9), and urticaria infectiosum (9). Given the rare nature of these various malaria-associated skin pathologies, it can be quite difficult to truly ascertain accurate presentations of these cutaneous presentations as they vary from individual to individual. Had there been more literature on similar incidences of these cases, a more accurate depiction of the affliction could have been provided, leading to more accurate insights into the prognoses of the aforementioned manifestations. It is of the utmost importance that physicians stay vigilant of these possible, rare, cutaneous presentations as focusing solely on objective lab values may prevent optimal and early treatment measures.


Non-invasive diffusion-weighted imaging (DWI) to assess brain microstructural changes via cortical mean diffusivity (cMD) has been shown to be cross-sectionally associated with tau in cognitively normal older adults, suggesting that it might be an early marker of neuronal injury. Here, we investigated how regional cortical microstructural changes measured by cMD are related to the longitudinal accumulation of regional tau as well as to episodic memory decline in cognitively normal individuals harboring amyloid pathology.122 cognitively normal participants from the Harvard Aging Brain Study underwent DWI, T1w-MRI, amyloid and tau PET imaging, and Logical Memory Delayed Recall (LMDR) assessments. We assessed whether the interaction of baseline amyloid status and cMD  was associated with longitudinal regional tau accumulation and with longitudinal LMDR using separate linear mixed-effects models.p = 0.044) but not the inferior temporal lobe, such that greater baseline cMD values predicts the accumulation of entorhinal tau in amyloid-positive participants. Moreover, we find a significant interaction effect of the amyloid status and baseline cMD in the entorhinal cortex  in predicting longitudinal LMDR (p < 0.001), such that baseline entorhinal cMD predicts the episodic memory decline in amyloid-positive participants.We find a significant interaction effect of the amyloid status and baseline cMD in predicting longitudinal tau in the entorhinal cortex (The combination of amyloidosis and elevated cMD in the entorhinal cortex may help identify individuals at short-term risk of tau accumulation and Alzheimer’s Disease-related episodic memory decline, suggesting utility in clinical trials. People with Alzheimer’s disease have problems with their memory and ability to acquire and process knowledge. Understanding the earliest brain changes leading to these problems helps identify those likely to develop Alzheimer’s disease early in the disease process. This study used a marker that measures the mobility of water in the brain to investigate how these changes can predict development of a protein named tau and changes in people’s memory. The participants showed no signs of memory impairment at the beginning of the study, but some developed memory decline during follow-up. Greater mobility of water in certain brain areas predicted future increase in tau and decline in memory, indicating this measure could be used to identify people at risk of developing Alzheimer’s disease. Gagliardi, Rodriguez-Vieitez et al. image brain microstructural changes over time in cognitively-normal older adults. Cortical mean diffusivity was associated with accumulation of tau and episodic memory decline in those with amyloid deposits. Using several methods, including cerebrospinal fluid (CSF), positron emission tomography (PET), and magnetic resonance imaging (MRI), studies have consistently shown a biological signature that would start to develop years before the clinical diagnosis5. This accumulation would follow a sequential order, with amyloidosis being the first to aggregate1, and has led to the concept of the preclinical stage of AD3. Note that not all AD biomarkers become detectable at the same time. With tauopathy, for example, recent studies have shown that while abnormal CSF could be detected years before the diagnosis, PET measures of tau would show changes closer to the diagnosis6 with a distribution significantly correlating with clinical symptoms7.Since the development of various neuroimaging techniques in the last decades, the research on Alzheimer’s disease (AD) has notably focused on the search for early biomarkers. AD has been biologically defined by the presence of both amyloid and tau brain pathology8. While DWI is typically quantified in the white matter, recent developments allow exploring AD-related changes on a microstructural scale. Specifically, Montal and colleagues have developed a method to assess microstructural properties in the gray matter by means of the cortical mean