Increasing numbers of aging individuals with chronic co-morbidities travel to regions where falciparum malaria is endemic. Non-communicable diseases are now leading risk factors for death in such countries. Thus, the influence of chronic diseases on the outcome of falciparum malaria is an issue of major importance. Aim of the present study was to assess whether non-communicable diseases increase the risk for severe imported falciparum malaria.A retrospective observational study of all adult cases with imported falciparum malaria hospitalized between 2001 and 2015 in the tertiary care Charité University Hospital, Berlin, was performed.A total of 536 adult patients  were enrolled. Of these, 329 (61.4%) originated from endemic countries, 207 patients (38.6%) from non-endemic regions. Criteria for severe malaria were fulfilled in 68 (12.7%) cases. With older age, lack of previous malaria episodes, being a tourist, and delayed presentation, well-characterized risk factors were associated with severe malaria in univariate analysis. After adjustment for these potential confounders hypertension , cardiovascular diseases , and dyslipidaemia  were individual diseases associated with severe disease in multivariable logistic regression. Hypertension proved an independent risk factor among individuals of endemic  as well as of non-endemic origin .In imported falciparum malaria hypertension and its related diseases are risk factors for severe disease. Due to increasing international travel and changing patterns of migration malaria is still the most important tropical disease imported to Europe . The patNumerous risk factors for both severe and fatal imported falciparum malaria have been identified in recent years . Among tThe present study aimed to identify co-morbidities associated with severe falciparum malaria imported to Berlin, Germany, in a large adult cohort. The findings are discussed in the context of the current concepts of malaria pathophysiology and in regard to the resulting implications for supportive therapy.Only hospitalized patients with a first episode of acute falciparum malaria were enrolled. Since the effects of co-morbidities in an adult population should be investigated, only patients over 18 years of age were included. Diagnosis relied on thin and thick blood smears. Parasitaemia was expressed as percentage of parasitized erythrocytes with 1% parasitaemia corresponding to about 50.000 parasites/µl. Patients born in countries with malaria transmission were referred to as of “endemic origin”.As part of the admission routine data on sociodemographics, travel history, chemoprophylaxis, full medical history including prior malaria episodes, current medication, and physical examination were recorded for all patients. Past medical records were retrieved, where available. Prior to statistical analysis, laboratory results and outcome parameters such as severe malaria criteria or need of intensive care treatment were retrieved. All relevant parameters were then anonymized and transferred into a database (IBM SPSS version 24).Severe malaria was defined according to the 2014 WHO definition  with modHistory of or active malignancy as well as chronic cardiovascular, pulmonary, gastrointestinal, rheumatologic, renal, endocrine, metabolic, chronic infectious disorders such as hepatitis B, hepatitis C or human immunodeficiency virus (HIV) infections, psychiatric, and neurologic diseases were considered relevant chronic comorbidities. In an effort to weigh the seriousness of the underlying disorders an age-adjusted Charleson co-morbidity index (CA-CCI)  was calc2 test, while the Mann–Whitney-U-test was used for continuous data. Odds ratios were determined by univariate logistic regression. Based on biological plausibility, age and endemic origin were included as possible confounders in all subsequent multivariable analyses. Individual diagnoses associated with severe malaria in univariate analysis (p < 0.05) were included with potential confounders in separate multivariable models with the level of significance set at < 5%. Fit of these models was assessed by − 2 log-likelihood (comparing against the constant) and Hosmer–Lemeshow goodness-of-fit tests prior to assuring that all necessary assumptions were met  [Endpoint of the analysis  was presence of severe malaria on admission . Categorical data were compared by χstances) . All staThe study was approved by the institutional review board .Between January 2001 and December 2015 a total of 558 cases of imported falciparum malaria were hospitalized in the Charité University Hospital, Berlin. Seventeen patients presented more than once. The 22 subsequent episodes of these individuals were excluded from the analysis. The remaining 536 patients ultimately enrolled represented 6.8% of all cases notified in Germany during the study period. Of these, 168 (31.3%) were female and 368 (68.7%) male. Median age of the whole patient group was 37 years (range 18–78 years). A total of 329 cases (61.4%) originated from endemic countries, 207 cases (38.6%) from non-endemic regions  had at least one relevant chronic condition range 0–3)—the most common being hypertension, endocrine/metabolic disorders, and chronic infectious disorders, including 15 HIV-positive individuals (Table —the mostAge distribution differed significantly (p = 0.002) between patients of non-endemic and endemic origin Fig. : among pThe present study demonstrates that hypertension and its secondary diseases increase the risk for a severe course of imported falciparum malaria. The complex pathophysiology of falciparum malaria interferes with the mechanisms that compensate for the systemic effects of the infection on multiple levels. Pre-existing chronic medical conditions can further hamper these physiologic mechanisms, thereby facilitating development of severe malaria. The age-dependent increase in the prevalence of such non-communicable diseases presumably explains to a large extent the association of increasing age with severe falciparum malaria that has consistently been found in various studies.The prevalence of hypertension is higher in populations constantly exposed to malaria . TherefoIn areas of unstable transmission and in imported falciparum malaria, however, the situation is likely different. Numerous studies have demonstrated the diverse effects of falciparum malaria on the cardiovascular system in the setting of acute disease in recent years . These eA reduced pre-load is the result of fluid losses from fever, perspiration, vomiting, diarrhoea, insufficient oral fluid intake, and increased vascular permeability. Nearly all patients with severe falciparum malaria are hypovolemic on presentation , 24. By Direct myocardial impairment has been demonstrated in severe but not in uncomplicated falciparum malaria cases by elevated levels of N-terminal brain natriuretic peptide (NT-pro-BNP) and heart-type fatty acid-binding protein (H-FABP) . Pro-infReduced red-blood cell (RBC) deformability is a pathophysiologic hallmark of falciparum malaria resulting in increased clearance of parasitized and non-parasitized erythrocytes. Cell-free haemoglobin released from ruptured erythrocytes acts as a nitric oxide (NO) scavenger , therebyImportantly, the various pathophysiologic processes occur simultaneously and may augment each other, together leading to a reduced oxygen delivery to peripheral tissues and hence complicated malaria Fig. . On the For long, HIV infection is known to increase the risk for severe falciparum malaria . RecentlYet, there are alternative explanations for this association. In malaria pathophysiology obstruction of the microcirculation by parasitized red blood cells plays a central role . Pre-exiSimilar to diabetes and hypertension lipid disorders promote development of microangiopathy . HyperteIn addition to various well-characterized risk factors for development of severe falciparum malaria such as increasing age, lack of semi-immunity , or delayed presentation chronic co-morbidity was associated with complicated disease in the present cohort. Not only the number of chronic conditions but also the seriousness of underlying co-morbidity (assessed by age-adjusted Charleson co-morbidity index) influenced the risk for development of severe falciparum malaria. One of the individual diagnoses responsible for this association was hypertension. With a prevalence of 9.2% hypertension was the single most common chronic condition in the study population. Multivariable analysis was therefore robust and revealed that hypertensive individuals had a threefold increased risk of developing severe imported falciparum malaria compared to patients without hypertension. With increased after-load, hypertensive cardiomyopathy and, most importantly, pre-existing microangiopathy, there were plausible pathophysiologic explanations for this association  . The supThe present study has several limitations, the main being its retrospective nature. Due to misclassification of exposure, for example, the medical histories of patients with severe disease could have been more thoroughly investigated than those of uncomplicated cases. However, all patients enrolled were hospitalized, treated under identical conditions, and their courses were recorded in standardized electronic files. Thus, data capture was high. Long observation period and monocentric design further add to limited quality of the data. However, severe imported falciparum malaria is a rare condition in most non-endemic regions. Thus, high-quality prospective trials conducted in resource-rich settings will hardly become available in the future. Retrospective analyses, therefore, continue to provide an important source of evidence. Together with a case load of > 500 individuals this allowed for detailed statistical analyses. Since cohorts of patients with imported falciparum malaria will differ in regard to average age, represented ethnicities, immune status, comorbidities, destinations, reason of travel and other important aspects, however, the results cannot be generalized recklessly. Though obesity and diabetes did not prove risk factors in the present cohort the analysis nevertheless strongly supports the hypothesis that chronic medical conditions promoting microangiopathy or reduced cardiac output facilitate development of severe falciparum malaria.In addition to diabetes and obesity, hypertension is another previously unidentified risk factor for severe disease in adults with imported falciparum malaria. Due to its high prevalence this finding is not only of importance for travellers of non-endemic origin but also for expatriates originating from endemic areas with slowly waning semi-immunity. The study also gave evidence that related conditions such as cardiovascular diseases or lipid disorders may increase the risk for development of severe disease. Based on pathophysiologic considerations these associations are plausible.Additional file 1. Severity criteria among hypertensive and non-hypertensive patients with severe falciparum malaria imported to Berlin, Germany.Additional file 2. Prevalence of selected co-morbidities among 536 patients with and without hypertension and falciparum malaria imported to Berlin, Germany.


