Plasmodium vivax may present with asymptomatic malaria, only mild symptoms, or be severely ill. We and others have reported a diverse repertoire of immunopathological events that potentially underly susceptibility to disease severity in vivax malaria. Nevertheless, the combined epidemiologic, clinical, parasitological, and immunologic features associated with defining the disease outcomes are still not fully understood. In the present study, we perform an extensive outlining of cytokines and inflammatory proteins in plasma samples from a cohort of individuals from the Brazilian Amazon infected with P. vivax and presenting with asymptomatic (n = 108) or symptomatic (n = 134) disease , as well as from uninfected endemic controls (n = 128) to elucidate these gaps further. We employ highly multidimensional Systems Immunology analyses using the molecular degree of perturbation to reveal nuances of a unique profile of systemic inflammation and imbalanced immune activation directly linked to disease severity as well as with other clinical and epidemiologic characteristics. Additionally, our findings reveal that the main factor associated with severe cases of P. vivax infection was the number of symptoms, despite of a lower global inflammatory perturbation and parasitemia. In these participants, the number of symptoms directly correlated with perturbation of markers of inflammation and tissue damage. On the other hand, the main factor associated with non-severe infections was the parasitemia values, that correlated only with perturbation of inflammatory markers, such as IL-4 and IL-1β, with a relatively lower number of symptoms. These observations suggest that some persons present severe vivax regardless of pathogen burden and global inflammatory perturbation. Such patients are thus little tolerant to P. vivax infection and show higher susceptibility to disrupt homeostasis and consequently exhibit more clinical manifestations. Other persons are capable to tolerate higher parasitemia with lower inflammatory perturbation and fewer symptoms, developing non-severe malaria. The analytical approach presented here has capability to define in more details the determinants of disease tolerance in vivax malaria.Homeostatic perturbation caused by infection fosters two major defense strategies, resistance and tolerance, which promote the host’s survival. Resistance relates to the ability of the host to restrict the pathogen load. Tolerance minimizes collateral tissue damage without directly affecting pathogen fitness. These concepts have been explored mechanistically in murine models of malaria but only superficially in human disease. Indeed, individuals infected with  Plasmodium vivax infection can result in a broad spectrum of disease manifestations, ranging from asymptomatic malaria to severe life-threatening disease. Despite significant advances in the current understanding of the critical factors associated with the disease outcomes in vivax malaria, the immunopathological events responsible for the diversity of severe manifestations in the disease remain deeply unknown. Here, a large panel of cytokines/chemokines were assessed in plasma samples from a Brazilian cohort of P. vivax patients presenting with asymptomatic infection or symptomatic malaria at the time of diagnosis, as well as from uninfected endemic controls, to define the relationships between systemic inflammation, disease presentation, parasitemia, and epidemiologic characteristics. In-depth analyses using the molecular degree of perturbation were employed to reveal nuances of a unique profile of systemic inflammation and imbalanced immune activation directly linked to disease severity. Moreover, the discoveries diagrammed the occurrence of disease tolerance by narrowing down the interactions between the systemic degree of inflammatory perturbation and parasitemia values in vivax malaria patients. Plasmodium falciparum infection in African countries, Plasmodium vivax represents the predominant parasite in American regions, with noticeable incidence within the Brazilian Amazon, responsible for the vast majority of malaria cases in this area [P. vivax is historically thought to result in milder disease presentation than that caused by P. falciparum, mostly because the latter commonly results in high parasitemia, intense hemolysis and inflammation associated with cytoadherence to small capillaries . Nevert failure . The exaesponses –13. Furtesponses . UnderstThe capacity to restrict the infection burden and control inflammation and immune activation are major determinants of the malaria clinical outcomes . TheP. vivax malaria [P. vivax malaria. Here, we aimed to analyze the degree of systemic inflammatory imbalance in P. vivax patients from the Brazilian Amazon. We have compared individuals with asymptomatic, mild, or severe symptomatic disease, to define the complexity and multifactorial regulation subjacent to the malaria immunopathogenesis and the responses underlying the disease tolerance. To do that, we employed a recently reported approach named the molecular degree of inflammatory perturbation [P. vivax malaria is a tightly regulated process that integrates features highly influenced by age, previous malaria episodes, and a coordinated regulation of the systemic concentrations of mediators of inflammation.Furthermore, in experimental malaria models, control of the collateral tissue damage occurs through tightly regulated mechanisms such as glucose and iron metabolism, renal control, and adrenal hormones ,27–29. I malaria . Despiteurbation –34 usingurbation ,35–41. TFaculdade de Medicina, Faculdade São Lucas, Rondônia, Brazil, where the study was performed.Written informed consent was obtained from all participants or their legally responsible guardians, and all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The project was approved by the institutional review board of the vivax malaria that was finalized in 2010 [P. vivax infection. Diagnosis, recruitment details and clinical definitions were previous published [P. vivax infection in the molecular degree of perturbation only patients with P. vivax monoinfection  and healthy controls (n = 128) were included. The exclusion criteria for the present study were: P. falciparum infection documented by both microscopy and nested polymerase chain reaction (PCR), HIV-1, hepatitis B and C infection, yellow fever, leptospirosis, tuberculosis, cutaneous leishmaniasis, diabetes mellitus, allergic diseases as asthma, cancer, chronic degenerative diseases, sickle cell trait and the use of hepatotoxic or immunosuppressant drugs. Blood collection for nested polymerase chain reaction (PCR) and other measurements  were performed at the time of study enrollment, meaning that specimens were collected at diagnosis, during acute phase of disease in symptomatic patients, before treatment initiation. Individuals without symptoms were actively recruited in their residencies by active search, mostly in remote riverine communities, and had thick blood smear samples and a small aliquot of blood in EDTA tubes collected for diagnostic screening by nested PCR. The diagnosis in those individuals was performed after the clinical visit and if a positive test for malaria was found, a second visit within 30 days was performed in order to search for appearance of malaria symptoms. A new sample collection was made, and a second round of diagnostic tests was performed. Subjects that remained with positive nested PCR for Plasmodium within this period of 30 days and presented no malaria-related symptoms, such as fever (axillary temperature >37.8°C), chills, sweats, myalgia, arthralgia, strong headaches, nausea, vomiting, jaundice, and severe asthenia during this period were considered as asymptomatic malaria cases. During the first clinical visit (previously to the results of the diagnostic tests) all the asymptomatic individuals received counselling and were oriented to seek for health care in a malaria reference center in case symptoms appeared during the period between the 2 clinical visits. After the second clinical visit, all the patients that had positive malaria screening with microscopy and/or nested PCR were treated following the treatment guidelines of the Brazilian Ministry of Health. The plasma samples used to assess the biomarkers were collected during the second clinical visit, before the initiation of anti-malarial drugs, as previous described [P. falciparum infection, as previously published by our group [We performed multidimensional retrospective analysis of a databank containing clinical, epidemiologic and immunologic data from 530 individuals from the Brazilian Amazon  recruited between 2006 and 2007, as part of a project aimed at describing determinants of susceptibility to  in 2010 . In this in 2010 –41. Hereublished ,36–42. Tescribed . ClinicaFaculdade São Lucas, Brazil.Plasma levels of cytokines interleukin (IL)1-β, IL-4, IL-6, IL-10, IL-12p70, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and of C-C motif chemokine ligand (CCL)2, CCL5, C-X-C motif chemokine ligand (CXCL)9, and CXCL10 were measured using the Cytometric Bead Array—CBA , according to the manufacturer’s protocol. The measurements of aspartate amino-transferase (AST), alanine amino-transferase (ALT), total bilirubin, direct bilirubin, creatinine, fibrinogen and C-reactive protein (CRP) were performed at the Pharmacy School of the Federal University of Bahia and at the clinical laboratory of The molecular inflammatory perturbation is based on the molecular degree of perturbation (MDP) method, used and recently published by our group –34. In tn = Number of data pointsxi = Each of the value of dataσ = Standard deviationIn this study we applied the MDP scoring system using data on 21 biomarkers measured from three distinct groups of patients, with symptomatic vivax infection, asymptomatic vivax infection and uninfected endemic controls. The MDP was filtered by the absolute MDP scores below 2 modules and by the sum of all accumulated MDP deviations. To identify samples implicated in ‘perturbation’, all values above the cutoff of the average MDP score plus 2 standard deviations of the reference group were considered “perturbed”. In additional analyses, the median value of ssMDP in symptomatic patients was calculated , and individuals exhibiting values above this threshold (top 50% symptomatic participants) were considered highly perturbed.Descriptive statistics were performed to characterize the study population. Continuous variables were tested for Gaussian distribution using the D’Agostino-Pearson test. No variables exhibited normal distribution. The median values with interquartile ranges were used as measures of central tendency and dispersion, respectively. The Kruskal–Wallis test with the Dunn’s multiple-comparison were used to compare continuous variables whereas the Pearson’s chi-square test was used to compare variables displayed as percentages. Hierarchical cluster analyses (Ward’s method) of log10 transformed and z-score normalized data were employed to depict the overall perturbation of indicated biomarkers in the study subgroups. All comparisons were pre-specified and two-tailed. Differences with p-values below 0.05 after Holm-Bonferroni’s adjustment for multiple comparisons were considered statistically significant. Profiles of correlation between biochemical parameters were examined using network analysis of the Spearman correlation matrices. Correlations with p-value<0.05 were included in the network visualization. Spearman rank values (rho) were used to describe the strength of correlations.U test between symptomatic and asymptomatic vivax participants, as shown in A Kaplan-Meier curve adaptation  was emplShapes from R.Based on the mathematic formulas employed to visualize space-time deformity based on the general relativity theory proposed by Albert Einstein in 1915, we adapted the application of the proposed model to assess the overall impact of variables used here on homeostasis (defined as space-time membrane). In this model, homeostasis was defined by a plain p-brane which may be subjected to different degrees of deformities depending on the perturbation  caused by each inflammatory marker, tissue damage markers and from the pathogen itself (parasitemia). The calculations and plots were build using the package The statistical analyses were performed using GraphPad Prism 8.0 , JMP 14.0  and R statistical software.P. vivax infection or uninfected controls . No differences were noted in regard to sex distribution (P = 0.43). Of note, individuals with asymptomatic malaria referred a higher number of previous malaria episodes . Also, statistical discrepancies were found in the time living in