diffusivity (cMD)10. Using this approach, they demonstrated that cognitively healthy older participants with significant amyloidosis in the absence of tau demonstrated a decreased cMD in cross-sectional analyses9, a finding that is probably related to amyloid-related inflammation11. In contrast, participants with both amyloidosis and tauopathy showed increased cMD values9. These results have been replicated using pseudo-longitudinal modeling in autosomal dominant AD looking at individuals across the spectrum of disease as they approach the estimated year of onset of symptoms10. The results showed a biphasic trajectory of changes in which preclinical mutation carriers demonstrated first a decrease followed by an increase in cortical diffusivity in preclinical mutation carriers closer to their estimated time of onset10. More generally, increased mean diffusivity is observed only in the presence of both amyloidosis and tauopathy14, and may be explained by amyloid-related inflammatory processes10. However, since cMD is a relatively recently developed biomarker, more studies are needed to explore the relationships with the pathological AD profile over time.Combining different techniques, multimodal neuroimaging studies have started to explore the associations and synergies between early AD biomarkers. Unlike PET techniques using radioactive tracers, MRI has the advantage of having no radiation exposure, being more affordable, and being widely available in clinics. Among these MRI techniques, diffusion-weighted imaging (DWI) computes the spatial distribution of the diffusion of water molecules15. In their longitudinal analyses, the authors also found that baseline regional cMD was related to hippocampal atrophy as well as to the progression from CN to mild cognitive impairment (MCI). Similarly, in their pseudo-longitudinal study including familial AD participants, Montal and colleagues also showed a relationship between cMD and the evolution of cortical thickness. Specifically, they found a quadratic relationship between the two variables, starting with an initial increased cortical thickness associated with decreased diffusivity in the absence of detectable tauopathy. As the estimated years to symptoms onset decreases, and tau pathology becomes detectable, the opposite pattern is observed10. While these studies have shown an association between microstructural and neurodegenerative changes, as well as with tau cross-sectionally, the association between cMD and subsequent longitudinal accumulation of tau pathology has yet to be explored. Compared to CSF-Tau, tau PET shows tau changes chronologically closer to the diagnosis and is closely associated with clinical symptoms 7. Therefore, predicting future tau PET burden, especially in an at-risk-for AD population, would be clinically meaningful.More recently, in a cross-sectional design examining cognitively normal (CN) older adult participants, Rodriguez-Vieitez and colleagues explored the relationship between cMD and AD pathological hallmarks measured by amyloid and tau PET imaging, and demonstrated that cMD was positively associated with tau but not amyloid burden11C-Pittsburgh compound-B (PiB) PET, similar to our previous study15. As one of the pathognomonic biomarkers of AD3, brain amyloidosis accumulates decades ahead of clinical dementia5 and is part of the NIA-AA Research Framework’s biological definition of AD1. Previous studies demonstrated that the topographical pattern of amyloid lesions overlaps with some brain networks, especially with the default mode network (DMN)16—a network known to support several cognitive functions such as self-referential processes17, episodic memory (EM) or executive functioning19. These cognitive functions have been identified as showing the earliest subtle cognitive decline in the preclinical stage24. Recent studies have explored the relationship between cMD and cognitive decline. Fortea and colleagues, focusing on temporal regions , showed a cross-sectional association between an increased cMD and decreased EM performances25. Similarly, Rodriguez-Vieitez and colleagues showed that cMD in the temporal regions was associated with longitudinal cognition using a cognitive composite score15. So far, no study has examined this relationship focusing on EM. This information may be clinically important, as episodic memory is one of the earliest cognitive domains to decline in AD27.To distinguish between asymptomatic at-risk-for AD participants and healthy controls, we stratified our sample into two groups based on the The present work aimed at determining the association between baseline cMD and the longitudinal changes in tau and episodic memory in CN individuals