Plasmodium knowlesi have been found throughout Southeast Asia, most cases originated from Malaysian Borneo. In Thailand, P. knowlesi malaria was considered extremely rare. However, during October 2017–September 2018, there was a surge in the number of reported P. knowlesi cases. Here, a series of six cases of P. knowlesi malaria found during this period in Songkhla and Narathiwat provinces of southern Thailand are presented. All cases were confirmed by polymerase chain reaction. The unprecedented case number in the affected area is a warning sign of an increasing P. knowlesi burden in the south of Thailand.Although human infections of  Plasmodium knowlesi is increasingly recognized as a major cause of human malaria. The natural hosts of this parasite are the long-tailed and pig-tailed macaques of Southeast Asia.2 The transmission of P. knowlesi is generally considered to be from monkeys to humans through local anopheline vectors,5 with reported human-to-human transmissions confined to blood transfusion7 and experimental infection.8 The first documented natural infection of humans with P. knowlesi was in 1965 when a traveler acquired the parasite from a visit to Southeast Asia.9 More recently, in 2004 and 2008, a large number of naturally acquired P. knowlesi infections in humans were reported in Sarawak state of Malaysian Borneo. In addition to Malaysia, P. knowlesi infections have now been observed throughout Southeast Asia.17 A number of infections were also found in international travelers.24Plasmodium vivax and P. knowlesi.25 In 2000, a case of human P. knowlesi malaria infection was stated in Prachuap Khiri Khan Province.16 During October 2006 and September 2007, 10 P. knowlesi infections were identified by polymerase chain reaction (PCR) in 1,751 malaria patients.17 During October 2008 and September 2009, a survey identified 23 P. knowlesi infections in 3,446 patients from various parts of the country.25 A separate study provided evidence of P. knowlesi infection in two patients working near the Thai-Myanmar border in Ranong Province.26In Thailand, a retrospective study of blood samples obtained from malaria patients in northwestern Tak Province during 1996 uncovered a case of mixed species infection of P. knowlesi clinical cases were reported, for the first time, by the Thai National Malaria Control Program (NMCP), with the nationwide total of 23 cases during October 2017 –September 2018.27 Here, we present six of these cases from Songkhla and Narathiwat provinces of Southern Thailand.Recently, Six blood samples were collected from malaria patients who sought treatment at the Vector-Borne Disease Control Center (VBDC) in Songkhla Province or at Naradhiwas Rajanagarindra Hospital in Narathiwat Province from November 2017 to April 2018. Thin and thick smears were prepared, stained with 10% Giemsa, and examined under a microscope. Genomic DNA was extracted from dried blood spots on filter paper or blood pellets and used in nested PCR and quantitative PCR (qPCR) to confirm the parasite species. This study received human use exemption (certificate no. MUTM-EXMPT 2018-009) by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand.Plasmodium species.28 The first round of nested PCR was performed with Plasmodium genus–specific outer primers . The reaction was set using GoTaq® Green Master Mix  in a 25-µL reaction volume under thermocycling conditions 95°C for 20 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. One microliter of 1:50 dilution of the first-round product was used as the template for the second round of PCR with the same forward primer (ACGATCAGATACCGTCGTAATCTT) and a reverse primer specific to each species . The second PCR was set in a 25-µL reaction volume using GoTaq Green Master Mix with 0.4 µM concentration of each primer and thermocycling conditions 95°C for 20 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The final PCR products were analyzed on 2% agarose gel. Dye-terminator sequencing of the nested PCR products was performed in both directions using the nested PCR primers through commercial service . Positive P. knowlesi detection was further confirmed by P. knowlesi–specific TagMan 18S qPCR which used primers  and a probe  with iTaq™ Universal Probes Supermix  and two-step thermocycling conditions 95°C for 15 seconds and 60°C for 60 seconds.28Nested PCR targeting the 18S rRNA genes was performed to detect the P. knowlesi infections, five from Songkhla Province and one from Narathiwat Province .From November 2017 to April 2018, we identified six malaria patients with Province . These cod films  of all cproducts , and qPCP. vivax malaria, and the patient was treated with chloroquine (25 mg/kg over 3 days) and primaquine (0.25 mg/kg/day for 14 days) and recovered fully. The patient had worked in a rubber plantation and camped out in the forest at Ban Na Kha, Malaysia. The camping cottage was surrounded by a wooded area populated by wild macaques, the potential reservoir of the parasite.The first case, a 22-year-old male resident of Na Thawi district, Songkhla Province, presented himself at the malaria clinic of the VBDC in Na Thawi in November 2017 with six days of fever, chills, and headache. The initial diagnosis by light microscopy was P. falciparum histidine rich protein 2 antigen but positive for Plasmodium lactate dehydrogenase. The patient was considered to have P. vivax infection and treated with chloroquine (25 mg/kg over 3 days) and primaquine (0.25 mg/kg/day for 14 days) according to the standard P. vivax treatment. The patient reported to have worked in palm plantations and traveled to work in Garuntun, Malaysia, before becoming sick. His work place was near a damp rainforest inhabited by monkeys.The second case, a 45-year-old man, presented at the Naradhiwas Rajanagarindra Hospital in Narathiwat Province in January 2018. He displayed symptoms including a 6-day history of daily fever, shivering, headache, nausea, vomiting, jaundice, pallor, and dark urine. His temperature during admittance to the hospital was 39.3°C. He did not have a history of either malaria or blood transfusion. A malaria rapid diagnosis test  was negative for P. vivax infection by light microscopy, the patient was treated with chloroquine (25 mg/kg over 3 days) and primaquine (0.25 mg/kg/day for 14 days) and recovered fully. The patient was a rubber plantation worker and reported to have had camped out for 4 days in the forest at Baan Keun Nam, Kedah state of Malaysia.The third case was a 50-year-old man in Na Thawi district, Songkhla Province. He had fever for 9 days with a mild headache. He was initially diagnosed as having influenza, but did not recover after treatment with antiflu medication. The patient returned to seek medical aid at the malaria clinic of VBDC in Na Thawi in February 2018. After being diagnosed as having P. vivax malaria and cured by the standard chloroquine (25 mg/kg over 3 days) and primaquine (0.25 mg/kg/day for 14 days). The patient lived in a rubber plantation and was a rubber tapper, herdsman, and nontimber forest product finder. She frequently visited the forest along the Thai-Malaysian border.The fourth case was a 48-year-old woman in Sadao district, Songkhla Province. The patient presented at the Sadao Hospital in April 2018 after having experienced fever, chills, abdominal pain, and severe headache for 4 days. The patient was initially diagnosed as having P. falciparum malaria due to the predominance of ring-stage parasites in blood smears. The patient was cured by standard 3-day dihydroartemisinin (2.5 mg/kg/day)–piperaquine (20 mg/kg/day) treatment with a single dose of primaquine (30 mg) according to Thailand’s national guideline. The patient was a rubber plantation worker and nontimber forest product finder. He reported to have had regularly visited the forest inhabited by wild monkeys near Satun Province of Thailand.The fifth case was a 32-year-old male resident of Sadao district, Songkhla Province. The patient presented at the malaria clinic of VBDC in Sadao in April 2018 with 5-day fever, chills, and severe headache. Initial diagnosis was P. vivax malaria and treated with chloroquine (25 mg/kg over 3 days) and primaquine (0.25 mg/kg/day for 14 days). The patient was a rubber plantation worker and wild animal hunter. He reported to have spent 4 days in a hilly forest at Baan Keun Nam, Kedah state of Malaysia, before becoming sick.The sixth case, also detected in April 2018, was a 35-year-old man in Saba Yoi district, Songkhla Province. The patient came to the malaria clinic of VBDC in Saba Yoi with 2-day fever and a mild headache. The patient was initially diagnosed by light microscopy as having P. knowlesi malaria in Songkhla and Narathiwat provinces of Southern Thailand. To our knowledge, this is the first report of P. knowlesi malaria in Songkhla Province, and the first from the Thai-Malaysian border area with blood smear evidence. The travel history of all patients revealed travel to an area inhabited by wild monkeys.We report a series of cases of Plasmodium knowlesi infection is considered extremely uncommon in Thailand. During October 2017 and September 2018, a total of 10 cases of P. knowlesi malaria from the four border provinces  were reported for the first time by the Thai NMCP, despite the fact that the current national reporting system had been deployed since 2012. These cases forewarn the potential emerging threat of P. knowlesi in the southernmost area of Thailand. It is noteworthy that all reported P. knowlesi cases in this study were mistakenly diagnosed as P. vivax or P. falciparum during admission to the clinics or hospitals, suggesting that the hidden burden of knowlesi malaria might be much higher. It is important that blood smears of all suspected cases of P. knowlesi in Thailand and Peninsular Malaysia are confirmed by expert microcopy and molecular diagnosis to closely track the disease burden in the near future.