the endemic area, with, on average, fewer years reported by symptomatic patients (P<0.001). Noteworthy, the vast majority of MDP values calculated with the biomarkers measured in the study exhibited significant differences among groups. Additional findings are detailed in the Here, we performed multidimensional analysis using previous collected samples from 128 endemic control participants, 108 asymptomatic malaria cases and 134 symptomatic patients ,36–42. OP. vivax infection is associated with overall disturbances in the systemic inflammatory profile which degree is proportional to the clinical presentation.To assess potential differences in the inflammatory activation according to the presence or absence of symptoms in vivax infection, we employed the molecular degree of perturbation  in each study subpopulation . IndividP. vivax infection , we found an important discrepancy in the network densities among the study groups. The group of symptomatic e lowest . Moreovee lowest . Curiouse lowest . In the e lowest . Importae lowest . Next, te lowest . These oP. vivax [P. falciparum infection in other regions of the globe [We have previously demonstrated that persons living in malaria endemic areas from the Brazilian Amazon for more than 10 years tend to exhibit increased odds of developing asymptomatic malaria once infected with P. vivax . Such fihe globe ,45. HereP. vivax parasitemia values failed to show a profile that could distinguish the individuals based on time living in the endemic area in both groups of malaria patients  , and in the individual degree of perturbation of fibrinogen, ALT, AST, TNF-α, IFN-γ, haptoglobin and also parasitemia  and lower frequency of symptomatic malaria cases   , reflectnfection . A clearnfection . Overallnfection . Asymptonfection . Furthernfection . Furthernfection . Our finAmong the biomarkers investigated in the present study, there were mostly two classes of molecules: (i) those that more closely infer inflammatory activity and (ii) those intrinsically related to tissue damage/injury .P. vivax, we used a discriminant algorithm based on sparse canonical correlation analysis (sCCA). We performed three models (i) one with all the markers  a  markers  and  (We further tested whether disease tolerance could be observed and characterized in the study participants infected with Methods) .P. vivax malaria. A principal component analysis (PCA), using the same parameters inputted in the hierarchical clustering, validated the idea that the number of symptoms, systemic inflammation assessed by ssMDP, and parasitemia were all independently influencing the distribution of the participants with symptomatic malaria into different subgroups of individuals. The PCA groups were similar to the clusters identified in the clustering analysis  approach, a statistical tool that led us to infer the global inflammatory status, and which has been recently proposed by our group to help to understand the pathophysiology of a range of infectious and non-infectious diseases –33,43,47P. vivax infected participants exhibited a higher degree of inflammatory perturbation than the control groups. Importantly, symptomatic patients displayed the highest global MDP scores. In fact, we have previously reported that the clinical manifestation in P. vivax infection is strongly associated with an inflammatory imbalance in peripheral blood [P. vivax infection [P. vivax [Plasmodium infections, directly affecting dampening the inflammation-driven cytokine storm, modulating the immune response, and limiting the tissue damage, reducing odds of severe malaria [Plasmodium infection was identified in our study. The levels of this cytokine were positively correlated with parasitemia in severe cases. IL-10 concentrations were also higher in asymptomatic participants and exhibited a positive correlation with the time living in endemic area, when all malaria cases were included in the analysis. Using the MDP assessment in malaria patients led us to define the nuances of the inflammatory imbalance associated with P.vivax infection.Using the MDP, we found that regardless of the symptomatology, al blood . Here, wnfection . Polymornfection , as wellnfection  were linP. vivax . IL-10 h malaria ,51,52. O malaria . This duP.vivax infection using the Spearman correlation matrices [P.vivax infection [P. vivax is associated with a potential uncoupling of the perturbed inflammatory response, probably due to lack of immune activation in peripheral blood. A curious finding was the relative dissociation between tissue inflammatory damage markers and immune factors. This dissociation argues that, except for the liver, the symptoms caused by P.vivax are strongly associated with the cytokine storm in response to infection and may be also linked to local tissue reaction, assessed by measuring levels of liver transaminases for example, but without direct interaction between these MDP values. This dissociation can be a consequence of the immune evasion mechanisms presented by the parasite, which alter the red blood cell (RBC) structure through expression and export of molecules encoded by Plasmodium genome [Plasmodium recognition and enables the tissue damage directly mediated by the pathogen.In some infectious diseases, such as malaria, the balance between immune activation, regulatory cytokine responses and degree of tissue damage is a critical determinant of disease outcomes –56. We ematrices ,31,35,57nfection ,59. Impom genome . The intP.vivax, reflected throughout time living in endemic area and age, the host is thought to develop clinical immunity against Plasmodium. Still, a controlled inflammatory activation can limit the parasite burden [P. vivax infection in the past gradually mitigates the inflammatory activation in later infection episodes. The indications of this phenomenon are several. First, we found a robust negative correlation between the time living in an endemic area and the MDP values for all markers measured, except for IL-10 MDP levels. Our results also revealed a higher incidence of symptomatic individuals and fewer symptoms among those who reported living less than ten years in the endemic region. Next, we showed negative correlations between the number of previous malaria episodes and the inflammatory disturbance detected during the current infection, except for, again, IL-10 MDP levels. It is important to note that we found a tendency to decrease systemic inflammation and a lower probability of being highly perturbed in individuals with a greater number of previous malaria episodes.Epidemiologic aspects have largely been described to influence clinical presentation and outcomes in malaria ,30,31,48e burden  without Plasmodium infections in the past tend to tolerate the current P. vivax infection through a decrease in perturbation of all pro-inflammatory and tissue damage markers, and increase in MDP levels of IL-10, which may limit tissue damage and minimize sickness [Regarding age, we found that aging is associated with less intense inflammatory activation. Reinforcing the role of hyper-inflammatory status in symptomatology, we demonstrated that the number of symptoms was positively associated with increased MDP levels. Precisely, levels of tissue damage markers, such as AST, ALT, bilirubin, fibrinogen, and creatinine, and the pro-inflammatory markers IL-6 and IFN-γ. Additionally, the ‘global’ systemic inflammation, measured by ssMDP levels, was higher in polysymptomatic participants. Our observations reported here suggest that older individuals who lived for long time in malaria-endemic area and had been exposed to several sickness ,51,52.P. vivax infection. Using data on parasitemia, ssMDP values, and the number of symptoms, we were able to evaluate the interplay between the main factors associated with unfavorable outcomes in an infectious disease: (i) pathogen load; (ii) host response to infection and (iii) clinical consequences of this interaction. Our analysis reveals that the main factor associated with severe cases in P. vivax infection was the number of symptoms. Of note, these participants exhibited relatively lower parasitemia and ssMDP values, arguing that some persons present severe vivax cases regardless of pathogen burden and inflammatory activation, being little tolerant to infection and showing higher susceptibility to homeostasis disruption and consequently more symptoms. Furthermore, some persons with a low number of symptoms had high parasitemia and high ssMDP values, showing that even in a smaller number, there may be symptoms directly related to molecular perturbation. Thus, the analyses suggest that there is a disruption between parasitemia, MDP and the clinical presentation of the disease. In converse, we identified a subgroup of individuals who presented with higher parasitemia and augmented ssMDP values but lower symptomatology, with reduced number of severe malaria cases. This latter group of individuals may be activating the immune response to counteract the parasite load, but without resulting in substantial tissue damage and symptomatology. We also found another group of P. vivax-infected study participants who seemed to have controlled infection without inducing substantial inflammatory disturbance, and presented with low parasitemia, lower ssMDP values and few symptoms. Importantly, we found that persons with severe vivax malaria lost the effective control of parasitemia resulting in tissue damage and symptoms, exemplified here by absence of a statistically significant correlation between parasitemia and number of symptoms.Finally, our study tried to dissect the determinants of disease tolerance to P. vivax infection. To do that, we employed networks inputting correlations between degree of molecular perturbation of tissue damage or inflammatory markers with parasitemia and number of symptoms. We described that higher parasitemia in severe vivax malaria was associated with a more robust tissue response, and that the degree of symptomatology was directly related to degree of perturbation in cytokine concentrations. Such scenario may significantly contribute to tissue damage through systemic inflammatory activation and also tissue inflammation, reinforcing our previous results reported above. Nevertheless, in non-severe cases, parasitemia was not associated with MDP of tissue damage markers and the number of symptoms were more linked to systemic inflammatory activation. This latter result reinforces the hypothesis that some patients manage to restrict P. vivax infection in a balanced fashion, despite presenting some symptoms but without substantial tissue damage. This ‘tolerant state’ may be associated with epidemiologic factors such as those reported here, confirming previously published results [Our final results disclosed the determinants of diseases tolerance in  results .P. vivax malaria and by identifying the major determinants of this phenomenon.Our study has some limitations. We did not have measurements of inflammatory markers after the malaria therapy, limiting our insights about the systemic inflammation and immune restoration in our participants. Additionally, all our study participants were recruited in Brazil, and additional studies in multiple sites/endemic areas are needed to confirm our findings. Despite these limitations, our study adds to the current knowledge in the field by demonstrating the occurrence of disease tolerance in S1 File(XLSX)Click here for additional data file.S1 FigAdjusted multinomial logistic regression analysis was performed with symptomatic malaria as the primary outcome. The model was composed with variables that were statistically significant (p < 0.05) in univariate comparisons (see univariate comparisons in (TIF)Click here for additional data file.S2 FigA Spearman correlation analysis was employed to identify the perturbation of each individual marker that contribute to changes in the systemic inflammatory imbalance, assessed by ssMDP score values, in each group as indicated. Colored bars infer the correlation with P-value<0.05 after adjustment for multiple comparisons. Abbreviations : ALT: alanine aminotransferase; AST: aspartate aminotransferase; CCL: C-C motif chemokine ligand; CXCL: C-X-C motif chemokine ligand; CRP: C-reactive protein; IFN: interferon; IL: interleukin; TNF: tumor necrosis factor.(TIF)Click here for additional data file.S3 FigHierarchical cluster analysis of Log-10 transformed and z-score normalized using Ward’s method with 100X bootstrap was employed to depict the overall perturbation of inflammatory and biochemical markers in study population. The participants were grouped based on age (A) and number of previous Malaria episodes (B).(TIF)Click here for additional data file.