at risk for AD. Here, we show that increased cMD at baseline is associated with a higher rate of tau accumulation and a steeper decline in episodic memory over time. We further demonstrate that amyloid moderates our findings, such that these associations are more pronounced in cognitively normal individuals harboring amyloid pathology.https://habs.mgh.harvard.edu), a single-center observational study conducted at the Massachusetts General Hospital. HABS is a longitudinal study focused on older adults, CN at enrollment, aiming to improve our understanding of AD’s preclinical stages. The inclusion criteria for participants to be considered CN at the screening included normal performances (adjusted for age and education) on the Mini-Mental State Examination (MMSE)28, Logical Memory (LM)29, and Clinical Dementia Rating Scale (CDR = 0)30, as well as the absence of significant depression as measured by the Geriatric Depression Scale (GDS < 10/30)31. Clinical Status and progression to either MCI or AD dementia were determined in clinical consensus meetings. In addition, all participants underwent brain imaging, including structural MRI (T1-weighted and DWI), as well as PET with amyloid (PiB)32 and tau (18F-AV-1451/flortaucipir [FTP])33 tracers. Apolipoprotein E4 (APOE-ε4) status , was also collected. The protocol was approved by the Mass General Brigham institutional review board, and study procedures were carried out only after participants reviewed and signed the consent form. The study was carried out following the ethical standards defined in the Declaration of Helsinki (2000).Participants were selected from the Harvard Aging Brain Study . Cognitive assessments were performed in annual visits . With these criteria, 122 participants were included in the study. Demographic characteristics at baseline are summarized in Table http://surfer.nmr.mgh.harvard.edu)34, and visually checked to correct potential processing errors. Cortical mean diffusivity was computed by processing DWI data using a surface-based approach combining FSL (FMRIB Software Library)  and Freesurfer 6.0 (http://surfer.nmr.mgh.harvard.edu)9. Briefly, the methods sample the mean DTI metric in the mid-point of the cortical ribbon and sample it to each individual cortical surface reconstruction. The DTI processing details for this sample were explained in a previous publication15. Measures of cMD were computed based on baseline DWI data. For this study, similarly to our previous publication15, we focused on regional cMD assessed in two regions of interest (ROI): the entorhinal (EC) and inferior temporal (IT) cortices.All participants underwent a 3-Tesla MRI imaging procedure on a TimTrio scanner  using a 12-channel phased-array head coil. Structural T1-weighted magnetization-prepared rapid-acquisition gradient-echo  = 2200 ms, echo times (TE) = 1.54, or 3.36, or 5.18, or 7 ms, flip angle = 7°, 4× acceleration, 1.0 × 1.0 × 1.2 mm voxels), as well as DWI sequences, were registered. Structural segmentation was performed using FreeSurfer 6.0  using FreeSurfer 6.037. PET images were co-registered to the participant’s native T1-MRI using FreeSurfer (mri_coreg).PET scans were performed on an ECAT EXACT HR+ scanner  at the Massachusetts General Hospital PET facility. Acquisition parameters and methods have been previously detailed38, and IT tau, a region in which tau accumulation has been proven to be characteristic of AD as opposed to normal aging40.FTP-PET values were corrected for partial volume (PVC) using the Geometric Transfer Matrix approach, and standardized uptake value ratios (SUVr) were obtained using the cerebellar gray matter as a reference. Based on previous literature, EC tau burden was selected as an early affected region in the course of AD37. Logan Distribution volume ratios (DVR) were computed using Logan models with the cerebellar gray matter as a reference. PVC was also applied. Participants were then assigned to two groups, amyloid-positive and negative, based on their baseline PiB-FLR DVR, using a 1.2 value (derived from a Gaussian mixture model) as a cutoff41. Out of 122 participants, our sample included 36 (29.5%) participants above this threshold who were considered amyloid-positive (A+).PiB-PET was computed in a cortical composite including frontal, lateral-temporal, parietal, and retrosplenial (PiB-FLR) regions29. The Logical Memory test is widely used in the context of AD in both clinical practice and research and consists of a paragraph read to the participant, who must recall as many relevant elements as possible, both immediately and after a delay . The score ranges from 0 to 25, with a greater score meaning a better memory performance. The LMDR score was