The Mindray BC-6800 haematology analyzer (BC-6800) provides a dedicated flag ‘Infected RBC’ (InR) and the number of InR (InR#)/the permillage of InR (InR‰) in routine blood testing as a screening tool for malaria in endemic areas. This study sought to evaluate the effectiveness of the BC-6800 flag parameter for aiding the diagnosis of malaria.Plasmodium vivax and Plasmodium falciparum. The receiver operating characteristic (ROC) curve analysis was developed using Analyse-it v4.92.3. The Kappa value was determined to evaluate the agreement between BC-6800 and light microscopy.A total of 181 samples were tested using the Mindray BC-6800 haematology analyzer, including 117 malaria-infected samples collected from Yunnan, China, and 64 samples from healthy controls. Microscopy examination was conducted as reference when stained thick blood film revealed the presence of malaria parasites identified as P. vivax and P. falciparum was 88.3 and 24.1%, respectively; specificity of InR‰ for malaria parasites was 84.3 and 84.3%, respectively; positive predictive value and negative predictive value was 89.4 and 82.7% for P. vivax, and 52.8 and 60.3% for P. falciparum. There was a strong correlation between ΔWBC and InR‰ . There was also a significant correlation between parasitaemia and InR# in P. vivax-infected samples (R2 = 0.734). InR# was evaluated using ROC curve analysis, the area under the ROC curve is 0.95 with a 95% confidence interval of 0.926 to 0.974, and the cut-off value is 0.01 × 109/L for P. vivax. However, the ring stage and the early trophozoite stage of Plasmodium cannot be detected easily on BC-6800, possibly because of the small size and low nucleic acid content of these stages.The sensitivity of InR‰ generated by BC-6800 for The findings suggest that the flag ‘InR’ and the parameters ‘InR#/InR‰’ provided by the BC-6800 haematology analyzer could be used to screen for malaria in a clinical setting. Malaria is a vector-borne infectious disease that continues to have high morbidity and mortality globally . The priLight microscopy, malaria nucleic acid amplification (PCR) test and malaria rapid diagnostic tests (RDTs) are used for parasitological diagnosis of malaria. Malaria PCR is not commonly used due to its high cost; RDTs are now more common, but not yet the regular test in non-endemic areas; microscopic examination of stained blood films remains the standard and most commonly used diagnosis method. Although this is the standard method, it is labour intensive and requires a high level of expertise to scan thick blood films for the presence of malaria parasites and thin blood films to determine the type of malaria , 4.Plasmodium falciparum [Malaria is prevalent in parts of Southeast Asia, South Asia, Africa and South America and, therefore, there is a demand for malaria screening in these areas –7. Haemalciparum . AlthougFor clinicians without extensive technical experience or expertise, the BC-6800 automated haematology analyzer  could be used to detect malaria parasites and to calculate parasitaemia in blood samples through cytometry analysis. The BC-6800 analyzer provides CBC parameters, reticulocyte and its fractions, and nucleated red blood cells (NRBC) value. It also provide a dedicated flag for ‘InR’ and ‘InR#/InR‰’ parameters. These are research use only parameters roughly equivalent to the number of InR (InR#)/the permillage of InR (InR‰) for the malaria iRBCs in a sample . BC-6800P. vivax and P. falciparum.The present study aimed to evaluate the utility of the infected RBC flag and parameters InR#/InR‰ in routine blood testing as a malaria screening tool in endemic areas. Furthermore, a comparison study was carried out for the flag information of CBC parameters between control group and the malaria group with P. vivax-infected patients  and 21 from P. falciparum-infected patients . Sixty-four healthy subjects from a non-endemic area in China were collected as the control group . Samples were classified as malaria positive only when microscopy examination of stained thick blood film revealed the presence of at least one of the four malaria parasite forms: ring form, trophozoite, schizont and/or gametocytes. All blood samples were examined using both light microscopy and BC-6800 haematology analyzer using the manufacturer recommended reagents, calibrator and controls. Analyzer performance was monitored daily using three levels of quality control material.EDTA anti-coagulated blood samples were collected from Tengchong, Yunnan Province (an endemic area in the border region of China and Myanmar) between May and August in 2016 according to physicians’ request. Some 117 malaria-infected samples were analysed, including 96 from Plasmodium-infected erythrocytes, that would result in resistance to lysis [Plasmodium-infected RBC, and eosinophils, as well as identifies and flags abnormal cells such as immature granulocytes, abnormal lymphocytes and blast cells.The BC-6800 haematology analyzer used sheath flow impedance, laser scatter and SF Cube analysis technology. The SF Cube analysis technology is three-dimensional using information from laser light scatter at two angles and fluorescent signals for cell differentiation and counting . In the to lysis , 17. ThrThin and thick blood films were prepared for staining and parasite investigation. Blood films were sent to Tengchong Laboratory in a standard slide box for staining with 3% Giemsa and microscopic examination . ParasitThe malaria microscopist was blinded to BC-6800 results. Similarly, healthy controls were blinded to microscopy results. In order to assure malaria form quality of the microscopic examinations, the entire positive and 10% of the negative slides were sent to a senior malaria microscopist and re-examined at the Tengchong Laboratory. An experienced reader was assigned to re-examine the discrepant slides.This study obtained ethical approval from the ethics committee of the General Hospital of the People’s Liberation Army. Individual informed consent was obtained from adults and from the parents or guardians of children under 18 years old when they presented in hospital. In addition, verbal assents were given to minors.P. vivax group and P. falciparum group). Sensitivity, specificity and predictive values for the detection of different Plasmodium species were calculated and compared the P. vivax group and P. falciparum group. InR# representing the number of ‘iRBC’ was evaluated using ROC curve analysis, reporting the area under the curve (AUC) and its confidence interval (CI). ROC analysis was completed using Analyse-it v4.92.3. The Kappa value was calculated to evaluate the agreement between BC-6800 and light microscopy. A P value less than 0.05 was considered statistically significant.Data entry was performed using Microsoft Office Excel. Statistical analysis was performed using SPSS 22.0  analyzer Microsoft tool box. It was compared on the blood cell parameters and suspect flags between control group and iRBC group in BC-6800 analyzer. Pearson’s correlation or Spearman’s rank correlation was also used to determine the association between control group and malaria group  in various InR groups were unequal, and the difference was statistically significant. In addition, these results displayed that the infection density in InR group II and group III were higher than that in group I.As shown in Table P. vivax-infected patient group and in the P. falciparum-infected group. The result showed that there was a clear relationship between ΔWBC (WBCDIFF–WBCBASO) and InR‰ (P. vivax group R2 = 0.973). . A better relationship was present between ΔWBC and InR‰ in the P. falciparum group, however, the InR flags were only flagged in 5 of all 21 P. falciparum patients by the BC-6800 analyzer .Automated RBC counts and suspect flags from BC-6800 haematology analyzer are shown in Table P. vivax/P. falciparum patient groups and control group (P < 0.05).Automated platelets (PLT) counts and suspect flags from the BC-6800 haematology analyzer are shown in Table Plasmodium parasites and the causative agent of malaria infection. In this study, the parameter ‘infected RBC’ was used to analyse the diagnosis performance in patients with malaria, as well as routine parameters and suspect flags.The BC-6800 haematology analyzer uses SF-Cube technology to achieve three-dimensional cell analysis for WBC, reticulocyte (RET) and NRBC with clinically relevant suspect flags , 20. It Plasmodium was evaluated. Samples with malaria parasites show a distinct cluster and unique location on three-dimensional cell plots. The characteristics of the cluster are dependent on the species of Plasmodium as well as the size and number of the parasite present. The sensitivity and specificity for detecting P. vivax was not significant for P. falciparum. This may be a result of early trophozoites of P. falciparum that developed over 10 h in the peripheral blood concealed in the microvasculature, sinusoids or other slow blood flow before being developed into late trophozoites and schizonts [P. falciparum are rarely seen in the peripheral blood of infected patients. The ring and the gametocyte stage are the most commonly seen in a peripheral blood smear. However, the ring and the early trophozoites of Plasmodium cannot be detected easily on BC-6800, the main reason possibly being the small size and low nucleic acid content.The diagnostic performance of the InR flag for the diagnosis of chizonts , 22. TroPlasmodium. Second, the number of intra-erythrocytic phase Plasmodium should surpass a certain threshold [P. vivax specimens were tested as false negatives because they did not meet these two conditions. This may be because the majority of samples among the P. vivax group are trophozoite and ring form, which are too small to be detected easily. Both microscopy and malaria RDT are very effective at identifying ring infections. In addition to its poor sensitivity for detecting P. falciparum, this study suggested the shortcoming should be improved in BC-6800 haematological analyzer. At present, this is the major limitation of broader use for P. falciparum in particular. The machine could be more effective for identifying P. falciparum in future.InR flag being triggered by BC-6800 should meet two conditions. First, the malaria-infected specimens should be collected during the intra-erythrocytic phase hreshold . Some P.P. vivax-infected patients [P. vivax infection, and was observed in up to 39% P. vivax-infected patients [P. vivax 6.25% and P. falciparum 14.29%). In P. vivax and P. falciparum-infected patients, results found that WBC count was higher in the DIFF channel (mild lyse) compared to the WBC/BASO channel (severe membrane destruction). A strong correlation was found between ∆WBC (WBCDIFF–WBCBASO) and the number of iRBC (R2 = 0.973) for P. vivax-infected patients (Fig. P. vivax-infected patients (R2 = 0.734; Fig. P. vivax after samples are tested using the BC-6800.Several studies report that DIFF scattergram suspect flags can provide assistance in the diagnosis of malaria in patients , 24. Respatients . In thisP. vivax or P. falciparum parasitaemia are associated with abnormal reticulocyte parameters, such as RET scattergrams and IRF. Results also show platelet-associated flags, including PLT clump, IPF and PLT abnormal in malaria-positive samples were increased compared to the control group. These findings are in accordance with the study of Dubreuil et al. [Anaemia a common symptom of malaria and is associated with dyserythropoiesis and ineffective erythropoiesis . The BC-l et al. .Except for cost, malaria PCR and RDTs are obviously ahead of the BC-6800 in sensitivity. It is necessary to further improve the sensitivity, especially for the ring infections, in the apparatus. In this study, the associations were limited between the haematological parameters and the species of infection considering patient age, symptoms and prior treatment.A dedicated ‘Infected RBC’ flag on a CBC result print-out provides an objective record and a trigger for further examination if malaria is suspected. Establishing a work protocol to further examine all flagged samples and use thick blood film microscopy and/or malaria RDTs to confirm or rule out malaria may reduce therapeutic intervention time and improve patient care outcomes. In malaria-endemic zones, ‘Infected RBC’ flag could serve as a rapid decision support tool when screening for malaria.