Plasmodium malaria  and hepatitis E virus (HEV) is a rare phenomenon.There is a high prevalence of malaria and viral hepatitis in South Africa. Co-infection with Plasmodium parasite burden of 33% on the blood smear. Interestingly, he also had a positive result for hepatitis E IgM. The patient was effectively treated with aggressive hydration and intravenous (IV) artesunate.A 33-year-old African American male with no past medical history developed altered mental status on his return from Ivory Coast. His blood tests were significant for renal and liver failure and a high Our report is the first to our knowledge in the cerebral malaria literature on a patient with hepatitis E co-infection. This exciting case emphasizes the importance of considering all kinds of endemic infectious diseases when evaluating sick returning travelers presenting to the emergency department. Plasmodium malaria, a mosquito-borne parasitic disease, has been known to humankind since early times. It presents mainly as high-grade fevers, chills, nausea, gastrointestinal symptoms, and anemia. Its high morbidity and mortality are significant health concerns for third world countries even in the current times of advanced treatment and prophylactic options.Plasmodium falciparum infection. Cerebral malaria presents mainly as a neurological sequela secondary to microvascular inflammation from sequestered blood cells, interaction between adhesive proteins and endothelial surfaces, generation of cytokines, and disruption of the blood-brain barrier [Cerebral malaria is one of the deadliest manifestations of  barrier , 2. AlteThe prevalence and severity of cerebral malaria are higher in children with life-long neurological complications . PediatrIn adults, multiorgan failure usually accompanies the state of altered mental status and coma. Furthermore, neurological deficits are generally long lasting in adults. The treatment of choice for cerebral malaria is intravenous artesunate, including the pediatric and pregnant subgroups .Plasmodium and HEV have been reported with an abysmal prognosis. For interest and awareness, we report an unusual case of a co-infection of hepatitis E in a patient who developed cerebral malaria from Plasmodium falciparum infection [Sub-Saharan Africa is the hub of several infections due to the low socioeconomic status, lack of hygiene and sanitation measures, and inadequate health resources. Hence, both the incidence and prevalence of malaria, human immunodeficiency virus (HIV), and hepatitis are exceedingly high in this region, accounting for the high regional morbidity and mortality. Recent studies have been done to document and understand the co-infection trends of hepatitis B, hepatitis C, and HIV in the Ivory Coast . Interesnfection .A 33-year-old African American man with no past medical history was evaluated in the emergency room (ER) 3 weeks after returning from Ivory Coast. He was born in Ivory Coast and came to the USA as a toddler. Since then, he had been visiting Abidjan, Ivory Coast, every few years to see family. Recently, he spent 1 uneventful month in Abidjan and returned to the USA. He denied any history of malaria in the past. He did not take any anti-malarial prophylaxis during the recent visit. He confessed to smoking a few tobacco cigarettes daily but denied any illicit drug abuse. He developed a fever after 7 days. The fever was high grade (103 °F) in intensity and intermittent, with each episode lasting several hours. The patient took multiple tablets of over-the-counter Tylenol and Motrin for the fever at home without any significant improvement.The patient had visited the ER twice in the last week for a similar complaint of low-grade fevers. Thrombocytopenia and transaminitis were seen on blood testing. However, given the COVID-19 pandemic, recent international travel, and blood testing results bearing similarity to COVID-19, he was promptly tested for COVID-19. He was discharged home after the COVID-19 polymerase chain reaction (PCR) test result was negative. The fevers persisted at home for the next 3 days, nonetheless. Two days before the presentation, he developed chills, nausea, and vomiting. The vomiting was initially bilious and later became dark-colored. The patient's family noticed that his mental status had gradually worsened over the past week, and they decided to bring him to the ER again.On this visit to the ER, he had an initial temperature of 101.4 °F, blood pressure of 122/78 mmHg, respiratory rate of 18 breaths per minute, and oxygen saturation of 98%. Physical examination revealed scleral icterus. The patient was oriented only to himself with a Glasgow coma scale (GCS) score of 13 out of 15. The physical examination did not reveal any hepatosplenomegaly, flapping tremors, or focal neurological deficits.The initial blood testing was significant for hypoglycemia, pancytopenia (microcytic anemia and thrombocytopenia), elevated lactate level, and transaminitis  . A lumbar puncture was recommended to rule out cerebral malaria as the patient was confused. The blood alcohol, acetaminophen, and salicylate levels were normal. Hepatitis E IgM was positive, and the rest of the acute hepatitis panel was negative , Cryptococcus neoforms, herpes simplex virus , human herpesvirus 6 enterovirus, parechovirus and varicella zoster virus (VZV) came out negative . The patient was started on intravenous artesunate . A dramatic improvement was seen in his blood work and mental status from hospital day 3. He was fully alert, awake, and oriented to himself, time, and place by hospital day 5. Parasitemia was also entirely resolved by hospital day 5 (0.02%). The liver enzymes trended down, and the platelet count was back to normal  after approximately 3 weeks. The CSF PCR was positive for Plasmodium falciparum, which established a cerebral malaria diagnosis. There was no evidence of cerebral edema, mass effect, or midline shift on CT head. The fundoscopic examination was unremarkable. The response to artesunate was rapid with prompt resolution of parasitemia on the peripheral smear. The only limitation in this study was the inability to repeat hepatitis E IgG analysis on outpatient blood testing as it would have left no stone unturned in confirming the diagnosis of acute HEV based on the projected timeline. Unfortunately, we were limited in our testing by financial constraints of the patient.Similarly, the incubation period of Anopheles mosquito transmits Plasmodium into human blood, which reaches the liver, undergoes asexual reproduction, and completes development prior to infecting red blood cells and multiplying [The two infections  have different vectors and different mechanisms of causing harm to human hosts. HEV spreads through the fecal-oral route. The female tiplying . The conThe initial clinical presentation of altered mental status can be very misleading for the physician and unfortunate for the patient. Fever, thrombocytopenia, and abnormal liver enzymes paint a clinical picture remarkably familiar to COVID-19 in the current circumstances heightened by recent travel . Delay iWhile reviewing the literature, a study done in Afghanistan demonstrated a high incidence of hepatitis A and E and malaria due to a lack of health resources and sanitation measures . Barcus Furthermore, co-infection with HEV and malaria has been reported with fatal consequences , 23. So Plasmodium malaria affects the hepatic microvasculature by endothelial injury from the parasitized erythrocytes. A case of sporadic HEV in a patient with a history of malaria has been reported in the USA before, and physicians should be reminded of this association in relevant clinical settings even in first world countries [Potential synergistic hepatotoxicity was suggested by Nasir et al. when they studied hepatitis C and malaria co-infection . Analogoountries . Cliniciountries , 26.Malaria and viral hepatitis are indeed not the first thought in a physician's mind when a patient comes in with vague complaints in an emergency room in a first world country. Particularly in the current circumstances, the threat of COVID-19 infection is real and is one of the top differential diagnoses in travelers. The liver enzyme levels in these patients are usually elevated, and suspicious personnel is managed conservatively with quarantine even if they test negative for COVID-19. COVID-19 should not hinder the thought process from ruling out other infectious etiologies. The suspicion of malaria and hepatitis should be high in patients with a recent travel history to endemic areas, particularly South Africa and South Asia. There is an exceedingly high mortality rate for malaria, particularly cerebral malaria, and efforts should be made to achieve prompt diagnosis and treatment. A detailed history should be taken from all available sources, and relevant blood testing should be ordered. Transaminitis and hyperbilirubinemia should raise the suspicion of potential endemic infections, such as malaria and viral hepatitis . We prop


Mild elevation of serum amino-terminal pro-B-type natriuretic peptide (NT-pro-BNP) is associated with myocardial dysfunction. A significantly lower Smith–Madigan inotropic index (SMII) has been shown to accurately represent cardiac contractility among heart failure subjects. We aim to monitor the effect of fluid resuscitation on cardiac function among paediatric patients by measuring serum NT-pro-BNP and SMII.  This is an observational study on 70 paediatric shock patients. NT-pro-BNP and noninvasive bedside haemodynamic monitoring were done by using an ultrasonic cardiac output monitor . The presence of cardiac diseases was excluded. SMII was obtained from the USCOM. An increase in the stroke volume index (SVI) of ≥15% indicates fluid responders. Measurements were taken before and after fluid loading. p=0.001 and p=0.004, respectively. Higher median NT-pro-BNP (preloading NT-pro-BNP of 1175.00 (254.50–9965.00) ng/mL vs. 196.00 (65.00–509.00) ng/mL, p=0.002) was associated with fluid nonresponders (subjects >12 months old). Preloading NT-pro-BNP <242.5 ng/mL was associated with fluid responders (AUC: 0.768 (0.615–0.921), p=0.003), 82.1% sensitivity, and 68.7% specificity for subjects >12 years old. Delta NT-pro-BNP in fluid responders (15.00 (−16.00–950.00) ng/mL) did not differ from fluid nonresponders (505.00 (−797.00–1600.00) ng/mL), p=0.456. Postloading SMII >1.25 W·m−2 was associated with fluid responders (AUC: 0.683 (0.553–0.813), p = 0.011), 61.9% sensitivity, and 66.7% specificity, but not preloading SMII. Fluid responders had a higher mean postloading SMII compared to nonresponders .  