used for this study. Previous research found that CN participants with amyloidosis demonstrated a significantly greater LMDR decline than a control group42. As mentioned earlier, at screening, all participants had to perform in the normal range on the LMDR (adjusted for education).Episodic Memory (EM) was measured using the Logical Memory from the Wechsler Memory ScaleAPOE-ε4 carrier status, and race; continuous variables included age, education, LMDR, as well as brain regional FTP-PET and cMD values evaluated in both the EC and IT regions.We compared the two amyloid groups at baseline using t-tests and chi-square tests for continuous and categorical variables, respectively. Categorical variables included sex, EC or IT ~ Age + Sex + Baseline Tau EC or IT + Baseline cMD EC or IT × Baseline Amyloid Group × Time + (1|Subject).We performed two linear mixed-effects models to explore the effect of brain regional cMD at baseline on the longitudinal evolution of tau burden and EM performance. The first set of models used regional tau (FTP-PET) SUVr burden as the dependent variable, with one model for EC and another for IT tau. These models were corrected for age and sex and included the baseline amyloid group, baseline cMD value, and time (years from baseline), as well as the interaction between those three variables and all lower-order interaction terms, as fixed-effects independent variables, and incorporating random intercepts at the subject level. For each model, both the cMD and FTP-PET imaging measures were evaluated in the same ROI, i.e., the EC and IT, respectively. The models were computed as follows: Tau EC or IT × Baseline Amyloid Group × Time + (1|Subject). Type II likelihood ratio was used to test the main and interaction effects. P-values were corrected for multiple comparisons using the Bonferroni method43 for the four models. Effect sizes for the models were computed through Cohen’s f2 method44. Continuous values were standardized for the different predictors to be put on similar scales.The second set of models was similar, except that LMDR was included as the dependent variable. The formula was the following: LMDR ~ Age + Sex + Baseline LMDR + Baseline cMD EC or IT × Baseline Amyloid Group × Time + (1|Subject). Additionally, we ran the following model: LMDR ~ Age + Sex + CTh + Baseline LMDR + Baseline cMD EC or IT × Baseline Amyloid Group × Time + (1|Subject). These results are presented as Finally, additional models were performed using baseline cortical thickness , to investigate the specificity of the cMD results. That is, first we ran the following model: LMDR ~ Age + Sex + Baseline LMDR + Baseline CTh 45 were conducted to assess the differential effects of baseline cMD in each group (A+ vs. A−). SSA is a post hoc analysis that can be used to help interpret significant interactions. When two or more terms interact, it means that the regression for each of them depends on the value of the other. SSA computes and examines the regressions using several values of the terms. For example, since our interactions consist of two continuous variables and one categorical , SSA was performed by computing the regression lines separately using the mean and standard deviation (SD) of cMD values  and the amyloid group over time. This procedure allows us to determine the directionality of each regression line in this three-way interaction.Simple slope analyses (SSA)https://www.R-project.org/). Linear mixed-effect models were computed using the lme4 package46.All statistical analyses were performed using R4.1.0 (Further information on research design is available in the p < 0.001). Among our sample, 37 (30.33%) participants were APOE-ε4 carriers, with a higher proportion of APOE-ε4 carriers seen in A+ participants when compared to A− participants . At follow-up (mean number of follow-up visits = 2.4 ± 0.75), 8.2% (N = 10) of the participants had clinically progressed. There was a significant difference between the amyloid groups, with A+ participants demonstrating a higher proportion of progressed individuals then the A− group .Our sample included 122 participants with 2.4 ± 0.55 (up to 4) follow-up observations. The majority were women (64.8%) with 16 ± 2.9 years of education; their baseline age was 71 ± 9.7 years old. Amyloid groups were equivalent on most demographic variables, including sex, education, race, and ethnicity . However, A+ participants showed greater IT cMD as well as tau burden (in both EC and IT regions) when compared to the A− group .We did not find any statistically significant differences between amyloid groups for baseline LMDR, cortical thickness in the EC and IT regions, and baseline EC cMD  both FTP-PET and baseline