To the editor: We read with great interest the rapid communication by Zammarchi et al. [Plasmodium falciparum malaria infection in a Moroccan man living in Tuscany, Italy. The authors described that the case was unlikely to be travel-associated or a malaria relapse. Moreover, they ruled out other possible risk factors, such as transfusion, transplant or nosocomial exposure. Thus, the authors reported the occurrence of a sporadic autochthonous P. falciparum infection in Italy, almost 50 years after the elimination of malaria in southern Europe in the 1970s.i et al.  describiP. falciparum diagnosed in October 2018 in Malta. A previously healthy man in his early thirties was admitted to Mater Dei Hospital in mid-October 2018 with an 8-day history of high fever, headache, vomiting, loss of appetite and dark urine. Blood tests showed anaemia , thrombocytopenia , low eosinophils , low lymphocyte count  and increased serum CRP .In light of this unexpected case, we wish to report a cryptic case of P. falciparum in peripheral blood and PCR results confirmed P.falciparum infection with high viraemia. The patient was treated with three doses of atovaquone/proguanil (1g/400mg per day) and was discharged 4 days after hospital admission. The patient made a full recovery within 7 days.Examination of blood smears showed the presence of The epidemiological investigation revealed that the patient, a resident of central Malta, was born in Burkina Faso and has been living in Malta for over 10 years. The patient reported having had malaria in 2007, when they were still living in their home country. Later that year, the patient arrived as a migrant to Malta. They had not travel abroad since then. The patient works discontinuously, mainly outdoors, and reported being unemployed prior to symptom onset. The patient has no history of surgery, blood transfusion or invasive examination, denied being an injecting drug user and was not aware of having had contact with persons with similar symptoms. The patient’s residence is located ca 7 km from Malta International Airport. They noticed multiple mosquito bites during the weeks preceding illness. Extensive investigations failed to identify the potential source of this sporadic case and there was no evidence of further local transmission.P. falciparum and one P. vivax). All of them were imported cases associated with recent travel to Asia or Africa. No cases of locally acquired malaria have been reported in Malta since the early 1940s, and Anopheles spp. have not been detected since its eradication in 1943 [Culex pipiens and demonstrated that Aedes albopictus has been introduced and established in the Maltese Islands [Anopheles spp. were identified in these investigations.During 2018, another four cases of malaria were reported in Malta (three P. falciparum infection that persisted for over a decade. Recently, a case identified 13 years after initial exposure to P. falciparum malaria was reported [Although unlikely, a plausible explanation may be that the patient had asymptomatic reported .Even though Malta is a small country, the high number of visiting tourists, along with increased numbers of migrants and favourable Mediterranean climate, increase the risk of sporadic autochthonous cases and establishment of malaria in the region. There exists an urgent need, both in Malta and in other European countries, for increased awareness of imported malaria as well as potential autochthonous malaria cases; further efforts should be made to heighten awareness among clinicians to ensure prompt detection, notification and treatment of cases, as well as timely implementation of public health control measures.P. falciparum malaria case in Malta suggests that competent vectors for transmission may be present. This event, together with the recent P. falciparum malaria case in Italy, indicates that re-establishment of malaria in southern Europe may be possible and reinforces the need for improved surveillance and urgent vector-control actions.The unexpected finding of this cryptic


In malaria endemic areas such as sub-Saharan Africa, repeated exposure to malaria results in acquired immunity to clinical disease but not infection. In prospective studies, time-to-clinical malaria and longitudinal parasite count trajectory are often analysed separately which may result in inefficient estimates since these two processes can be associated. Including parasite count as a time-dependent covariate in a model of time-to-clinical malaria episode may also be inaccurate because while clinical malaria disease frequently leads to treatment which may instantly affect the level of parasite count, standard time-to-event models require that time-dependent covariates be external to the event process. We investigated whether jointly modelling time-to-clinical malaria disease and longitudinal parasite count improves precision in risk factor estimates and assessed the strength of association between the hazard of clinical malaria and parasite count.Using a cohort data of participants enrolled with uncomplicated malaria in Malawi, a conventional Cox Proportional Hazards (PH) model of time-to-first clinical malaria episode with time-dependent parasite count was compared with three competing joint models. The joint models had different association structures linking a quasi-Poisson mixed-effects of parasite count and event-time Cox PH sub-models.There were 120 participants of whom 115 (95.8%) had >1 follow-up visit and 100 (87.5%) experienced the episode. Adults >15 years being reference, log hazard ratio for children <5 years was 0.74  in the joint model with best fit vs. 0.62  from the conventional Cox PH model. The log hazard ratio for the 5–15 years was 0.72  in the joint model vs.0.63  in the Cox PH model. The area under parasite count trajectory was strongly associated with the risk of clinical malaria, with a unit increase corresponding to-0.0012  decrease in log hazard ratio.Jointly modelling longitudinal parasite count and time-to-clinical malaria disease improves precision in log hazard ratio estimates compared to conventional time-dependent Cox PH model. The improved precision of joint modelling may improve study efficiency and allow for design of clinical trials with relatively lower sample sizes with increased power. Malaria remains one of the most common parasitic infections globally with a disproportionately high burden in sub-Saharan Africa . In malaFor accurate estimation of the risk of clinical malaria, analytical methods are required that account for historical exposure and the relationship between clinical malaria and infection parasite count. To investigate the strength of this association, one approach would be including the parasite countas a time-dependent covariate in the model of time-to-clinical malaria episode. However, this approach may be inaccurate because it does not account for the fact that parasite count in this case is an endogenous covariate whose existence and future path can be directly related to the occurrence of the episodes . StandarA second approach is to fit a joint model of time-to-clinical malaria and longitudinal parasite count profile. Previous applications of the joint modelling framework are many. These include, for example, analysis of CD4 count jointly with time-to-development of AIDS –12 and mWe investigated whether modelling time-to-new clinical malaria disease jointly with parasite count trajectory data may improve precision in log hazard ratio estimates when compared to the conventional Cox PH model with time-dependent parasite count. We also assessed the strength of the association between the hazard of clinical malaria and a time-varying parasite count.Plasmodium falciparum parasite is high in Chikwawa , 6.3 years (IQR: 3.2–13.1) for males and 9.2 years (IQR: 5.3–18.5) females . The medAnalyses of time-to-new clinical malaria disease included 115 participants who had a least one follow-up visit post enrolment and together contributed a total 894 observations. The median follow-up time to new clinical malaria disease episode was 3.5 months (IQR: 1.1–7.9). Out of the 115 participants, 100 (87.5%) experienced the episode while 15 (12.5%) were administratively right censored . Among 1Regression coefficient estimates are log hazard ratios, log (HRs). Overall, the joint models gave larger log (HR) estimates with consistently smaller standard errors and narrower credible intervals when compared to the conventional Cox PH model with time-dependent parasite count . CompariThe MCMC sampler trace plots from the final joint model4 seemed to mix well for parameters of children under 5 years, 5–15 years and infrequent bed net use and never moved beyond 1.5 achieving convergence –2C. The In unadjusted analyses using Kaplan Meier failure estimator, the risk of experiencing new clinical malaria episode was higher in children under 5 years and the 5–15 years compared to adults above 15 years  . The risThis study has demonstrated that jointly modelling longitudinal parasite count and time-to clinical malaria episodes improves precision in risk factor estimates associated with clinical malaria disease. The joint model yielded larger parameter estimates with consistently smaller standard errors and narrower credible intervals when compared to a conventional Cox PH model with time-dependent parasite count. These results are consistent with findings from other areas including HIV ,12 and cP. Falciparum otherwise acquired with age [Factors associated with a high risk of experiencing a new clinical malaria episode were young age and infrequent use of bed nets as have been reported in other studies –28. Chilwith age . In thisThis study may be limited by focusing on time-to-first malaria episode only, thus estimates obtained here may not be applicable to analyses examining all clinical malaria episodes over a follow up period. This paper has established the optimal way of incorporating parasite count in estimating time-to-first new clinical malaria which may further be extended to study recurrent episodes over entire follow-up, but this may require different distributional assumptions. There were missing values for bed net use which may have affected the findings. Future studies should include multiple imputation of missing covariate data and also investigate the role of measurement error due to detection limit of parasite count in these joint models. Further work is required to consider joint modelling of parasite count with recurrent episodes and to predict risk of future clinical malaria episodes.In conclusion, jointly modelling longitudinal parasite count and time-to-new clinical malaria improved precision in log hazard ratio estimates for clinical malaria when compared with the conventional Cox PH model with time-dependent parasite count. The improved precision of joint modelling may improve study efficiency and allow for design of clinical trials with relatively lower sample sizes with increased power.


