Plasmodium ovale spp. and Plasmodium malariae significantly increased, which resulted in increased risks during the malaria elimination phase. The purpose of this study was to acquire a better understanding of the epidemiological characteristics of P. ovale spp. and P. malariae imported to Hubei Province, China, so as to improve case management.There have been an increasing number of imported cases of malaria in Hubei Province in recent years. In particular, the number of cases of P. ovale spp. and P. malariae malaria.Data on all malaria cases from January 2014 to December 2018 in Hubei Province were extracted from the China national diseases surveillance information system (CNDSIS). This descriptive study was conducted to analyse the prevalence trends, latency periods, interval from onset of illness to diagnosis, and misdiagnosis of cases of P. ovale spp.  and 18 P. malariae cases were confirmed. The latency periods of P. ovale spp., P. malariae, Plasmodium vivax, and Plasmodium falciparum differed significantly, whereas those of P. ovale curtisi and P. ovale wallikeri were no significant difference. The proportion of correct diagnosis of P. ovale spp. and P. malariae malaria cases were 48.3% and 44.4%, respectively, in the hospital or lower-level Centers for Disease Control and Prevention (CDC). In the Provincial Reference Laboratory, the sensitivity of microscopy and rapid diagnostic tests was 94.3% and 70.1%, respectively, for detecting P. ovale spp., and 88.9% and 38.9%, respectively, for detecting P. malariae. Overall, 97.7% (85/87) of P. ovale spp. cases and 94.4% (17/18) of P. malariae cases originated from Africa.During this period, 634 imported malaria cases were reported, of which 87 P. ovale spp. and P. malariae cases, long latency periods, and misdiagnosis pose a challenge to this region. Therefore, more attention should be paid to surveillance of imported cases of P. ovale spp. and P. malariae infection to reduce the burden of public health and potential risk of malaria.The increase in the number of imported  Plasmodium species  [P. ovale spp. and P. malariae compared with P. falciparum and P. vivax, since malaria caused by P. ovale spp. and P. malariae has a more benign clinical course than that caused by P. falciparum. Therefore, malaria caused by P. ovale spp. and P. malariae have been neglected and are understudied [Malaria is one of the major global public health threats and causes substantial morbidity, mortality, and human suffering. In 2017, an estimated 219 million malaria cases occurred worldwide, with 435,000 deaths reported [rstudied –8.P. ovale spp. and P. malariae malaria cases has increased yearly in China [P. ovale spp. malaria cases have ever been reported in Hubei Province, and no local P. malariae malaria cases have been detected since 1963 [P. ovale spp. and P. malariae, significantly increased, which resulted in increased risks during the malaria elimination phase [P. ovale spp. and P. malariae often had longer latency periods than those infected with P. falciparum, and they may present with malaria symptoms months or even years after returning from an endemic region [In recent years, the number of imported in China , 10. Hownce 1963 . Since 2on phase , 13. Patc region , 15. TheP. ovale spp. have been distinguished: P. ovale curtisi and P. ovale wallikeri [P. ovale spp. was identical on microscopy, but the two can be differentiated by genetic typing [P. ovale spp. in Hubei. In light of the epidemiology of P. ovale spp. and P. malariae, a comprehensive analysis was required to explore the changing epidemiology and challenges encountered in Hubei with respect to malaria elimination. Thus, in this study, the data were obtained from the China National Disease Surveillance Information System (CNDSIS) during 2014–2018. This descriptive study was conducted to analyse the prevalence trends, latency periods, interval from onset of illness to diagnosis, and misdiagnosis of cases of P. ovale spp. and P. malariae malaria. The evidence of this study will help in providing a reference for preventing and managing imported P. ovale spp. and P. malariae malaria cases in Hubei Province.In recent years, two sympatric species of allikeri . The morc typing , 17. To Data on malaria cases in Hubei Province from 2014 to 2018 were obtained from (CNDSIS). The following details of patients with malaria were extracted: gender, age, occupation, admission dates, symptom onset time, time of diagnosis, laboratory-confirmed results, travel history.Plasmodium