cMD were evaluated in the EC region, and 2) both FTP-PET and baseline cMD were assessed in the IT region .We observed a significant three-way interaction of amyloid status, baseline EC-cMD, and time in the model predicting EC-Tau  but no significant effect for higher values. In A+ participants, low baseline cMD values were not associated with greater tau accumulation over time. However, A+ participants demonstrating greater levels of baseline cMD, showed a significant increase of tau over time .To further understand the relationship between baseline EC-cMD and EC-tau over time in the amyloid-positive and negative groups, SSA were performed Table . The resThe model investigating whether baseline IT-cMD, and time predicted IT-Tau was not significant see Fig. .β = 0.593 ± 0.046, CI = 0.501–0.685, ES = 0.56, p < 0.001; IT β = 0.597 ± 0.046, CI = 0.506–0.689, ES = 0.57, p < 0.001). In both models, the main effect of time was significant (p < 0.001) but didn’t show a main effect of baseline cMD.Two models predicting the longitudinal evolution of EM using the LMDR value as the dependent variable were performed, using EC and IT cMD values at baseline as independent predictors, respectively Table . The modβ = −0.149 ± 0.05, CI = −0.249–−0.05, ES = 0.006, p = 0.013), and a trending effect in the IT model . However, in A+ participants, greater baseline cMD was associated with a decline in LMDR over time .We observed a three-way interaction between amyloid status, baseline cMD value, and time in the EC model , but a non-significant level when looking at the IT region  was associated with increased regional cMD9. One study, using a pseudo-longitudinal model of cMD in AD based on estimated years before the onset of clinical symptoms, showed that preclinical AD participants have a decrease followed by an increase in cMD in later stages10. Our current study confirms the previously reported positive association between cMD and tau burden cross-sectionally15 and further extends the findings to show that this relationship can also be observed concerning the longitudinal accumulation of tau in cognitively asymptomatic older adults with elevated amyloid but not in those without elevated amyloid. Our findings advocate for the specificity of cMD increased diffusivity in AD-sensitive areas for CN participants at risk for AD. This result also corroborates the previous proposal that a synergistic interaction between amyloid and tau determines the evolution of AD, and that the first triggers the toxicity of the second55.When modeling tau accumulation with time as predicted by the participant’s baseline regional cMD in both amyloid groups, we found a significant three-way interaction between amyloid status, time, and cMD in the EC region , as the measure itself uses the mean and standard deviation of the entire group to create the measure. The current study focused on EM, using the LMDR, an EM test widely used in both clinical practice and research. The models predicting tau and our findings showing that baseline regional EC-cMD predicted tau increase over time in CN participants with elevated amyloid serve as a potential explanation for the indirect effect of MTL dysfunction caused by tau accumulation and subsequent atrophy. Accordingly, previous research has linked AD patients’ cognitive performance, and especially EM, with tau accumulation in the MTL62. Our results suggest that amyloid-positive participants exhibit greater tau accumulation in the MTL over time, leading to regional cortical thinning, dysfunction, and ultimately a greater EM decline.This three-way interaction also confirms the similar relationship between medial-temporal cMD and a measure of global cognition  that was previously found16. Consequently, it has been proposed that brain amyloid accumulation could affect the functions supported by this network, including executive function and episodic memory19.The contribution of amyloid to longitudinal cognitive decline has been the topic of much research, and our results support a critical body of work. In our study, greater memory decline was observed in amyloid-positive participants as compared to amyloid-negative participants, and a significant interaction between amyloid status and time in both memory models was observed. Previous studies observed that the pattern of both amyloid and tau accumulation overlapped with the default mode network63. More recently, it has been shown that CN female participants with high amyloid load demonstrate greater cognitive decline than male participants64. In our current study, the absence of a sex effect may have several explanations. Future models are needed to answer this question more in detail.Our results also