Plasmodium falciparum (74 [48%]), P. vivax (69 [45%]), or both organisms (10 [7%]). Of 153 malaria patients, 80 were severe malaria patients presenting with sepsis, 70 were non-severe malaria patients presenting with sepsis, and three were non-severe malaria patients presenting without sepsis. The modified SOFA score  was strongly correlated with malaria severity determined by the number of World Health Organization severity criteria satisfied by the patient . Of 80 severe malaria patients, 2 (2.5%), 11 (14%), 62 (77.5%) and 5 (6%), presented with qSOFA scores of 0, 1, 2 and 3, respectively. Twenty eight-day mortality was 1.3% (2/153). In conclusion, qSOFA and SOFA can serve as markers of disease severity in adults with malarial sepsis. Patients presenting with a qSOFA score of 1 may also require careful evaluation for sepsis; including diagnosis of cause of infection, initiation of medical intervention, and consideration for referral as appropriate.Sepsis can be caused by malaria infection, but little is known about the utility of the quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) and SOFA score in malaria. We conducted a prospective observational study from March 2013 to February 2017 to examine adults admitted with community-acquired infection in a tertiary-care hospital in Ubon Ratchathani, Northeast Thailand (Ubon-sepsis). Subjects were classified as having sepsis if they had a modified SOFA score ≥2 within 24 hours of admission. Serum was stored and later tested for malaria parasites using a nested PCR assay. Presence of severe malaria was defined using modified World Health Organization criteria. Of 4,989 patients enrolled, 153 patients (3%) were PCR positive for either  Sepsis is a life-threatening acute organ dysfunction caused by a dysregulated host response to infection with any organism; including bacterial, fungal, viral and parasitic agents , 2. GlobIn malaria endemic areas, malaria patients are also at risk of developing and presenting as sepsis . In 2016Nonetheless, it is largely unknown whether a significant proportion of severe malaria patients may present with a qSOFA score of 0 or 1. Such patients may be considered not to have sepsis, and as a result not afforded further investigation of the cause of infection or close management [We conducted a four-year prospective observational study from March 2013 through February 2017 NCT02217592) to examine the epidemiology and outcomes of individuals with community-acquired infection and accompanying systemic manifestations of infection in a resource-limited setting of Sunpasitthiprasong Hospital in Ubon Ratchathani province, Northeast Thailand 2 to exam. Ubon RaFrom March 2013 to February 2017, we prospectively enrolled adult patients aged 18 years and older who were admitted with a primary diagnosis of suspected or documented infection made by the attending physician (Ubon-sepsis) . For incThe study team of trained research nurses sequentially screened all medical patients by conducting ward rounds and reviewing admission logs in the emergency department, medical wards and medical ICUs twice daily (morning and afternoon) on each working day. Nurses in the emergency department also notified the study team directly about potentially eligible patients. Written, informed consent was obtained from participants prior to enrollment. For illiterate participants, the study information was read to the participant and their impartial witness, then fingerprinted and signed informed consent was obtained from the participant and their representative, respectively, before enrollment.Following enrollment, patients were evaluated by the study nurses at the bedside using four point-of-care assessments: a whole blood lactate Rapid Diagnostic test (RDT) , a whole blood glucose RDT , pulse oximetry  and the Glasgow Coma Scale (GCS). The results were reported to the attending physicians. The study did not involve any clinical interventions and all medical treatment was provided by the attending physicians and respective medical teams. Twenty eight-day mortality data were collected via telephone contact if subjects were no longer hospitalized and had been discharged alive.Plasmodium were used as described previously [The FilmArray Instrument designed by BioFire Diagnostic  and the Severe Acute Systemic Febrile Illness (SASFI) pouch containing various species of eviously . In shoreviously . The Fileviously . Then, wWe conducted the study in full compliance with the principles of good clinical practice (GCP), and the ethical principles of the Declaration of Helsinki. The study protocol and related documents were approved by Sunpasitthiprasong Hospital Ethics Committee (039/2556), the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (MUTM2012-024-01), the University of Washington Institutional Review Board (42988) and the Oxford Tropical Research Ethics Committee at the University of Oxford (OXTREC172-12).2/FiO2 indices were not available for the majority of subjects due to infrequency of arterial blood gas tests, the score was modified as follows: Subjects were scored a maximum of 2  if they received advanced respiratory support  and arterial blood gas test was not performed. For patients who required mechanical ventilation, the GCS verbal score was calculated by the following formula: (-0.3756) + GCS Motor*(0.5713) + GCS Eye*(0.4233) [Presence or absence of severe malaria was defined using modified World Health Organization (WHO) criteria . Malaria0.4233) . The max33 . The (0.4233) .th and 75th percentiles. Continuous variables and proportions were compared between groups using Kruskal Wallis tests and Fisher exact tests, respectively. A Spearman rank correlation coefficient (Spearman’s rho) was used to assess correlations between malaria severity and qSOFA and modified SOFA score. The McNemar exact test was used to compare sensitivity of different diagnostic criteria. Specificity of qSOFA and modified SOFA score to detect severe malaria was not estimated as the prospective study focusing on patients with community-acquired infection and sepsis [https://dx.doi.org/10.6084/m9.figshare.9765362).Data were summarized with medians and interquartile ranges (IQR) for continuous measures, and proportions for discrete measures. IQRs are presented in terms of 25d sepsis . All anaOver the 4 year study period, 4,989 patients with community-acquired infection were enrolled and evaluated . From MaPlasmodium falciparum (PF), 69 (45%) had P. vivax (PV), and 10 (7%) had mixed PF and PV infection  were positive for nfection . OverallOf 153 malaria-confirmed patients, 114 (75%) had a qSOFA score ≥2 and 150 (98%) met criteria for sepsis, with a modified SOFA score ≥2. Of overall, 80 (52%) met criteria for severe malaria, with at least one modified WHO criterion within the first 24 hours . All 80 Plasmodium organisms at the study hospital. Of the two patients without positive blood films at the study hospital, one patient  had blood film positive only at a primary care unit prior to referral, and data of parasite count was not available and blood films were repeatedly negative at the study hospital. His blood specimen was not tested with the FilmArray. The other patient  had no history of blood film positive for Plasmodium organisms, had a positive dengue rapid test for NS1, and was diagnosed and treated as dengue infection. She improved and survived. She had PCR positive for malaria by the FilmArray platform, which was later confirmed as PV infection by nested PCR assay. We identified another patient  with blood film and nested PCR assay positive for PF, who had a final diagnosis of both severe PF infection and dengue hemorrhagic shock. He had a negative dengue rapid test for NS1 and positive dengue IgM antibody. He was treated as co-infection, improved and survived. In total, only these two patients  probably had co-infection with malaria and dengue in our study.Of 153 malaria-confirmed patients, 151 (99%) had blood films positive for Escherichia coli bacteremia , a second patient had Pseudomonas spp. bacteremia , and a third had non-typhoidal Salmonella bacteremia . Those three patients were also treated with parenteral antibiotics and survived. The median modified SOFA score of those three patients with bacterial co-infection was not significantly higher than those who had no bacterial co-infection .All patients had blood cultures performed based on the study protocol. Three patients (2%) had blood cultures positive for bacterial pathogens: one patient had Of 153 malaria-confirmed patients, 2 (1.3%) died within 28 days of admission. 81% (65/80) and 59% (43/73) of patients with severe and non-severe malaria, respectively, received parenteral artesunate during hospital admission. Among 151 survivors, the median length of hospital stay at the study hospital was 3 days . All patients’ survival outcomes on day 28 were confirmed via telephone contact.The first fatal case  presented at a secondary-care hospital in Ubon Ratchathani province with high fever and a history of generalized convulsion for two times. He had no known underlying diseases. His blood film found PF >10%. He received parenteral diazepam and artesunate, and was transferred to the emergency room of the study hospital where he developed sudden cardiac arrest but was resuscitated. He had a qSOFA score of 3, modified SOFA score of 18 and malaria severity of 8. Parenteral artesunate plus oral mefloquine and parenteral ceftriaxone were prescribed at the study hospital. He died on day 2 after hospital admission. The other fatal case  presented at a secondary-care hospital in Ubon Ratchathani province with high fever, jaundice, dyspnea and hypoglycemia (blood glucose level of 30 mg/dL). He had no known underlying diseases. His complete blood count showed leukocytosis  and mild thrombocytopenia . No malaria parasitemia was mentioned in the medical record. He was treated with parenteral ceftriaxone and glucose, intubated and was transferred. On admission at the study hospital, the patient had a qSOFA score of 3, modified SOFA score of 18 and malaria severity of 9. His blood film found PF >10%. Parenteral artesunate plus oral mefloquine and parenteral ceftrixone were prescribed at the study hospital. He developed acute respiratory distress syndrome (ARDS) and acute kidney injury (AKI), and died the next day.The qSOFA score had a weak correlation with malaria severity  . Of 80 sA sensitivity analysis was conducted by including only the 80 severe malaria patients, and a moderate correlation between qSOFA and malaria severity was observed .The modified SOFA score  had a strong correlation with malaria severity  . All sevA total of 28 malaria-confirmed patients were tested with the FilmArray platform, and 24 of them were positive for the FilmArray. Compared with the nested PCR assay, the diagnostic sensitivity of the FilmArray was estimated to be 86% . The sensitivity was not significantly different by the type of Plasmodium infection .Of 152 malaria-confirmed patients who were tested with blood film, as described above, 151 were blood film positive. Compared with the nested PCR assay, the diagnostic sensitivity of blood film was estimated to be 99% .Of 28 FilmArray-tested cases, 27 were blood film positive and one was blood film negative but FilmArray positive as described above. Therefore, compared with blood film, the diagnostic sensitivity of FilmArray was estimated to be 85% .Our results show that all severe malaria patients presented as sepsis (defined as a modified SOFA score ≥2), suggesting that the current criteria for sepsis are highly sensitive for the identification of patients who are at high risk of death due to malaria infection. Our results also show that a moderate or higher qSOFA score (qSOFA score ≥1) was sensitive for the detection of severe malaria. Nonetheless, fourteen percent of severe malaria patients presented with qSOFA scores of 1. Based on this lower qSOFA score, these patients may not be considered to have sepsis, and therefore not be afforded the requisite further evaluation–including investigation of the etiology of infection–or management. These findings have important implications for triage and judicious resource allocation in the care of malaria patients. Our findings support the suggestion that any patients presenting with even one of three qSOFA criteria at bedside should prompt careful evaluation for sepsis, initiation of appropriate diagnostic testing and medical interventions, and consideration for referral as appropriate  and otheThe strong correlation between the modified SOFA score and malaria severity is because both criteria are based on five similar parameters as shown in Weak to moderate correlation between the qSOFA score and malaria severity is because qSOFA was designed to be simple and available at the bedside without the need for blood tests  on which the majority of malaria severity criteria are based. Therefore, 14% of severe malaria patients presented with a qSOFA score of 1, and these patients might have not been identified as sepsis or severe malaria patients if a range of blood tests were not performed at the study hospital. Although none of the severe malaria patients presenting with qSOFA score of 1 died in our study, this could be due to the standard of care at the study hospital and the study protocol which evaluated the modified SOFA score on all enrolled patients. The outcome of those patients could be poorer if early detection of sepsis and the identification of pathogenic organisms are delayed. Both fatal cases who met criteria for severe malaria presented with high modified SOFA scores. Parenteral artesunate plus oral mefloquine prescribed in one fatal case is not the treatment regimen recommended for severe malaria by the WHO .Our study also shows that severe PV infection defined by the WHO criteria  is not aOur study has limitations. First, although we conducted detailed assessments, specific