Preloading NT-pro-BNP and SMII category were significantly different between the fluid responsiveness group,  Higher NT-pro-BNP and lower SMII in the absence of cardiac diseases were associated with poor response to fluid loading. The SMII is affected by low preload conditions. Nowadays, paediatric resuscitation guideline recommends the use of haemodynamic monitoring to guide fluid resuscitation . Most haSerum level of amino-terminal pro-B-type natriuretic peptide (NT-pro-BNP) is found to increase in structural cardiac diseases, as well as cardiac failure , 4. Its The ultrasonic cardiac output monitor (USCOM) is a noninvasive, bedside haemodynamic monitoring device that has been widely used to study haemodynamics in adults and paediatric patients , 8. Its The use of serum NT-pro-BNP and USCOM during fluid resuscitation has never been investigated. This study aims to assess the value of NT-pro-BNP and SMII in assessing myocardial response to fluid loading in paediatric shock patients.We performed a prospective observational study at Pasar Rebo Regional Referral Hospital, a secondary general hospital in Jakarta, Indonesia, from March 2020 to March 2021. All data were recorded following written informed consent from parents or guardians. Ethical approval was obtained from the Ethics Committee of Faculty of Medicine, Universitas Indonesia .®; USCOM Ltd., Sydney, Australia), and peripheral venous blood was collected for serum NT-pro-BNP and other laboratory examinations.We enrolled paediatric patients older than 1 month old, indicated for fluid resuscitation due to the presence of hypotension and/or signs of impaired perfusion  , 10. SubResearcher team who collected the sample and recorded the data did not make any clinical decision for subject's treatment. However, attending physicians were not blinded to the USCOM parameters measured and were able to use it for clinical purposes. Following fluid resuscitation (subjects no longer received fluid loading), vital signs and haemodynamic assessment were repeated by the respective attending clinician. One hour after fluid resuscitation ends, blood samples were collected for serum NT-pro-BNP measurements, during which subjects only received maintenance infusion. The flow of the study is presented in Mean arterial pressure (MAP) was defined as (systolic blood pressure (BP) + 2 × diastolic BP)/3 and further categorized into percentiles . Patient®; USCOM Ltd., Sydney, Australia) by trained professionals and measured three times at the aorta. We did not perform central venous pressure (CVP) measurement, and values were assumed to be 0. The inotropic index was expressed as the Smith–Madigan inotropic index (SMII) [× 100%. SMII was further classified “low” percentage of SMII < −35% [× 100%. Fluid responsiveness was defined as delta SVI percentage ≥+15%. Left ventricular end-diastolic volume index (LVEDVI) was calculated using inotropy 2009 software .The haemodynamic assessment was performed using the USCOM (USCOMx (SMII) . “PercenI < −35% , otherwiSerum NT-pro-BNP was quantified using the electrochemiluminescence immunoassay (ECLIA) method and reagent pro-BNP II CalSet  on an Elecsys Modular E170 platform , performed at a local commercial laboratory partner, Prodia Clinical Laboratory . Serum NT-pro-BNP values were categorized as “high” if they exceed the upper limit of the normal range according to the age group . “Delta T-test. Bivariate correlation analysis was performed using Spearman's correlation, while the multivariate linear model was used for multivariate analysis. The area under the curve (AUC) was obtained by constructing a receiver operating characteristic (ROC) curve, and optimum cutoff was identified using the intersection point by plotting sensitivity against specificity.Data were computed into Excel and analysed using SPSS IBM version 24.0 . Variable frequency was expressed as frequency and percentage. Shapiro–Wilk test was used to assess data distribution, in which nonparametric data were expressed as median . Statistical significance was set at 5%. Chi-square, Fisher's exact test, and independent Kruskal–Wallis test were used to analyse categorical data association. Between-group comparison of numerical data was conducted using Mann–Whitney U or unpaired p=0.026); among all subjects, 15 (22.06%) were diagnosed as septic shock, 29 (42.65%) were diagnosed as dengue shock, 24 (35.29%) were diagnosed as other types of shock , and none were diagnosed as anaphylactic or neurologic shock. Normal preloading serum NT-pro-BNP was associated with fluid responsiveness (p=0.001), while a low preloading SMII was associated with fluid responsiveness (p=0.004)  .p=0.005), a specificity of 75%, and a sensitivity of 65.4%. Excluding subjects under 12 months old, we obtain an optimum cutoff of NT-pro-BNP of <242.5 ng/mL to discriminate fluid responders with an AUC of 0.768 , a sensitivity of 82.1%, and a specificity of 68.7% , with a significant negative correlation between percentage preloading SMII from the lower limit and percentage of delta SVI ((postloading − preloading SVI)/preloading SVI × 100%) . On the contrary, despite the nonsignificant correlation, percentage of postloading SMII from the lower limit had a positive correlation coefficient with a percentage of delta SVI . We observed that delta SMII percentage had a moderate positive correlation with delta SVI percentage  (A low preloading SMII was associated with fluid responsiveness (≤ 0.001) .p=0.001). However, postloading SMII was able to discriminate fluid responders  with an optimum cutoff of >1.25, sensitivity of 61.9%, and specificity of 66.7%  transformation of preloading NT-pro-BNP and used ln(242.5) = 5.49 as a y-axis reference line. There was no significant correlation between preloading NT-pro-BNP and postloading SMII in subjects aged >12 months old. Under categorical analysis, the postloading SMII category was not associated with the preloading NT-pro-BNP classification (p=0.596).Since NT-pro-BNP values vary greatly, we performed logp ≤ 0.001), median preloading SVI (p=0.001), and lower mean preloading SMII (p ≤ 0.001). However, using postloading values, only higher SMII was associated with fluid responders (p=0.006) (Fluid responders were associated with a lower median preloading CI (p=0.006) .Since preloading SMII had a significant correlation with preloading SVI, we included the percentage of preloading SVI into the multilinear regression. None of the variables were significantly associated with delta SVI percentage, apart from preloading SVI (percentage from the lower limit) . Upon coNT-pro-BNP has been widely used to diagnose congestive heart failure in children . SMII haSimilar to previous findings, we also found a negative correlation between NT-pro-BNP and age . This caAn interesting observation in this study was that the lower NT-pro-BNP value was consistently associated with fluid responders among subjects >12 months old. NT-pro-BNP is associated with not only HF of cardiac structural origin but also other conditions such as sepsis and fever , 16. FurAnother novel observation in this study was the value of postloading SMII as an inotropic index in discriminating fluid responders among shock subjects. Preloading SMII could not be used as we found that SMII is preload dependent under hypovolemic conditions. This was proven as lower preloading SMII percentage had a strong positive correlation with preloading SVI percentage, and delta SMII percentage had a strong positive correlation with delta SVI percentage. We think that this is due to contractility measured by the SMII which was derived from the SVI measured at the aortic notch via the Doppler ultrasound velocity-time integral. Postloading SMII with a cutoff of >1.25 was able to discriminate fluid responders. This cutoff is lower than the normal SMII range for age 3 months old and older according to the manufacturer's reference range. Furthermore, we showed that postloading SMII was significantly different between fluid responders and nonresponders compared to other markers such as postloading CI . This imDespite both preloading NT-pro-BNP and postloading SMII being able to discriminate fluid responders, we found that preloading NT-pro-BNP was not associated with postloading SMII. On the Frank–Starling curve, both preload and inotropy affect the curve independently . Based oUpon multiple linear regression, we identified that the preloading SVI percentage (percentage of the lower limit − preloading SVI) was the only significant factor in determining the positive response to fluid resuscitation. This is especially true as a lower preloading SVI would yield a higher percentage of SVI increase upon loading. We also found a moderate positive linear correlation between preloading SVI and preloading SMII even after adjusting for other variables. This further supports the finding of SMII calculation to be affected by low preload status.In summary, this study found that NT-pro-BNP was able to identify fluid responders among paediatric shock patients. However, since it is a serum biomarker, its use in aiding clinical decisions is limited. Using bedside haemodynamic monitoring, we also found that the inotropic index, SMII, could not be used to predict a positive response to fluid resuscitation since it is affected by the SVI. SMII was not useful in identifying potential fluid responders. However, we found that the SMII can potentially be used for further study revolving around inotropy (cardiac contractility) as it may be a good representative of the inotropic index exclusive of hypovolemia. There were also some limitations to this study. Firstly, the small sample size was only able to determine the association between categorical variables. A bigger sample size is needed to obtain a more accurate cutoff for NT-pro-BNP and SMII across age categories. Secondly, USCOM was performed by a team of trained paediatricians; however, interoperator variability was still unavoidable. Thirdly, we did not adjust for the subjects' acute clinical conditions that might have affected NT-pro-BNP values. A previous study demonstrated that acute, noncardiac diseases and sepsis were linked to increased NT-pro-BNP, albeit much lower than the cutoff for HF; this might affect the cutoff obtained in the study due to heterogeneity of shock , 16. LasOur study was the first to demonstrate the use of NT-pro-BNP and SMII in monitoring response to fluid resuscitation in the paediatric population in the absence of cardiac diseases. We found that elevated NT-pro-BNP was associated with a poor response to fluid loading. We also show that a higher SMII value, as a measure of cardiac inotropy, was associated with a desirable outcome to fluid resuscitation; however, its value during low preload condition may be falsely low.


Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. This study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia.A health facility-based, cross-sectional survey was conducted in selected malaria sentinel sites. Malaria-suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood spot (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at Ethiopian Public Health Institute (EPHI) malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other.Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI (93–100)) and 83.2% (95% CI (77.6–87.9)), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI 46.9–68.4) and 67% (95% CI 56.2–76.7); and 100% (95% CI 98–100) and 98.9% (95% CI 96–99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65.Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as Multiplex real-time PCR had an advanced performance in parasite detection and species identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination programme, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities. Plasmodium and transmitted by the bite of infected female Anopheles mosquitoes. Currently, there are five species of the genus Plasmodium known to cause human malaria: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi [Malaria is a life-threatening disease caused by the protozoan parasite genus knowlesi . Despiteknowlesi . In 2017knowlesi .Plasmodium falciparum is the predominant parasite (65%) that is known to cause the most serious infection than other species, followed by P. vivax (34%). Malaria transmission peaks during the harvesting season which poses a serious impact on the country’s socio-economic development [Malaria has been the major cause of illness and death for thousands of peoples for several years in Ethiopia; this is mainly due to variable climatic changes such as altitude and rainfall which favour the proliferation of mosquitoes . Around parasite 5% that iPlasmodium-specific protein antigen targets [P. falciparum infection; they also require no electricity source and minimum training to perform the test, compared to microscopy. Reader bias when reading result bands and/or false negative results during hyperparasitaemia due to antigen prozone effects are limitations of RDT tests. Moreover, during mixed and low parasitaemia both microscopy and RDTs have shown reduced performance for detection of Plasmodium infection [Light microscopy using Giemsa-stained blood film is a primary malaria diagnostic tool and considered as gold standard for malaria parasite identification and confirmation at hospitals and health centres all over Ethiopia. In malaria-endemic rural areas rapid diagnostic tests (RDTs) are the malaria diagnostic tool . Histidi targets . RDTs hanfection , 10.Since 2005, malaria morbidity and mortality in Ethiopia has declined due to the implementation of malaria prevention and control programmes . EncouraPlasmodium parasites within communities, which demands early and accurate diagnosis followed by prompt treatment and case management of patients [To achieve the elimination of malaria requires the halting of every possible transmission of patients . The imppatients .Plasmodium species in low transmission settings, which can lead to an underestimation of infection prevalence. Application of malaria RDT testing in the malaria elimination programme is threatened due to the emergence of P. falciparum pfhrp2 and pfhrp3 gene deletion [The currently employed malaria diagnostic approaches throughout Ethiopia, microscopy and RDT, have poor sensitivity for detection of deletion . To guiddeletion , 14.Plasmodium species. These molecular tests have been developed based on real-time quantitative PCR, with qualities of quantitative and closed systems that reduce time, labour, reagents, cost, and risk of contamination compared to conventional PCR [Plasmodium infections and detection of Plasmodium species in low parasitaemia cases, with the detection limit of fewer than 2 parasites per ml and has an advantage of simultaneous detection of multiple targets in a single run to increase sensitivity and specificity of the test, compared to microscopy, RDTs and conventional PCR [Nowadays, different molecular tests are in use for the detection of onal PCR . Multiplonal PCR , 18.The study was conducted in two malaria sentinel sites in Ethiopia: Shewa Robit health centre in the northeast and Metehara health centre in eastern Ethiopia. The healthcare facilities are located in the targeted malaria elimination areas. Ethiopian Public Health Institute (EPHI) in collaboration with Federal Ministry of Health established 25 malaria sentinel surveillance sites, representing regional malaria cover in Ethiopia. These sites are eco-epidemiologically representative and focal areas of malaria infection.A facility-based, cross-sectional study was conducted by collecting data from malaria-suspected febrile outpatients referred for malaria testing to the laboratory within the period December, 2019 to March, 2020.All malaria-suspected febrile patients of any age referred to the laboratory for malaria testing were included in this study. Patients who had anti-malarial therapy in the 4 weeks before sample collection, and any critically ill patients were excluded from the study.Sample size was calculated using Buderer’s formula  as follo1−α/2  = 1.96, SN (anticipated sensitivity) = 0.9, Prevalence = 13%, L (absolute precision desired on either side of sensitivity) = 0.1. Because no previous malaria prevalence studies using multiplex real time PCR were found, an estimated, intellectual guess was made of malaria prevalence of 13% and anticipated sensitivity of 90% (95% CI 80–100%) for P. falciparum, compared to conventional PCR. A total of 271 participants were enrolled into this study.ZConvenient sampling technique was used on a consecutive basis to recruit study subjects referred to the laboratory for malaria testing by attending clinical staff, according to the usual standard of care, within the period December 2019 to March 2020.After obtaining patient consent, demographic profiles and clinical data were collected using a structured questionnaire. Using capillary blood taken from each patient, thick and thin smears were prepared for microscopy. RDT testing was performed at spot and five circles of dried blood spot (DBS) were collected on Whatman filter paper and transported by cold chain to the EPHI laboratory for molecular tests.RDT testing was performed as per manufacturer instruction using CareStart™ malaria HRP2/pLDH combo test. This test detects HRP-2 proteins specific to falciparum and pLDH. The tests were performed in the field laboratory by the health centres’ laboratory personnel as a routine malaria testing.After preparation of thick and thin blood films, slides were allowed to air-dry at room temperature, and the thin smear fixed by using absolute methanol then stored at 2–8 °C until being transported to EPHI. In EPHI parasitology laboratory, slides were stained with 10% Giemsa solution for 10 min, after being air dried both thick and thin smear were examined by an experienced laboratory technologist. An expert microscopist re-checked all positive slides, and 10% of negative slides. According to WHO malaria microscopy standard operating procedure, at least 100 high power fields (HPFs) were examined for parasite detection.® 96 DNA Blood Kit (QIAGEN Inc.) from DBS sample. Briefly, three 3-mm circles of the DBS punched out and placed into a 1.5-ml tube for processing as per manufacturer instructions. Finally, with 100 μl volume of elution buffer the DNA was eluted and stored at − 20 °C until assayed.Genomic DNA extraction was performed using QiagenQIAampPlasmodium-specific and P. falciparum-specific primers and the second were by multiplexing P. falciparum- and P. vivax-specific primers. Briefly, each reaction mixture was prepared by mixing 2 µl of purified DNA template, 5 µl Luna Universal Probe qPCR Master Mix , 2 µl PlasQ Primer Mix and 1 µl molecular biology grade water with a final reaction mixture volume of 10 µl. Amplifications were carried out using thermal cycling conditions: for the first PCR run 95 °C for 1 min, followed by 45 cycles of 95 °C for 15 s and 57 °C for 45 s and for the second run 95 °C for 1 min, followed by 45 cycles of 95 °C for 15 s and 53 °C for 45 s. The 3D7 DNA standard was run in each experiment and used as a positive control and nuclease free water used as a negative control. For PCR run, the positive control has 25 to 30 Ct values and all samples have Ct values < 30.0 for HsRNaseP taken as qualified run. Samples with Ct values between 12 and 40 and sigmoidal shape amplification curve were considered as positive . Official letter was written to sentinel sites. The confidentiality of patient-related data was maintained by avoiding possible identifiers, such as name of patient. Throughout the whole process of data collection and research work, all data were kept safe.From a total of 271 study participants, more than half of them were females (54.6%). The mean age was 24.12 years (± 14.83 SD), with 4 months minimum age and 90 years maximum age  had chills and headaches. Sweating and muscle pain was diagnosed in 207 (76.38%) and 199 (73.43%), respectively.From the total 271 study participants, 26.2% (71) were enrolled from Shewa Robit and the remaining 73.8% (200) from Metahara health centre. Among 271 study participants, malaria-positive cases by microscopy, RDT and multiplex real-time PCR were 51 (19%), 61 (22.5%) and 88 (32.5%), respectively. Comparing the three methods, the positivity rate was highest for multiplex real time-PCR. The positivity rate by all three methods was increased among the age group 16–25 years old, and in male participants , specificity of 83.2% (95% CI 77.57–87.87) and RDT sensitivity of 98.9% (95% CI 96.1–99.87) and specificity of 95% (95% CI 91.23–97.5), respectively Table . Using mon Table .Table 4PP. falciparum when microscopy was used as a reference test. RDT showed sensitivity and specificity of 100% and 95.38% for P. falciparum identification when microscopy was used as a reference test (Table P. vivax (16) identified by the three methods were not statistically sufficient to compute performance, and were omitted from the analysis.Multiplex real-time PCR had shown sensitivity and specificity of 100% and 83.61% for the identification of st Table . The numAll samples that tested positive by microscopy were positive by multiplex real-time PCR. Additionally, 37 samples, that had missed microscopy testing, tested positive by multiplex-real time PCR. Except for two samples, all RDT-positive samples were also positive by multiplex real-time PCR and multiplex real-time PCR detected 29 samples missed by RDT.P. falciparum and P. vivax) were detected by multiplex real-time PCR, one by RDT, none by microscopy. There was little difference among the three methods in detecting P. vivax . However, significantly more P. falciparum cases were detected by multiplex real-time PCR than RDT and microscopy .Three mixed infections  