species and two sympatric subspecies of P. ovale spp. at the molecular level, nested PCR was performed by amplifying the 18SSU rRNA gene of the malaria parasite. The nested PCR protocol was followed: DNA isolation was extracted by using the QIAamp DNA Mini Kit (QIAGEN®) from the blood sample following the protocol. The first reaction of DNA amplification was performed by using rPLU5 and rPLU6 primers (Thermos®) [®), 1.1 μl of each primers (10 μmol/l), 1.6 μl of dNTPs (2.5 mmol/l), 4.0 μl of 5× Buffer and 9.0 μl of ddH2O. For the second reaction, there are five pairs of primers were used: rVIV1/rVIV2 (P. vivax), rFAL1/rFAL2 , rMAL1/rMAL2 , rOVA1/rOVA2  and rOVA1v/rOVA2v . These were used to determine the Plasmodium species [®), 1.0 μl of each primers (10 μmol/l), add enough ddH2O to make up the volume to 20 μl. The incubation conditions of the two PCR rounds were as follows: 3 min at 94 °C; followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, 1 min at 72 °C, and a final extension for 5 min at 72 °C. The PCR products of the second reaction were run on standard 1.5% agarose gels.All cases were initially diagnosed using thick and thin blood film microscopy at hospitals or low-level Center for Disease Control and Prevention (CDC). Thereafter, all results were then sent to the Hubei Provincial Reference Laboratory for Malaria Diagnosis. All cases were confirmed by the Provincial Reference Laboratory for using thick and thin blood film microscopy, malaria rapid diagnostic tests (RDTs), and nested polymerase chain reaction (nested PCR). Thick and thin blood films were stained using a standard technique with Giemsa solution for 35 min; they were then examined by trained microscopists to diagnose and identify malaria species. Next, 5 μl of whole blood of each patient was analysed using the Ag Pf/Pan RDTs  following the manufacturer’s protocol. As nested PCR is more sensitive and specific in determining the malaria parasite species , 19; forhermos®) . The PCR species , 20. ThePlasmodium infections. Because the latency periods and interval from the onset of illness to diagnosis had non-normal distribution, the differences were compared using Kruskal–Wallis H tests and two independent sample Mann–Whitney U tests, with p-values < 0.05 considered statistically significant. The ArcGIS software version 10.0  was used to produce figures of the geographic distribution of the countries of origin of the imported P. ovale spp. and P. malariae cases.Statistical analyses were performed using the SPSS software version 16.0 . Owing to the lack of the exact date of malaria infection, the date of arrival in China was used as the date of malaria infection. The latency period, in days, was calculated for each case of malaria by subtracting the date of arrival in China from the date of symptom onset. The confirmed diagnosis interval in days was calculated from the onset of symptoms to laboratory confirmation in provincial CDC. The latency period and confirmed diagnosis interval were calculated and compared for all malaria cases except mixed P. falciparum (440), P. vivax (84), P. ovale spp. (87), P. malariae (18) and mixed infection (3) accounted for 69.4%, 13.2%, 13.7%, 2.8%, and 0.5% of all reported cases, respectively. The P. ovale spp. incidence ranged from 11 to 24 cases per year and that of P. malariae from 0 to 7 cases per year for the period 2014–2018. Among the 87 patients infected P. ovale spp. and 18 patients infected with P. malariae, there was only one patient infected with P. ovale spp., who was a Nigerian, the rest were all Chinese who worked in a malaria-endemic countries as migrant workers.During 2014–2018, a total of 634 imported malaria cases were reported in Hubei Province . For all P. malariae patients, the median and interquartile range of duration of stay overseas was 360.5 days (222.5–667.8). In total, 87 P. ovale spp. cases, including 61 P. ovale curtisi and 26 P. ovale wallikeri   days, P. malariae was 20.5 (2.5–54.25) days, P. vivax was 37 (9.75–89) days, and P. falciparum was 6 (2–10) days. There was a significant difference in latency periods of four Plasmodium species . The median (IQR) latency periods of P. o. curtisi and P. o. wallikeri were 62 (24–195) days and 33 (14.25–129) days, respectively. There was no significant difference in the latency periods between P. o. curtisi and P. o. wallikeri .The latency periods from returning to the country to the onset of symptoms are shown in Fig. P. ovale spp. was 9 (4–43.5) days, P. malariae was 