tests were not always available or conducted  and data of doses of adrenergic agents were not recorded. This could lead to an underestimate of the prevalence of sepsis. Second, due to the study design, specificity of qSOFA and modified SOFA scores to detect severe malaria among patients presenting with community-acquired infection was not estimated. This was because most non-severe malaria patients, who should be the main numerator for estimating specificity, were not transferred to the tertiary-care hospital  and not This study fills a gap in our knowledge about the presenting manifestations of sepsis among patients with non-severe and severe malaria in LMICs, how qSOFA and SOFA score may be useful in the clinical setting, and how these scores correlate with the criteria of severe malaria defined by the WHO.S1 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Plasmodium knowlesi, a malaria parasite typically found in long-tailed and pig-tailed macaques, is the most common cause of human malaria in Malaysian Borneo. Infections in humans result in a spectrum of disease, including fatal outcomes. Spontaneous splenic rupture is a rare, but severe complication of malaria and has not been reported previously for knowlesi malaria.P. knowlesi malaria infection at Kapit Hospital. He was in compensated shock upon arrival to the hospital. He had generalized abdominal tenderness, maximal at the epigastric region. Bedside focused abdominal ultrasonography revealed free fluid in the abdomen. He underwent emergency exploratory laparotomy in view of haemodynamic instability and worsening peritonism. Intraoperatively, haemoperitoneum and bleeding from the spleen was noted. Splenectomy was performed. Histopathological examination findings were suggestive of splenic rupture and presence of malarial pigment. Analysis of his blood sample by nested PCR assays confirmed P. knowlesi infection. The patient completed a course of anti-malarial treatment and recovered well post-operation.A 46-year-old man presented with fever and acute surgical abdomen with concomitant P. knowlesi malaria infection. Detection of such a complication requires high index of clinical suspicion and is extremely challenging in hospitals with limited resources.Spontaneous splenic rupture is a rare complication of malaria. This is the first reported case of splenic rupture in  Plasmodium  until a large focus of human infections with Plasmodium knowlesi was reported in 2004 in the Kapit Division of Sarawak, Malaysian Borneo [Plasmodium knowlesi infections in humans have subsequently been reported throughout Southeast Asia and result in a spectrum of disease from very mild to fatal outcomes [P. vivax although there have been rare cases associated with other Plasmodium species [P. vivax was the most common (15 patients), followed by P. falciparum (5 patients) and P. malariae (2 patients) [P. knowlesi.Malaria in humans was thought to be caused by four species of n Borneo . Plasmodoutcomes . Most of common 1 patientsA 46-year-old man, previously well, presented at Kapit Hospital, Sarawak, Malaysian Borneo with fever, chills and rigors for 2 days. It was associated with severe epigastric and left hypochondrium pain and loose stool. There was no preceding history of trauma. Upon arrival to the emergency unit, his general condition was stable. Physical examination revealed blood pressure of 123/86 mmHg, pulse rate of 114 beats/min and temperature of 39 °C. His respiratory rate was 23 breath/min and the oxygen saturation on room air measured by pulse oximetry was 97%. The abdomen was generally tender and guarded, maximal at the epigastric region. Bedside focused abdominal ultrasonography revealed free fluid in the abdomen. Chest radiograph did not reveal any obvious sign of pneumoperitoneum.3/μL and platelet count of 77 × 103/μL. His creatinine level was 89 μmol/L and electrolytes were within the normal range. The results of the liver function tests were as follows: aspartate aminotransferase 15 U/L, alanine aminotransferase 11.8 U/L and total bilirubin 22.9 μmol/L. Serum amylase was normal. The arterial blood gas revealed good oxygenation and absence of metabolic disturbance . The serum lactate measured was 0.8 mmol/L.Haematological analysis showed haemoglobin of 11.5 g/dL, white blood cell count of 8.2 × 10Plasmodium knowlesi was identified by examination of a Giemsa-stained blood film and the parasitaemia was estimated to be 240 parasites/μL blood. Finger prick blood samples from the patient were spotted on filter paper and sent to the Malaria Research Centre at Universiti Malaysia Sarawak where DNA was extracted as described previously [P. knowlesi, P. falciparum, P. vivax, P. malariae and P. ovale, which indicated the patient was infected with P. knowlesi [eviously . The DNAknowlesi .P. knowlesi malaria.There was no formal radiological imaging done, such as abdominal ultrasonography or computed tomography, since this service is unavailable at Kapit hospital. Four tablets of artemether–lumefantrine were given upon laboratory diagnosis of The patient later developed hypotension despite fluid resuscitation and required single inotropic support, noradrenaline at a dose of 0.2 mcg/kg/min. He simultaneously experienced worsening peritonism. The surgical team was consulted, and he was posted for emergency exploratory laparotomy after stabilization. The anti-malarial was changed to intravenous artesunate in view that the patient was vomiting.Intraoperatively, haemoperitoneum was observed upon entering the abdomen, with blood clots seen overlying the splenic region. Gross examination of the spleen revealed tear of splenic capsule and subcapsular haematoma at the superior pole Fig. . OtherwiStreptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. One month later, he was well upon review at the surgical outpatient clinic.Histopathological examination revealed splenic tissue with focal breaching of the capsule with areas of haemorrhage, consistent with splenic haemorrhage. There were a few brownish pigments within the spleen indicative of malarial pigment. The patient recovered well after the operation and was discharged after 9 days of hospital stay. He was given routine follow up post-malaria treatment and later administered with the routine post-splenectomy vaccinations against P. falciparum is recognized as the most common cause of severe complications in malaria [P. knowlesi with approximately 1 in 10 patients developing severe complications, including fatal outcomes, at a district hospital in Sarawak, Malaysian Borneo [Malaria can present with numerous clinical symptoms and complications.  malaria . Howevern Borneo .P. knowlesi malaria was made in less than 2 h from presentation. After thorough assessment, suspicion of splenic rupture was raised based on clinical grounds and prompt operative intervention was performed. Therefore, early diagnosis of malaria, recognition of splenic rupture and prompt intervention have contributed to the favourable outcome in this patient.Due to the relatively high prevalence of malaria in the Kapit division of Sarawak, there is a high suspicion for malaria in patients who came with febrile illness. In the present case, early diagnosis of Presentation of acute abdominal pain in malaria patients poses a great diagnostic challenge, especially in hospitals with limited resources. Left hypochondrium pain and circulatory shock are the commonest presentation of splenic rupture . ImagingPlasmodium knowlesi parasitaemia upon hospital admission has also been shown to be an independent determinant of respiratory distress, renal and liver impairment [Hyperparasitaemia is one of the criteria of severe malaria and the risk of severe knowlesi malaria increases 11-fold with parasitaemia > 20,000/μL and 28-fold with parasitaemia > 100,000/μL [pairment –14.Traditionally, spontaneous splenic rupture due to malaria is treated with splenectomy . HoweverMalaria remains as a major health problem worldwide. Spontaneous rupture of malarial spleen is rare. Nevertheless, failure to recognize and manage this complication early can adversely affect the patient’s outcome. Malaria management in poorly accessible healthcare services is extremely challenging and greatly dependent on clinical competence and experience.


EMPs) are reported to be increased in acute coronary syndrome (ACS). However, it remains unclear whether EMPs from dysfunctional endothelium participate in the initiation and progression of ACS and what the underlying mechanisms might be.Circulating endothelial‐derived microparticles  stressed by serum‐starvation or hypoxia were compared to the EMPs from healthy HUVECs. Confocal and fluorescent microscopy was used to visualize the incorporation of EMPs into monocytes and the translocation of NF‐кB. Monocyte adhesion, cell proliferation, and phagocytosis were detected by PKH26 red fluorescent labelling, Ki67 immunostaining, and Sudan IV staining for uptake of oxidized low‐density lipoprotein, respectively.Plasma EMPs was significantly increased in ACS patients compared to controls. EMPs were incorporated into monocytes and EMPs from stressed HUVECs produced more pro‐inflammatory cytokines compared to vehicle control, which was depended on NF‐кB and IL‐1β signal pathways. EMPs from dysfunctional endothelium promoted monocyte adherence via NF‐кB and IL‐1β‐mediated MCP‐1 and CCR‐5 signals, as well as proliferation via the NF‐кB and IL‐1β‐mediated Cyclin D1 signals. Finally, EMPs from dysfunctional endothelium showed greater promotion of macrophage phagocytosis forming foam cells to produce more pro‐inflammatory cytokines.Plasma MPs might be involved in the inflammatory process in patients with ACS via NF‐κB and IL‐1β‐dependent signals. Targeting EMP‐mediated inflammatory responses may be a promising therapeutic strategy to limit the progression of disease in ACS. Dysfunctional endothelial cells (ECs) promote leukocyte adhesion and migration, which is a critical step in the initiation and progression of atherosclerotic lesion development leading to coronary artery diseases.Microparticles (MPs), the sub‐cellular particles of a size <1 μm, are derived from the plasma membrane of many eukaryotic cells in response to various biological processes such as cellular activation and apoptosis. It was reported that MPs were also present in excess numbers in disease states such as ACS,EMPs have recently emerged as a surrogate marker of endothelial injury and cardiovascular risk, and high circulating EMP levels were observed in patients with ACS.Although many studies confirmed the ability of EMPs to promote vascular inflammation,22.1A case‐control study was conducted to investigate the levels of EMPs in ACS, and 22 ACS patients complaining of chest pain in the internal medicine cardiovascular ward of Shanghai East Hospital were recruited, with 20 hospitalized patients with normal computer tomography coronary angiography acting as controls. The diagnosis of ACS was based on the American Heart Association criteria.The institutional review board of Tongji Medical School affiliated Shanghai East Hospital approved the study protocol, all studies were performed in accordance with relevant guidelines and regulations, and written informed consent was obtained in all cases.2.2g. The resuspended PPP 50 μL was incubated with 5 μL fluorescein isothiocyanate (FITC)‐labelled anti‐Annexin V  or 10 μL of phycoerythrin (PE)‐labelled anti‐CD144  for 20 minutes with gentle orbital shaking, then 50 μL standard MPs (Beckman Coulter) added followed by 1 mL phosphate buffered saline (PBS). The MP samples present in PBS were analyzed regarding size and fluorescence by flow cytometry in FACS Arial I . On a log forward scatter/log side scatter dot plot, we defined the upper size limit of the MPs using 1 μm calibrant beads and drew a gate around the MP population. Only the MPs included in this gate were further analyzed for their fluorescence, and 5000 standard beads were counted and used to calculate the concentrations of EMPs for each sample. EMPs were defined as elements with a size <1 μm that were positively labelled with FITC‐annexin V and PE‐CD144.The EMPs was measured by flow cytometry as previously described.2.3http://www.kangchen.com.cn/ support/Supportmain.asp?ID=129). Twofold and greater changes were considered significant. Hierarchical clustering was used to generate heatmap in order to improve the understanding of the systematic variations in pro‐inflammatory