was reported between microscopy and multiplex real-time PCR with Kappa value of 0.65. There was an observed agreement of 88.56 (Kappa: 0.72) between RDT and multiplex real-time PCR; almost perfect agreement was reported between microscopy and RDT test results   and Moges et al. (92.9%) using similar RDT format [Although identifying malaria to species level has a crucial impact on patient management and transmission interruption, RDT testing is incapable of detecting non-falciparum malaria . In the T format , 32.. [Among the total febrile study participants in this study, a larger proportion of positive cases were identified by multiplex real-time PCR than by RDT and microscopy. However, the study was conducted in non-endemic settings comparing the three methods, microscopy and RDT missed fewer cases, reported by Nijhuis et al. . This miPlasmodium infection could be ruled out with high certainty if individuals test negative using multiplex real-time PCR. This quality of PCR makes it an ideal diagnostic tool to be used in malaria elimination, rather than RDT and microscopy which were found to have high positive predictive value and low negative predictive value, in the current study. RDT and microscopy increases the probability of missing Plasmodium infection, which has negative impact on transmission interruption in elimination settings, and potentially to contribute to ongoing transmission [The negative predictive value of multiplex real-time PCR was found to be 100% using microscopy as a reference test. This means that the multiplex real-time PCR is good in ruling out malaria. smission .P. falciparum infection, 16 as P. vivax and three as mixed infections from a total of 271 symptomatic malaria-suspected patients in the current study. Of these malaria cases, large numbers of P. falciparum were missed by both RDT and microscopy testing. However, all three methods showed perfect agreement in P. vivax species identification. This is probably due to in the case of P. vivax infection all developmental stages of parasites are found in the peripheral blood circulation that increases parasitic density in symptomatic patients compared to P. falciparum infection in which the parasite causes cyto-adherence and sequestration of infected erythrocytes which result in reduced parasite densities from peripheral blood [P. falciparum (100%) identification, which is closely related to the finding of a study in Bangladesh on clinically suspected-malaria patients where the sensitivity of real-time PCR for P. falciparum using microscopy as a gold standard was 97.1% [Multiplex real-time PCR identified 69 malaria cases as al blood . Using mas 97.1% .P. falciparum infection was detected by multiplex real-time PCR than microscopy and RDT methods. This might be explained as follows: in this study varATS Plasmodium gene was used for P. falciparum detection which is a more highly sensitive PCR primer for malaria than 18S rRNA-based PCR, which was also used in the current study for the detection of P. vivax [Substantial numbers of P. vivax . AnotherP. vivax .Plasmodium infection among febrile patients. This assay can be used for epidemiological and community-based prevalence studies and for verification of elimination in areas where malaria elimination is launched. However, microscopy and RDT still have an acceptable performance to be used as a malaria diagnostic tool in health facilities for patient treatment due their affordability and easy performance diagnostic methods.Multiplex real-time PCR is the most sensitive malaria diagnostic method that can be used in malaria elimination programmes. It had a more advanced performance in species identification and mixed infection detection than microscopy and RDT in low malaria transmission settings and showed better performance in detection of


Plasmodium falciparum malaria RDT  with the cRDT conducted in the same field conditions.To overcome the limitations of conventional malaria rapid diagnostic tests (cRDTs) in diagnosing malaria in patients with low parasitaemia, ultrasensitive malaria rapid diagnostic tests (uRDTs) have recently been developed, with promising results under laboratory conditions. The current study is the first meta-analysis comparing the overall sensitivity, and specificity of newly developed ultrasensitive th April 2021. Studies with enough data to compute sensitivity and specificity of uRDT and cRDT were retrieved. A random-effect model for meta-analysis was used to obtain the pooled sensitivity and specificity.PubMed, EMBASE, Cochrane infectious diseases group specialized register, and African Journals Online (AJOL) were searched up to 20P. falciparum RDT regardless of the reference test and the clinical presentation of participants, was 55.5% , while the sensitivity regardless of the reference test and the clinical presentation of participants, was 42.9%  for the cRDT performed in the same field conditions. When PCR was used as reference test, the sensitivity of uRDT was 60.4% , while the sensitivity was 49.4%  for the cRDT. The pooled specificity of uRDT regardless of the reference test and the clinical presentation of participants was 98.6% , and the pooled specificity of cRDT regardless of the reference test and the clinical presentation of participants was 99.3% . When PCR was used as reference test the specificity of uRDT and cRDT was 97.5%  and 98.2% . Regardless of the reference test used, the sensitivity of Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT in symptomatic patients was 72.1% , while sensitivity of cRDT was 67.4% .Overall, 15 data sets from 14 studies were included in the meta-analysis. The overall sensitivity of the Alere™ ultra-sensitive Malaria Ag P. falciparum RDT compared to cRDT performed in the same field conditions has higher sensitivity but lower specificity although the difference is not statistically significant.Findings of the meta-analysis show that Alere™ Ultra-sensitive Malaria Ag The online version contains supplementary material available at 10.1186/s12936-021-03783-2. Even though the global burden of malaria has been reduced since 2000, in 2018, nearly 228 million new cases of malaria were recorded globally, and there were close to 405.000 excess death caused by malaria  women rePlasmodium falciparum to bind to the placenta, which can lead to parasite densities in peripheral blood below the detection threshold of the most used cRDTs and light microscopy, hence the need for ultrasensitive diagnostic tests (uRDTs)  OR "highly sensitive" [tiab] OR "hypersensitive"[tiab] OR "high-sensitive"[tiab] OR "high sensitive"[tiab] OR "RDT"[tiab]). The search strategy used in PubMed which was adapted to fit with other databases is presented in Additional file After bibliographic search, the titles and abstracts were screened for eligibility and duplicates were removed. Full texts of potentially eligible articles were retrieved and assessed for final inclusion independently by two reviewers, with discrepancy between both resolved by discussion.Both observational and experimental studies reporting enough data to compute sensitivity and specificity of uRDT and cRDT in the same setting and comparing both with the same reference test were included in the meta-analysis. Only studies conducted on the field (not in the laboratory), regardless of the language and year of publication were retained. We excluded editorials, reviews, letters, commentaries, and studies lacking key data.The Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) was used independently by two reviewers to assess the quality of included studies . DisagreThe following information was retrieved on a preconceived data extraction form by one reviewer: the name of the first author, the country where the study was conducted, the year of publication, the characteristics of the study population in terms of symptoms and age, the presence of pregnant women in the sample, the commercial name of the uRDT and reference test used in the study, the storage condition of the uRDT, the number of true positive (TP), true negative (TN), false positive (TP), false negative (FN).Articles reporting on diagnostic performance of uRDT in different age categories  or in which uRDT was compared with more than one reference test were divided in separated data sets. Thereafter, a second reviewer checked the concordance between data extracted and the content of the article prior to the data synthesis and analysis.The “meta” package within R software version 4.0.2 was used for analysis , 13. A rThe bibliographic search yielded 1440 articles. The screening based on title and abstracts and full text allowed to retain 15 data sets from 14 studies for the quantitative synthesis as depicted in the Prisma flow diagram . The reference test was mainly PCR (10 data sets), and ultrasensitive PCR (4 data sets) (Table Eight studies 57.1%) were conducted in patients regardless of the presence of symptoms, while two (14.3%) were conducted in symptomatic patients and four 28.6%) in asymptomatic patients. The mean/median age of the study population ranged from 4 to 36 years as reported by seven studies. Four (28.6%) studies were conducted in pregnant women  while the figure was 42.9%  for the cRDT  while it was 49.4%  for the cRDT. When the ultra-sensitive PCR was used as reference test, sensitivity of uRDT was 60.3%  and of cRDT was 44.1%   and 44.9%  respectively. Regardless of the reference test used, the sensitivity of Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT in symptomatic patients was 72.1% , while sensitivity of cRDT was 67.4% . In asymptomatic patients the sensitivity of Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT was 42.1% , and sensitivity of cRDT was 26.0% .The sensitivity of Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT was lower than the cRDT . The pooled specificity of Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT was 98.6% , and of cRDT was 99.3% . When PCR was used as reference test the specificity of uRDT and cRDT was 97.5%  and 98.2%  respectively, and when the ultra-sensitive PCR was used as reference test the specificity was 99.5%  and 99.8% , respectively  and 98.7% , respectively. Regardless of the reference test used, the specificity of Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT in symptomatic patients was 99.5% , and specificity of cRDT was 99.7% . In asymptomatic patients the specificity of Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT was 98.4% , and specificity of cRDT was 99.3% .The specificity of Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT) highlights the higher sensitivity of the Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT compared to the cRDT when performed on the same field conditions and confirmed findings observed in the laboratory conditions [This meta-analysis assessing the field performance of malaria uRDT  and is higher than 95% regardless of the reference test used in blood. Importantly its specificity is not statistically significantly different from the one