13.5 (7.5–77.25) days, P. vivax was 4 (2–9) days, and P. falciparum was 3 (1–5) days. There was a significant difference in the interval from the onset of illness to diagnosis among four Plasmodium species . The median intervals from the onset of illness to diagnosis (IQR) of P. o. curtisi and P. o. wallikeri were 7 (3–34) days and 15 (4.25–62.75) days, respectively. There was no significant difference in the time from the onset of illness to diagnosis between P. o. curtisi and P. o. wallikeri .The intervals from onset of the illness to diagnosis are shown in Fig. P. ovale spp. and P. malariae is shown in Table P. ovale spp. cases, only 42 had an accurate initial diagnosis, accounting for 48.3% of the P. ovale spp. cases. Similarly, of 18 P. malariae cases, only eight had an accurate initial diagnosis, accounting for 44.4% of all P. malariae cases.The misdiagnosis of P. ovale spp. and P. malariae are shown in Table P. ovale spp. cases and 18 P. malariae cases were diagnosed by nested PCR. Of the 82 P. ovale spp. cases, 5 were misdiagnosed as P. vivax on microscopy. Of the 18 P. malariae cases, 2 were misdiagnosed as P. vivax on microscopy. RDT sensitivity of infections resulting from P. ovale spp. and P. malariae were 70.1% and 38.9%, respectively.The sensitivity of microscopy and RDTs for diagnosing P. ovale spp. cases and 18 imported P. malariae cases were acquired from 24 countries located in Africa, Asia, and Oceania. Overall, 97.7% (85/87) of P. ovale spp. cases and 94.4% (17/18) of P. malariae cases were from Africa, with only two P. ovale spp. cases from Asian countries and one P. malariae case from Oceania (Papua New Guinea). The countries of origin of cases imported from African countries are shown in Fig. During 2014–2018, the 87 imported P. o. curtisi and 96.2%(25/26) of P. o. wallikeri were from Africa. P. o. curtisi cases were mainly imported from Democratic Republic of Congo , Nigeria , Equatorial Guinea , Angola , and Congo (Brazzaville) , and one P. o. curtisi case from Turkey. P. o. wallikeri cases were mainly imported from Democratic Republic of Congo , Cameroon , Nigeria , Angola , Congo (Brazzaville) , and Uganda , and one P. o. wallikeri case from Bangladesh. Plasmodium malariae was mainly imported from Angola , Congo (Brazzaville) , Cameroon , Liberia , and Nigeria .98.4% (60/61) of P. falciparum, the number of patients with imported P. ovale spp. and P. malariae significantly increased, especially the number of patients with imported P. ovale spp. was more than that with P. vivax from 2014 to 2018. The increase in the number of imported P. ovale spp. and P. malariae cases presented a new challenge for achieving the long-term goal of malaria elimination [Hubei Province has achieved remarkable success in controlling the local malaria situation; however, the number of imported malaria cases has increased. Although the majority of imported malaria cases were associated with mination .Plasmodium species. However, the difference between the two sympatric species of P. ovale spp. was not significant in this study, which is similar to the finding of Cao et al. [P. o. curtisi and P. o.e wallikeri were 733 and 793 days, respectively. The longest latency periods of P. o. curtisi in the study by Nolder et al. [P. ovale spp. and P. malariae were longer than those of P. vivax and P. falciparum. Because of the rare occurrence of indigenous P. ovale spp. and P. malariae malaria in Hubei Province, these Plasmodium species have been largely neglected. Moreover, some patients did not visit the hospital immediately because of their mild symptoms. However, delayed diagnosis of these two Plasmodium species will lead to worse clinical outcomes [In this study, there was a significant difference in the latency period of the four o et al.  and Rojoo et al. . In thisr et al.  and Zhour et al.  were 108r et al. . The intoutcomes .P. ovale spp. and P. malariae malaria cases were 48.3% and 44.4%, respectively, in hospitals or lower-level CDC. The misdiagnosis were driven by two factors, one was the health technicians’ lack of knowledge about these two species of malaria [Plasmodium parasites at all levels of CDC and hospitals; furthermore, a standard malaria testing procedure should be established at a lower level [In this study, the rates of correct diagnosis of  malaria , and the malaria –31. Therer level .Plasmodium species is one of the important steps in malaria elimination. Common methods for the diagnosis of malaria include thick and thin blood film