cytokines levels between ACS and non‐ACS patients.Serum samples from three ACS and three non‐ACS patients were collected and total protein concentrations were quantified by BCA protein Assay Kit . The samples were performed for RayBio® C‐Series Human Atherosclerosis Antibody Array C1  of KangChen Bio‐Tech, Shanghai, China, according to the manufacturer's manual by blocking, incubation and detection. Data from these arrays were analyzed  were cultured in RMPI 1640 culture medium  supplemented with 10% of FBS and the cell density was kept between 1 × 106 and 5 × 106 cells in a 25 cm2 flask.Human umbilical vein endothelial cells  were cultured in EC growth media with SupplementMix  at 37°C, 5% of CO2.5g for 15 minutes to remove cell debris. Then the supernatant was further centrifuged at 20 000 g for 40 minutes and the EMPs collected for further characterization. A 50 μL of EMPs were mixed with 5 μL of FITC‐labelled anti‐Annexin V and 10 μL of PE‐labelled anti‐CD144. After incubation at room temperature for 20 minutes, the labelled EMPs were washed two times and resuspended in 100 μL PBS for flow cytometry according to the method described in quantification of patient EMPs.EMPs were generated from HUVECs as previously described.HUVECs cultured under normal condition rarely produce EMPs. HUVECs cultured with starvation or under hypoxia condition produce significantly increased EMPs compared to the HUVECs under normal culture condition, similar to the significantly increased circulatory EMP levels in ACS patients compared to non‐ACS patients.g for 15 minutes to remove cell debris. Then, centrifuged at 20 000 g for 40 minutes and the EMPs collected, which were resuspended with 100 μL PBS and added to the THP‐1 culture in 1:50 dilution. The same procedure of EMPs preparation was performed in HUVECs under normal culture conditions which were used as vehicle control to compare with EMPs from HUVECs with starvation and under hypoxia condition, which were considered as EMPs from dysfunctional ECs.For functional study of the EMPs, the supernatant from serum‐starved or hypoxia‐exposed HUVECs cultured in a 10 cm dish was collected and centrifuged at 1500 2.6To visualize the incorporation of EMPs on monocyte/macrophages in a time‐dependent manner, EMP‐derived from HUVECs were labelled with PKH26 red fluorescent cell liner Mini Kit  for 30 minutes, washed twice, and resuspended in PBS, and these red fluorescence‐labelled EMPs were added to the THP‐1 culture medium. Images were taken at 2 and 4 hours by a confocal microscopy  to visualize the incorporation of EMPs into the membrane of THP‐1 monocytes. Further studies were performed using the same amount of empty PKH26 red fluorescent dye but without EMPs as a control for dye‐labelled EMPs derived from HUVECs to rule out the non‐specific lipophilic binding of non‐incorporated dye to the membrane of monocyte/macrophage . Reverse transcription was carried out by a TaKaRa Primescript™ RT reagent kit  according to the manufacturer's instructions. The quantification of gene expression was performed by real time quantitative PCR on a QuantStudio™ 6 Flex Real‐time PCR system . The Ct values were normalized for the housekeeping gene GAPDH, and then the relative gene expression was calculated using expression of vehicle treated cells as a control. Primers used for quantitative real time PCR were included in Supplementary Table 2.8THP‐1 cells pellets were extracted in RIPA lysis buffer  containing protease inhibitor cocktail . Protein concentrations were determined by a BCA kit . Equal amounts of cell extracts were electrophoresed on sodium dodecyl sulfate‐polyacrylamide gels, and then transferred onto polyvinylidene difluoride membranes . The membranes were blocked in 5% non‐fat dried milk in TBS‐T at room temperature for 2 hours and incubated with indicated primary antibodies at 4°C overnight. The primary antibodies used in the study were: anti phospho‐NF‐кB (Ser468) , anti‐TNFα , and anti β‐actin . The membranes were washed three times with TBS‐T for 10 min each time, and then incubated with IRDye‐680LT Goat Anti‐Mouse IgG (H+L)  or IRDye‐800CW Goat Anti‐Rabbit IgG (H+L)   at room temperature for 1 hour. After washing in TBS‐T for another three times, the membranes were analyzed by the Odyssey infrared imaging system .Levels of IL‐1β were determined in EC supernatants by an ELISA assay kit  according to the manufacturer's instruction.2.9THP‐1 Cells cultured on sterile glass cover slips in 24‐well plates were fixed with 4% of paraformaldehyde (PFA) in PBS, washed, and permeabilized with 0.1% of Triton X‐100 PBS. The cells were blocked with 1% goat serum for 1 hour and incubated with primary antibodies: rabbit anti‐NF‐кB  or rabbit anti‐Ki67 . After washing, the cells were incubated with Alexa Fluor 488‐conjugated donkey anti‐rabbit secondary antibodies  for 45 min. Then, the cover slips were mounted with Prolong Gold Anti‐fade reagent with 4′,6‐diamidino‐2‐phenylindole (DAPI) . The immunostaining images were captured under a Leica fluorescent microscopy .Monocyte/macrophages are phagocytic cells, which engulf oxidized low‐density lipoprotein (ox‐LDL) to form foam cells and produce pro‐inflammatory cytokines. Actin cytoskeletal remodelling is required for the process of phagocytosis of monocyte/macrophages, thus actin immunostaining was performed using Alexa Fluor 488 conjugated‐phalloidin  and quantification by calculating the relative cell area as the actin filament cytoskeletal spreading, which reflected the capacity of phagocytosis as previous described.2.105) were plated in 24‐well plates and treated with vehicle control and EMPs, as well as EMPs with NF‐кB inhibitor ammonium pyroolidinedithiocarbamate (PDTC) and IL‐1β inhibitor diacerein for 48 hours, and then ox‐LDL  was incubated with these cells for 9 hours. After being fixed with 4% PFA for 10 min, the cells were stained with Sudan IV for 6 min, and rinsed with 80% ethanol 3 times. The cell images were captured under a Leica fluorescent microscopy and the percentage of the Sudan IV positive cells of the total cells was used for quantification of macrophage phagocytosis activity.Oxidized low‐density lipoprotein (ox‐LDL) uptake by THP‐1 monocytes was used to determine the phagocytosis activity as previously described.2.114 THP‐1 cells were treated with dysfunctional EMPs or vehicle control for 48 hours, then labelled with 5 mM PKH26 red fluorescence cell linker Mini kit  and added into 70%‐80% confluent HUVECs in a 96‐well plate. The non‐adherent cells were removed by rinsing with PBS three times after incubation at 37°C for 1 hour. Monocyte‐endothelial adhesion was quantified by measuring the fluorescence at excitation and emission wave lengths of 485 and 535 nm, respectively, and counting the number of attached monocytes in 10 random high‐power (400×) microscopic fields of images taken by Leica fluorescence microscopy. Wells containing ECs without monocytes served as blank control. To investigate whether the adhesion is dependent on the NF‐кB or IL‐1β pathway, NF‐кB, or IL‐1β specific inhibitors PDTC (1 μM) and diacerein (50 μM) were added to see whether PDTC or diacerein reversed the EMP‐induced increased monocyte adhesion to ECs.Adhesion of monocytes to ECs was performed as previously described.2.125 THP‐1 cells per well were seeded in 12‐well plates and treated with EMPs. The cells of each well were harvested at 24, 48, 72 hours and the number of trypan blue negative live cells were counted under a hemocytometer. PDTC (1 μM) and diacerein (50 μM) were added to test whether monocyte/macrophage proliferation is dependent on the NF‐кB and IL‐1β pathways.Ki67 immunostaining and cell count assay were used to evaluate the cell proliferation. In the cell count assay, 1 × 102.13Unless indicated, all chemicals and solvents were from Sigma‐Aldrich. NF‐кB inhibitor ammonium pyroolidinedithiocarbamate (PDTC)  was dissolved in PBS at 1 mM stock solution and used at a final concentration of 1 μM, IL‐1β inhibitor diacerein  was dissolved in DMSO at 2.5 mM stock solution and used at a final concentration of 50 μM. Ox‐LDL  was used at a final concentration at 50 μg/mL. All stock solutions were protected from light and kept at −20°C, except ox‐LDL which was stored at 4°C. The solvent was used as a control vehicle in all experiments.2.14P < 0.05 was considered statistically significant.Data were presented as mean ± SEM of the indicated number of independent experiments. All data were analyzed by one‐way analysis of variance (ANOVA), and the least significant difference (LSD) post‐hoc test was used for multiple comparisons. Statistical analyses were performed using the SPSS software version 19.0 . 33.1P > 0.05). In contrast, the high‐sensitivity troponin T  and CK‐MB (P = 0.045) in ACS patients was significantly higher than that in non‐ACS patients, and ACS patients had significantly higher hs‐CRP level (P = 0.018) which indicates higher inflammatory status.Basic clinical characteristics of patients were shown on Table 3.2P < 0.001).The plasma EMPs were determined by flow cytometry using CD144 and Annexin V antibody and the double positive MPs with diameters under 1.0 μm considered as EMPs  and PE‐labelled anti‐CD144  to identify and quantify EMPs and the result confirmed increased circulatory EMP levels in patients with ACS who also tended to have higher serum levels of hs‐CRP, as well as increased TNF‐α and IL‐1β in the plasma.Recent studies found that EMPs can affect inflammation, blood coagulation, and vascular functions and are closely related to the occurrence of cardiovascular disease.Flow cytometry can assess EMPs in patients’ plasma, but unfortunately it is technically difficult to separate EMPs from patients for further functional evaluation. Recently one study collected EMPs from the supernatant of apoptotic human coronary arterial endothelial cells (HCAECs) after starvation for functional study of EMPs.Dysfunctional ECs promote leukocyte adhesion and migration, which is a critical component in the initiation and progression of atherosclerotic lesion development.The NF‐кB transcription factor family is a pleiotropic regulator of many cellular pathways, providing a mechanism for the cell to respond to a wide variety of stimuli and environmental challenges.Beyond their roles in monocyte adhesion and proliferation to increase the inflammatory cell number in the blood vessels, EMPs also influence macrophage functional status which contributes to vascular inflammation and atherosclerotic lesion development. Our study demonstrated that EMPs were incorporated into the monocyte membrane and promoted the NF‐кB p65 subunit phosphorylation and further translocation to the nucleus to increase the production of pro‐inflammatory cytokines.The phagocytosis of ox‐LDL by macrophages and the subsequent formation of foam cells are crucial for atherosclerotic lesion development.This study has limitation including relative small sample size and usage of EMPs from dysfunctional endothelial cell line. It would be ideal to use EMPs from ACS and non‐ACS patients to assess the different functions ex vivo. However, there may also be many other interacting factors in ACS patients, such as different background cardiovascular risk factors and differences in treatments used that may complicate such an analysis. It was also not practical to separate EMPs from patients’ samples that had been collected, the patient plasma has many MPs from different origins that will not reflect the function of EMPs, and more reproducible EMPs from endothelial cell line was adopted for further functional study.In summary, ACS patients have higher circulatory EMP levels, which may participate in the regulation of NF‐кB and IL‐1β signal pathways of monocytes/macrophages and influence cell adhesion, proliferation, and macrophage phagocytic capability, contributing to the vascular inflammation associated with atherosclerosis progression in ACS patients. Therefore, future targeting of EMP‐mediated inflammatory responses in vessel wall may be a promising therapeutic strategy to limit the progression of atherosclerotic lesion formation post‐ACS.The authors declare that they have no conflicts of interest.YF W, J L, XL C, and S P conceived and designed the study, together HM S, YS K, JJ P, and T Z performed the experiments; L Z, ZR Y, B T, P C, and YH C guided the study; YZ Z and Y L designed the study and wrote the manuscript. Click here for additional data file.