obtained for the cRDT in the current meta-analysis.P. falciparum RDT is more sensitive than cRDT and could help to capture additional low parasite density malaria cases that escape the current cRDT. These results call for the assessment of additional criteria, namely the stability at high temperature, cost and shelf life of uRDTs, before Alere™ Ultra-sensitive malaria Ag P. falciparum RDT could be integrated into the already available malaria diagnostic arsenal. Furthermore, it is essential that a correlation be established in the field between the parasitaemia observed in patients and the positivity of uRDTs to confirm the results obtained in the laboratory conditions. Nevertheless, given that cRDTs typically do not consistently detect parasite densities lower than 100p/µL, the definition of an ultrasensitive malaria test needs to be clarified and the conditions to fulfil for a test to be considered ultrasensitive need to be consensually adopted to compliment the current WHO definition, which is based solely on the parasite density detection threshold (below 100 parasites/μl) [From a public health perspective, the findings of this study suggest that Alere™ Ultra-sensitive malaria Ag ites/μl) .P. falciparum RDT and a specific brand of cRDT test. Nevertheless, this study is the first to assess by the mean of a meta-analysis, the performances of one of the most recent diagnostic tools of malaria diagnostic in the field conditions. Furthermore, recent guidelines were used for the assessment of quality of included studies and the reporting of the review.The results of the current study must be interpreted considering some drawbacks. Most of the studies were conducted in WHO African region (sub-Saharan Africa), which is the region with the highest burden of malaria. This can limit the generalizability of the results to other malaria endemic regions of the world. Data does not allow for stratified analysis according to parasite density, which is one of the key elements in the performance of malaria diagnostic tests. None of the studies specified the storage conditions of uRDT and cRDT, and only seven reported clearly that the test was conducted by trained laboratory technician/nurse, this may have impacted on the quality of the results. Several brands of cRDT with different performances were used as comparators to the Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT in the current study. The diversity of these tests could be an additional source of heterogeneity in the results and may limit a direct comparison between Alere™ Ultra-sensitive Malaria Ag P. falciparum RDT compared to cRDT performed in the same field conditions has higher sensitivity but lower specificity although the difference is not statistically significant.Findings of the meta-analysis show that Alere™ Ultra-sensitive Malaria Ag Additional file 1: Table S1. Search strategy for PubMed.


Anopheles mosquitoes. It remains a significant problem for the most vulnerable children and women. Recent research has helped establish the relationship between microRNAs (miRNAs) and many other diseases. MiRNAs are the class of small non-coding RNAs consisting of 18–23 nucleotides in length that are evolutionarily conserved and regulate gene expression at a post-transcriptional level and play a significant role in various molecular mechanisms such as cell survival, cell proliferation, and differentiation. MiRNAs can help detect malaria infection as the malaria parasite could alter the miRNA expression of the host. These alterations can be diagnosed by the molecular diagnostic tool that can indicate disease. We summarize the current understanding of miRNA during malaria infection. miRNAs can also be used as biomarkers, and initial research has unearthed their potential in diagnosing and managing various diseases such as malaria.Malaria is a severe life-threatening disease caused by the bites of parasite-infected female  Plasmodium, a protozoan parasite. The bite of infected female Anopheles mosquitoes facilitates the transition of the parasite to humans and other animals. According to the 2019 WHO report, 229 million malaria cases and an estimated 409,000 deaths occurred worldwide. The most susceptible groups affected by malaria are children under five and pregnant women. Malaria is caused by five species of the protozoan parasite, namely, Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae and newly discovered Plasmodium knowlesi. Over 90 per cent of malaria infections are caused by P. falciparum compared to the other species. The Apicomplexa parasite like Plasmodium involves two different hosts in their life cycle, including female Anopheles mosquitoes as vectors  and vertebrates as the final host . Malaria infection begins with the injection of sporozoites into the skin of the vertebrate host from malaria-infected female Anopheles mosquitoes. Within 30–60 min, these sporozoites migrate through the blood to the liver and infect the hepatic cells, during which asexual multiplication occurs. These sporozoites are maturing subsequently into schizonts over 6–15 days. Eventually, each schizont multiplies to thousands of merozoites released into the bloodstream. These merozoites target specific proteins on the surface of erythrocytes and invade the cells. Through this stage, the merozoites develop to form ring stage or mature trophozoites, and then mature trophozoites develop into schizonts. This results in four to 36 new parasites in each infected cell in a 44 to72 h period. On maturation, the infected red blood cells burst, releasing the merozoites, which infect other uninfected healthy RBCs. This progression produces malarial symptoms, including chills, headache and fever and so forth. Instead of replicating, some merozoites from blood transform into gametocytes. Another mosquito then ingests these gametocytes during a blood meal. Gametocytes further develop in male and female gametes, and these gametocytes fuse inside the mosquito and form diploid zygotes, which additionally become ookinetes. These ookinetes transfer to the mosquito’s midgut and form the oocysts on the exterior wall of the midgut. Consequently, sporozoites are formed through the meiotic division of the oocysts, which further moves to the salivary glands of the female Anopheles and are injected into a new human host during the mosquito bite ..10].MiRNAs have a primary role in regulating gene expression in the human body by partially binding to mRNA, usually within the 3′ untranslated region and thus preventing their translation to protein . InteresMiRNAs can be tissue-specific or species-specific. Such miRNA-122 and miRNA-124 is highly distributed in the liver and neurological tissues, respectively . ResearcAnopheles mosquitoes and humans. At both transcriptional and post-transcriptional levels, this complex life cycle progress requires tight regulation of gene expression [plasmodium parasites invade into erythrocytes (RBCs) using cytoadherence ligand of P. Falciparum Erythrocyte Membrane Protein-1  and regulatory (PKA-R), have been recognized, and the regulation of PKA-R levels is critical for plasmodium survival. MiR-451 integration into P. falciparum AMP-dependent protein kinase (PKA-R) transcript reduced the translation of regulatory PKA activity and reduced the parasite growth [The malaria parasites have a complex life cycle, including sexual and asexual stages in female pression . The asestration . Approxistration . Some chne Rad54 . Moreovee growth ,18.Reports revealed that high levels of specific human miR-451, miR-223 and let-7i are found in both HbAS (having sickle cell trait) and HbSS (having sickle cell disease) erythrocytes compared with normal red blood cells and negatively regulate the malaria infection . RathjenPlasmodium vivax-infected patients while let-7a-5p was upregulated in both Pv and pf infected patients in Thailand [Plasmodium vivax infected patients compared to normal individuals but no significant difference for pf infected patients with normal individuals [Plasmodium parasite in in-vitro [Plasmodium falciparum [MiRNAs are significantly involved in several biological processes by regulation of gene expression. These are secreted in biological fluids like plasma, serum, cerebrospinal fluid, milk, urine, saliva, seminal fluid, tears and blood . These aThailand . A recenThailand . In contividuals ,28. Therin-vitro . EVs relin-vitro . Anotherlciparum . AGO prolciparum .Plasmodium infection, mostly affecting children. MiRNAs play an important role in regulating immune cell responses against infection. The infection of Plasmodium chabaudi and P.ANKA in the mice experiment model showed changes in the miRNA expression and caused the development of protective immunity against the Plasmodium parasite. The up-regulation ofmiR-27a, miR-150 and let7i was seen in the brain of Plasmodium berghei infected CBA mice that cause cerebral malaria when compared to non-cerebral malaria (NCB) and non-infected (NI) samples [PlasmodiumANKA and P.yoelii infected CBA mice that cause cerebral malaria and severe malaria and found miR-146a and miR-193b were rich in microvesicles from cerebral malaria-infected mice when compared with non-cerebral malaria and non-infected mice [Several studies were also performed on an animal model for indicating miRNA expression in the malaria model. Cerebral malaria (CM) is a fatal complication of  samples . Anotherted mice . MiRNAs ted mice . MiRNA-1ted mice .Diagnostic methods for malaria include microscopy, rapid diagnostic test (RDT), molecular diagnosis including polymerase chain reaction (PCR), quantitative PCR (qPCR) or reverse transcriptase PCR(RT-PCR), semi-nested PCR (n-PCR), multiplex PCR(m-PCR), indirect fluorescent antibody test and isothermal methods, viz, loop-mediated isothermal amplification (RPA) etc. . While mGiven the limitations of the current malaria diagnostic tools, miRNAs, which are non-coding RNA molecules present in tissues, most cell types and all body fluids, have emerged as an up-and-coming diagnostic tool for infectious diseases . MiRNAs The research on microRNAs has evolved; the significance of small non-coding RNA molecules in malaria and other diseases has been well documented. The advent of new techniques, such as deep sequencing and microarray analysis, has revealed the altered expression patterns of the specific host miRNAs during the virus, bacteria and other pathogenic infections. MiRNAsare also significantly involved in intercellular communication and regulate host cell activity, which plays a vital role in the host immune response to the malaria parasite. Moreover, miRNAs expression assessment in body fluids likes plasma, serum and so forth can be used as a promising biomarker for detecting malaria. This review has tried to summarize the changes reported in host miRNAs and their response after malaria infection based on current literature. Other miRNAs research on malaria infection may help as a malaria diagnostic tool in the early detection of severe malaria and its prognosis.