showed an absence of sex effect on cognition. Previous research has shown sex differences in the episodic memory profile of MCI and AD dementia patients15, and provide further evidence that cMD independently explains variance in cognitive decline, beyond cortical thickness in these regions.We also ran supplementary analyses looking at cortical thickness, rather than cMD and found a significant interaction in the EC, but not in the IT, such that decreased CTh at baseline predicted a faster decline in EM (Supp. Tab. 1). More importantly, when adding CTh as a covariate in the cMD models, the three-way interaction between cMD, time and baseline amyloid status remained significant for both EC and IT cMD (Supp. Tab. 2). These results are in line with our previous findings investigating cognition using a cognitive composite score (PACC) as our main outcome variableThis study has several limitations. First, our current sample, highly educated and predominantly white, is not representative of the global population. Also, the relatively short follow-up period could prevent us from observing some changes, both in tau and cognition, in the context of preclinical AD. Similarly, this also prevented us to include random slopes in the models. Subsequently, longer follow-up time and with more data, is needed to confirm our observations in a more diverse population with random slopes included.60. However, episodic memory is not the only measure that has shown a subtle decline over the course of the preclinical AD phase3. Other cognitive domains may involve a distinct network61 that does not necessarily include the MTL. Further research involving other brain regions and/or cognitive functions might show some interesting results.In this research, we used two MTL regions for tau (EC and IT). MTL functioning and integrity, especially in AD, have been mostly related to EM performanceIn this current study, we used baseline cMD to predict future tau accumulation. However, future studies should also explore the longitudinal evolution of tau and cMD, including longitudinal paired DWI measures along with tau scans over time.38. The same relationship was observed with episodic memory decline, showing that CN participants at risk for AD have greater memory decline over time associated with higher baseline diffusivity. These results confirm previous cross-sectional12 as well as findings obtained with cMD and longitudinal global cognition (PACC)15.Our results show a relationship between baseline cMD and longitudinal accumulation of tau in CN participants at risk for AD. This was particularly the case in the entorhinal cortex, a region known to be affected very early over the course of ADAPOE genotyping, as an efficient early screening biomarker.As compared to tau PET tracers, DTI-MRI does not entail radiation exposure and is used more widely in the clinical setting. Therefore, these results support the use of this technique, along with other methods such as PET or Supplemental MaterialReporting Summary


Plasmodium cytochrome c oxidase subunit III (cox3) gene. Of the 1,954 individuals who appeared healthy, no malaria parasites were found using microscopy. However, nPCR revealed seven cases of Plasmodium knowlesi mono-infection (0.4%), and six out of seven infections were in the group of 19 to 40 years old (P = 0.026). No human malaria species were detected by nPCR. Analysis of the DNA sequences also showed high similarity that reflects common ancestry to other P. knowlesi isolates. These findings indicate low submicroscopic P. knowlesi infections among indigenous communities in Malaysia, requiring PCR-based surveillance to support malaria control activities in the country.Malaysia has maintained zero cases of indigenous human malaria since 2018. However, zoonotic malaria is still prevalent in underdeveloped areas and hard-to-reach populations. This study aimed to determine the prevalence of malaria among remote indigenous communities in Peninsular Malaysia. A cross-sectional survey was conducted in six settlements in Kelantan state, from June to October 2019. Blood samples were tested for malaria using microscopy and nested polymerase chain reaction (nPCR) targeting the  Anopheles mosquito bites. Five species of the Plasmodium parasite cause malaria in humans, with Plasmodium falciparum being the most lethal. In 2020, about 241 million people in 85 malaria-endemic countries were affected by malaria, resulting in 627,000 deaths globally.Plasmodium knowlesi remains a public health concern, particularly in underdeveloped areas including remote regions of Malaysian Borneo and among indigenous populations in Peninsular Malaysia.Malaria is a life-threatening infectious disease