Anaemia and malaria are common and life-threatening diseases among preschool-aged children in many tropical and subtropical areas, and Malawi is no exception. Accordingly, this study aimed to examine the association of referral clinical malaria with anemia (hemoglobin [Hb] < 110 g/L) in preschool-aged children in Malawi.P.f/Pan test histidine-rich protein (HRP-II)™), Hb (using HemoCue 301), α-1-acid glycoprotein (AGP), and serum ferritin biomarkers  and inherited blood disorders from dry blood samples (DBS) using polymerize chain reaction (PCR). Diagnosis of clinical malaria was made on the basis of fever and a positive rapid diagnostic test (RDT).Using cross-sectional data obtained from the 2015–2016 Malawi Micronutrient Survey (MNS), multivariate logistic regression models were constructed using surveylogistic to account for the complex survey design. Blood samples of 1051 children aged 6–59 months were evaluated for malaria , P <  0.0001.Of the 1051 PSC analysed, 29% had anaemia while 24.4% had a referral to the hospital due to malaria. After adjustments for known confounders, PSC with a history of referral clinical malaria had increased odds of being anaemic (adjusted odds ratio [aThis study found that clinical malaria increased the risk of anaemia in PSC. Thus, elimination of malaria-causing parasites from the PSC’s blood should be rapid and complete in order to prevent the progression of uncomplicated malaria to a chronic infection that can lead to the development of malaria-related anaemia. Please see Additional file According to the World Health Organization (WHO), anaemia is a condition characterized by the reduced number of red blood cells (RBCs) and consequently their oxygen-carrying capacity is insufficient to meet the body’s physiologic needs . GloballPlasmodium parasites which are transmitted to people through the bites of infected female Anopheles mosquitoes [Malaria is also a major cause of morbidity and mortality among preschool-aged children in sub-Saharan Africa . It is rsquitoes . In Malasquitoes . This desquitoes . Unfortusquitoes . Generalsquitoes .Plasmodium falciparum involves an increased removal of parasitized and unparasitized RBCs through cytokine-mediated dyserythropoiesis and bone marrow suppression to iron delocalization [Malaria parasites constitute one of the most complicated and multifactorial life cycle . The Plalization . Preciselization . As suchlization –20 howevThis study was conducted in Malawi, a sub-Saharan African country located south of the equator. Malawi is bordered by the United Republic of Tanzania, the People’s Republic of Mozambique, and the Republic of Zambia. Malawi has a tropical continental climate with maritime influences. Rainfall and temperature vary depending on altitude and proximity to the lake . MalariaThe current study utilized data taken from the 2015–2016 Malawi Micronutrient Survey (2015–16 MNS) . The MNSData were collected from women aged 15–49 with children younger than five years of age prior to the survey using standard questionnaires. In the DHS data, of the 25 146 eligible households, 24 562 were successfully interviewed representing a 98% response rate. Information on sociodemographic, household and economic factors were collected through face-to-face interviews. As per WHO recommendations, analysis of the current study was restricted to all PSC aged 6–59 months [P.f/Pan histamine-rich protein (HRP-II)™ RDT) – Standard Diagnostics Inc., Suwon City, South Korea [3.7) was rated using a copy-number variant TaqMan assay with custom TaqMan probes, as described elsewhere [An axillary temperature of every child was documented where by fever was defined as an axillary temperature of equal to or greater than 37.5 °C. Regarding to laboratory, approximately 7 ml of blood samples were collected from PSC for biochemical and haematological tests. About 5 ml of the blood sample was transferred into trace free elements  test tube and 2 ml into Purple Top – Ethylenediaminetetraacetic acid (EDTA) test tube . About 1th Korea , 24. Theth Korea . A combith Korea . To identh Korea . To detelsewhere . The prolsewhere . In brieChildhood anaemia was the dependent variable of this study. Using the WHO recommendations, anaemia in PSC was characterized as children with Hb concentration < 110 g/L after adjusting for altitude .Plasmodium falciparum positive blood smear [Plasmodium falciparum result was confirmed using the RDT [Clinical malaria was the main independent variable assessed in this study. Clinical malaria was defined as any PSC with malaria-related symptoms such as fever – an axillary temperature ≥ 37.5 °C, chills, severe malaise, headache or vomiting at the time of examination or 48 h prior to the examination in the presence of a od smear . However the RDT .The covariates included in this study were; age of the child in months, sex of the child, and fever in last two weeks, malaria test result, α-1-acid glycoprotein (AGP), serum ferritin, α-thalassemia, household hunger scale, type of place of residence, and region of residence were used as covariates. The sex was grouped as , the age of the children was categorized as 6–11, 12–23, 24–35, 36–47, and 48–59 months. As regards fever in the last two weeks, the respondents were asked if the child had a fever (yes/no) in the last two weeks, while malaria test result was categorized as positive or negative. AGP and serum ferritin were grouped as normal or abnormal. Abnormal value for AGP was AGP levels of greater than 1 g/L and abnormal levels for serum ferritin were less than 12 μg/L. Serum ferritin adjusted for inflammation using internal regression approach . The houP-values from the bivariate analyses were reported using Rao-Scott Chi-Square to test the differences between groups anaemic (yes/no). All factors that showed significance at P ≤ 0.25 in the bivariable analyses were fitted into the final models of the regression analyses in order to have the best fit statistical model. The multivariate regression analyses were performed using Surveylogistic where anaemia was the dependent variable and characteristics such as sociodemographic, clinical, biochemical, and inherited blood disorders were the independent variables. Adjusted odds ratio (aOR) with their p-values and 95% confidence interval (CI) were reported.All statistical analyses were conducted using SAS software version 9.4 . To account for the complex survey design, the survey-specific SAS procedures for weighting, clustering, and stratification in the survey design (SurveyFreq) was used to report the basic statistics. Baseline characteristics were reported as weighted frequency and percentages. The https://dhsprogram.com/What-We-Do/Protecting-the-Privacy-of-DHS-Survey-Respondents.cfm). The survey was implemented by the National Statistics Office (NSO) and the Community Health Sciences Unit (CHSU). At the beginning of each interview and prior to blood collection, informed consent from MNS eligible households and the survey participants were sought. Furthermore, community leaders provided the consent of survey activities. ICF Macro IRB ensures that the survey is in line with the U.S. Department of Health and Human Services regulations for the protection of human subjects (45 CFR 46), while the NHSRC ensures that the survey was conducted with laws and norms of the Malawi.Protocols for the MNS 2016 were approved by the National Health Sciences Research Committee (NHSRC) and the Institutional Review Board (IRB) of ICF Macro . The prevalence of clinical malaria was 27.4%. Similarly, the prevalence of P <  0.0001) – Fig. P <  0.0001), in PSC with a positive RDT (P <  0.0001), in PSC with referral clinical malaria (P <  0.0001). Furthermore, the prevalence of anaemia was high in PSC with abnormal levels of AGP (P <  0.0001) and serum ferritin (P <  0.0001).Table OR = 4.63; 95 CI: 2.90–7.40); P <  0.0001, compared to those PSC without a history of clinical malaria even after adjusting for known factors associated with anaemia. Additionally, the odds of anaemia were high in PSC of the age group 6–11 months ; P = 0.0003, in PSC with a history of fever in the last two weeks ; P = 0.0324, in children with high levels of AGP ; P = 0.0010, those with iron deficiency ; P = 0.0014, and those with alpha-thalassemia disease ; P = 0.0004.Table The aim of this study was to examine the likelihood of clinical malaria and the development of childhood anaemia in Malawi. This is the first study to report the association of clinical malaria and the likelihood of anaemia in PSC using a nationally representative sample in Malawi. Accordingly, this study revealed that children who had clinical malaria were more likely to develop anaemia.Anaemia occurs when RBCs are destroyed at an increased rate than the rate at which are supposed to be replaced or when RBC production falls below the rate at which the body requires to maintain a steady state . The patAnopheles mosquito [Malarial infection begins when sporozoites are inoculated together with anti-coagulant saliva in the course of a blood meal of an infected mosquito , 35, 36.mosquito , 37. Durmosquito .Plasmodium species induce homeostatic imbalance and lower Hb concentration thus resulting in anemia [Infection with n anemia . It has n anemia . Iron den anemia , 40. Genn anemia .Another pathway by which an altered cytokines balance and inflammation can induce anemia is through bone marrow depression (dyserythropoiesis) and erythrophagocytosis following low IL-10 and IL-12 or excess of T helper cell type 1 (th1), cytokines follicular helper T cells (TFH), TNF- α and nitric acid (NO) . Often tIn addition to clinical malaria, the other characteristics such as the age of the child, a history of fever in the last two weeks, AGP, serum ferritin, and alpha thalassemia were significantly associated with anaemia in PSC. The results of age are in line with the past research where it was reported that anaemia is frequent among children around the time of the growth spurt, especially between the ages of 6 and 24 months , 55. DurThe findings can be generalized in the Malawian context due to the use of the nationally representative sample. The use of malaria RDTs was helpful since they can assist in making a rapid, accurate diagnosis in circumstances where the microscopy-based diagnosis may be unreliable or available. The use of serum ferritin and alpha-thalassemia makes our results on clinical malaria to be robust and valid. However, the use of cross-sectional study design cannot provide a causal relationship between the explanatory variable and anaemia. The HemoCue system was used for assessment of Hb levels. Additional studies may need the use of other Hb indices to define anaemia. The RDT may not be able to detect some infections with lower numbers of malaria parasites circulating in the patient’s bloodstream. In addition, all positive RDTs should be followed by microscopy.Plasmodium parasite from the PSC’s blood should be rapid and complete in order to prevent progression of uncomplicated malaria to a chronic infection that leads to malaria-related anaemia. The results of this study have some policy implications, i.e. programmes that aim at combating anaemia in infants and young children should not focus on iron supplementation, deworming treatment, and complementary feeding practices as strategies to manage anemia, diagnosis and case management of malaria should also be considered promptly as it has been proved by this study that clinical malaria is associated with anaemia in PSC thus, posing threats to the strategies that were out in place to tackle anaemia in Malawi.In this study we aimed to examine the likelihood of clinical malaria and the development of anaemia among PSC in Malawi. Indeed, the results of this study have demonstrated that clinical malaria is a potential risk factor for anaemia in PSC. Thus, clearance of the Additional file 1: Multilingual abstracts in the five official working languages of the United Nations.