Imported malaria and recurrent infections are becoming an emerging issue in many malaria non-endemic countries. This study aimed to determine the molecular patterns of the imported malaria infections and recurrence. Blood samples were collected from patients with imported malaria infections during 2016–2018 in Guangxi Zhuang Autonomous Region, China. Next-generation amplicon deep-sequencing approaches were used to assess parasite genetic diversity, multiplexity of infection, relapse, recrudescence, and antimalarial drug resistance. A total of 44 imported malaria cases were examined during the study, of which 35 (79.5%) had recurrent malaria infections within 1 year. The majority (91.4%) had one recurrent malaria episode, whereas two patients had two recurrences and one patient had three recurrences. A total of 19 recurrence patterns  were found in patients returning from malaria epidemic countries. Four parasite species were detected with a higher than usual proportion (46.2%) of non-falciparum infections or mixed-species infections. An increasing trend of recurrence infections and reduced drug treatment efficacy were observed among the cases of imported malaria. The high recurrence rate and complex patterns of imported malaria from Africa to non-endemic countries have the potential to initiate local transmission, thereby undermining efforts to eliminate locally acquired malaria. Our findings highlight the power of amplicon deep-sequencing applications in molecular epidemiological studies of the imported malaria recurrences. Plasmodium falciparum is the predominant parasite species  in the African region, followed by South-East Asia (3%) . In addi species . The inc species .msp1 and msp2), apical membrane antigen (ama1), circumsporozoite protein (csp), and microsatellites, have poor sensitivity for detection of less abundant parasite infections  is more sensitive and reliable for detection of minority clone infections and determination of MOI , genetic diversity in relapse and recrudescence, and antimalarial drug resistance are essential for malaria treatment, prognosis, and prevention of malaria transmission. To improve control, we require information about the parasite’s diversity, transmission dynamics, relapse frequency, and mechanisms of adaptation to environmental and interventional pressures . Traditifections , while nn of MOI , recruden of MOI , quantifn of MOI , and invn of MOI .msp1 and ama1 markers for understanding the genetic diversity between new and relapsing infections. Finally, we examined the gene mutations of the P. falciparum Kelch-domain protein (pfk13), chloroquine resistance transporter (pfcrt), and multidrug resistance protein-1 (pfmdr1) by Sanger sequencing. Since the effectiveness of primaquine (PQ) for the radical cure of vivax malaria is influenced by CYP2D6 activity  What is the species composition of malaria parasites that cause imported malaria cases? 2) What proportion of these infections represent recrudescence or relapse? It is needed to establish whether the recurrent/relapsing infections were due to a new or existing infection. 3) Which mutations were associated with recrudescent infection in imported and recurrent episodes? To answer the first question, we conducted PCR and ADS using the mitochondrial mtDNA cytochrome b (CYTB) gene and small-subunit ribosomal (18S rRNA), referred to here as SSU gene markers for species identifications. To answer the second question, we performed ADS using activity , we wantDuring 2016 and 2018, imported malaria infections in Shanglin County, Guangxi, were diagnosed by microscopic examination of Giemsa-stained thick and thin blood films. The questionnaires including gender, age, occupation, travel history, and date of diagnosis were recorded. Malaria patients were admitted for inpatient treatment following the guidelines of the Chinese Center for Disease Control and Prevention (CDC) and WHO recommendations on the diagnosis and treatment of uncomplicated and severe malaria   following the manufacturer’s instruction and eluted in 100 μl of water. The nested PCR method was used to identify the knowlesi . The ampmsp1) gene, and apical membrane antigen 1 (ama1) gene, were used to design seven sets of PCR primers based on the highly variable regions of SSU, CYTB, pvmsp1, pvama1, pomsp1, pfmsp1, and pfama1 genes  was used as control. Amplicons were cleaned and normalized to 1 ng/µl concentration using the SequalPrep Normalization Plate Kit . Amplicon deep sequencing was performed on an Illumina MiSeq platform in paired-end mode using a MiSeq Reagent Kit v3 PE300  with PhiX control  and the minimum target read depths of 10,000×.Four target genes, including mitochondrial cytochrome b (CYTB) gene, small subunit ribosomal RNA (SSU) gene, the merozoite surface protein 1 . The full-length pfk13 gene was amplified and sequenced using a previously published protocol  and the P. falciparum multidrug resistance protein-1 (pfmdr1) gene were amplified and sequenced using previously described primers  P. vivax, 9 (9.7%) P. ovale curtisi, 4 (4.3%) P. ovale wallikeri, 1 (1.1%) P. malariae, and 15 (16.1%) mixed-species infections  in single-species infections and 4.5 (9/2) in mixed-species infections. Six out of the 15 mixed species were identified by the SSU marker, which showed various frequencies of different haplotypes, and most of them had predominant P. ovale curtisi mixed with less abundant P. falciparum  in Africa. ADS of the CYTB and SSU markers was performed in 81 and 85 samples, respectively. An average sequencing coverage depth for the major haplotypes was 20,069 and 11,109 for CYTB and SSU markers, respectively  had a second malaria attack within the first week of returning to China. Thirty-five patients (79.5%) had recurrent malaria infections later, but within 1 year of their return. The majority (91.4%) had one recurrent malaria episode, whereas two patients had two recurrences and one patient had three recurrences  in the 27 predominant haplotypes within samples  of pfama1 haplotypes were new haplotypes compared to the sequences available in GenBank. Among the 20 predominant haplotypes of pfama1, a total of 31 segregating sites were found with a nucleotide diversity Pi = 0.025 and a haplotype diversity Hd = 0.960. Overall, the number of clones ranged from 1 to 6 with a mean MOI of 2.2 ± 0.2, 95% CI  determined for the 50 P. falciparum samples by combining the two markers. Of these, 44% (22/50) samples had multiclonal infections  and pvama1 amplicon (288 bp). Seven haplotypes were identified for pvmsp1 and 5 for pvama1. Two of the seven pvmsp1 haplotypes were new, whereas none of the five pvama1 haplotypes were new compared to those sequences in the GenBank  and five haplotypes (four for poc and one for pow). Four of the five pomsp1 haplotypes were novel haplotypes compared to those sequences in GenBank. Phylogenetic tree analysis indicated that Ghana had all five haplotypes, whereas Congo, Ghana, and Myanmar had one haplotype for each country  identified by pomsp1 with identical genotypes between second episode and third episode with a time length interval of 24 days, whereas the other patients had only one episode successfully genotyped.The deep sequencing of p > 0.05). The sample size for patients with recurrence may not be enough to exclude its association with mutations.A total of eight patients were successfully genotyped for mutations in the five antimalaria drug resistance genes  of non-falciparum infections cases in this study. Recently, P. ovale infections have become more common in Africa, suggesting that P. ovale is becoming an increasingly important malaria parasite, highlighting the need for attention toward non-falciparum malaria  by the SSU marker, five haplotypes  from the pomsp1 marker. Among these, four out of the five pomsp1 haplotypes were identified as novel haplotypes ; its potential role in antimalaria resistance needs to be studied further. The high proportion of P. vivax infections and relapse was observed in the study, possibly due to positive Duffy antigens elevating susceptibility to vivax malaria in Asian patients  and K189T/N; their roles in resistance are not yet determined , although this patient had an additional trip to Ghana. The short or long latency relapses after primaquine treatment for P. vivax infections could be explained by parasite drug resistance  can be found in the article/The studies involving human participants were reviewed and approved by the Ethical Review Committee of Shanglin People’s Hospital, Guangxi, China. The patients/participants provided their written informed consent to participate in this study.GY, ZY, and DZ conceived and designed the study. XH wrote the first draft of the paper. DZ and LC helped in performing data analysis and drafted the manuscript. XH, CZ, LP, LZ, YQ, MP, SW, WZ, ZX, XC, YW, YS, and YH conducted the sample collection and experiments. All authors contributed to the article and approved the submitted version.This work was supported by grants (31860604 and U1802286) from the National Natural Science Foundation of China and by a grant (2018ZF0081) from Major Science and Technology Projects of Yunnan and Technology Cooperation-Yunnan International Science and Technology Cooperation Base (202003AE140004). LC was supported by a grant U19AI089672 from the National Institutes of Health, USA. GY was supported by the National Institutes of Health USA (U19AI129326). CZ was funded by a grant from the Youth Fund Project of People’s Hospital of Guangxi Zhuang Autonomous Region, China (QN2017-10). MP was funded by a grant (ZC20153012) from the Science and Technology Bureau Programs of Nanning, Guangxi, China. YQ was funded by the Scientific Research Project of Health Committee of Guangxi Zhuang Autonomous Region (20191525). WZ was supported by the Education Department Fund of Yunnan Province (2019J1184). XC and ZX were sponsored by the Yunnan Applied Basic Research Projects-Union Foundation . YW was supported by the Hundred-Talent Program of Kunming Medical University (60117190439), and the Foundation of the Education Department of Yunnan Province (2018JS151) and the Creative Experimental Project of Kunming Medical University and Yunnan Province.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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