caused by parasites transmitted through P. knowlesi cases, resulting in 13 deaths.,P. knowlesi.In 2021, Malaysia reported 3,575 A cross-sectional survey with a convenience sampling strategy was carried out between June and October 2019. Six Orang Asli settlements were surveyed, namely Kuala Betis , Mendrop , Gob , Bihai , Tuel  and Brooke  in Gua Musang district, Kelantan state, Peninsular Malaysia . The stuPlasmodium infection (primary PCR) and species identification (secondary PCR) were conducted following a previously published method targeting the Plasmodium mitochondrial cytochrome c oxidase subunit III (cox3) gene,Plasmodium vivax (MtNst_vivF2: 5′-TATTATTGTCTATACTAGATACTATAGTT-3′ and MtNst_vivR: 5′-GTTCTTTTTCTATTCAGAATAATGAATATAT-3′) and P. knowlesi (MtNst_knoF: 5′- CTTAATTGTCTATACTAGATACTATGGAC-3′ and MtNst_vivR: 5′- GTTCTTTTTCTATTCAGAATAATGAATAT-3′). Species-specific primers for the secondary PCR for P. falciparum, Plasmodium malariae, and Plasmodium ovale were as described previously.Thin and thick blood smears were prepared on site, stored in slide boxes and transported daily to the main laboratory in Gua Musang, where thin blood smears were fixed with methanol. All smears were stained with 3% Giemsa solution  for 30 minutes and examined under oil emersion (10 × 100 magnification) by experienced microscopists. For malaria detection by nested polymerase chain reaction (nPCR), blood samples withdrawn by finger prick using BD Microtainer Contact-Activated Lancet  were spotted on Whatman ET31 Chr filter papers  and dried thoroughly at ambient temperature. The dried blood spots were put in individual zipped plastic bags and stored at −20°C. DNA was extracted from three 3-mm filter paper punches using the QIAamp Blood Mini Kit  according to the manufacturer’s instructions. 2 statistical test or Fisher’s exact test were conducted to study the relationship between malaria infection and sociodemographic variables such as gender, age group and location of the settlement. The DNA sequences generated were confirmed for Plasmodium DNA sequence using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI) website.All data were analyzed using Microsoft Excel software and double-checked to avoid errors. Then, the data were processed and analyzed using statistical software STATA/SE version 13.1 . χP. knowlesi, and none were positive for other malaria species. Of the 7 P. knowlesi-positive individuals, 5 (71.4%) were males, with no significant difference in malaria-specific prevalence observed between gender (P = 0.251). Furthermore, P. knowlesi prevalence differed significantly by age group (P < 0.001): highest in the 19 to 40 group (0.8%), followed by ≤ 18 (0.1%) and no parasite was detected in the > 40 group by nPCR. Plasmodium knowlesi cases were also observed in 2 out of 6 settlements, namely Brooke (n = 5) and Mendrop (n = 2). Furthermore, the cox3 gene of malaria parasites from seven P. knowlesi-positive isolates were successfully amplified and sequenced. Analysis of the DNA sequences based on BLAST from the NCBI database revealed high sequence similarity that reflects common ancestry to other P. knowlesi isolates for all seven samples. The pairwise identity ranges from 99.31% to 99.77%  out of 6 settlements. This finding is consistent with the clustering of human settlements similar to in Malaysia Borneo,–P. knowlesi infection came from peri-domestic exposure of competent specific Anopheles vector.P. knowlesi endemicity. A recent study on land use change along the Sungai Kelantan Basin in Brooke settlement showed that the expansion of deforestation for logging and agricultural cropland has significantly impacted the temperature variations in the area.P. knowlesi malaria transmission and other zoonotic disease outbreaks.,Higher proportions of P. knowlesi infection among Orang Asli communities in Malaysia. We found a high proportion of P. knowlesi infections, particularly among adult Orang Asli, to be a significant endemic disease with potential health implications in the country. To effectively detect the infection, highly optimized and sensitive molecular methods are required. A guideline on the importance of submicroscopic P. knowlesi infections and a treatment policy is necessary to improve strategies for knowlesi malaria control in Malaysia.Our study has two limitations. Firstly, we only examined a limited number of sociodemographic variables among participants. Secondly, although our BLAST searches suggested possible matches, we were unable to conclusively establish species validation through phylogenetic analysis of the sequencing data. Despite these limitations, our study provides new insights into the prevalence of submicroscopic