microscopy, RDTs, and PCR. The current gold standard for malaria diagnosis remains microscopy. However, this technique needs considerable training and experience. It was difficult to identify P. ovale spp. by only microscopic examination, because the morphology of this species is similar to that of P. vivax [P. ovale spp. isolates were misidentified as P. vivax in microscopic examination in the provincial reference laboratory. PCR has higher sensitivity than microscopy in diagnosing P. ovale spp. and P. malariae infections [P. ovale spp. and P. malariae infections [P. ovale spp. was 70.1% which was suboptimal [P. ovale spp. infections.Identification of P. vivax . This stfections , 33 whicfections . RDTs prfections , becausefections . In thisboptimal . Hence, P. ovale spp. and P. malariae were acquired from sub-Saharan Africa. Collins and Jeffery [P. ovale spp. cases was in sub-Saharan Africa and some western Pacific islands. Both species of P. ovale spp. are considered to be sympatric throughout their geographical distribution [P. malariae is mainly found in sub-Saharan Africa and southwest Pacific [In this study,  Jeffery  observedribution . However Pacific . With thPlasmodium species.This study had some limitations. First, the database relied on passive case detection and inevitably could underestimate the numbers of imported malaria cases. Second, owing to the lack of an exact date of infection, the latency period was calculated using the arrival dates of patients when they returned to China, which means that the latency period of each case used in the study is a minimum estimate; the date of symptom onset relied on patient reporting, which is may be subject to recall bias and may therefore affect the confirmed diagnosis interval. Third, the small number of patients limited the statistical power to show differences among infections with different P. ovale spp. and P. malariae cases increased significantly. It also indicated the number of P. ovale spp. cases overtook that of P. vivax cases in Hubei Province. Nevertheless, these two species were most often misidentified or missed at hospitals or low-level CDC. Thus, suggested that more attention be paid to surveillance of imported P. ovale spp. and P. malariae cases to reduce the burden of public health and potential risk of malaria in China.Imported malaria cases are the primary challenge for Hubei Province in achieving the long-term goal of malaria elimination. The study results demonstrated that the incidence of


Plasmodium falciparum malariais 7-40 days. We report a case of symptomatic P. falciparum malaria that manifested four years after a visit to a malaria-endemic area in a 51-year-old female patient. Our case illustrates the importance of a broad differential with regard to infectious disease including remote travel history and raises questions of the survival of P. falciparum for four years despite the administration of traditional chemoprophylactic agents.With increased global travel, unique diseases from different parts of the world pose a new category of differential diagnosis, and an incubation period of different infectious diseases helps narrow differential diagnosis to point to one specific etiology. The normal incubation period of  Plasmodium falciparum malaria is 7-40 days, unlike other species such as P. vivax and P. ovale, which may remain dormant in the liver for up to about four years. It is also responsible for the most serious form of the disease [P. falciparum malaria can lead to life-threatening complications, such as shock, lactic acidosis, seizures, and renal failure. The underlying pathology is explained by the visible clumping of the erythrocytes in deep capillary beds. We report a case of symptomatic P. falciparum malaria that manifested four years after a visit to a malaria-endemic area in a 51-year-old woman. This patient lives in the United States and had received appropriate malaria chemoprophylaxis four years prior to presentation. This surprisingly long incubation period raises questions regarding the survival and fitness of P. falciparum prior to disease manifestations and the rising resistance to traditional chemoprophylactic agents.Malaria is a mosquito-borne disease, transmitted by the Anopheles mosquito . The nor disease . HoweverClinical presentationA 51-year-old female from Liberia with a past medical history of hypertension, gastroesophageal reflux, and appendectomy presented to the emergency department with fever, generalized body aches, malaise, and headache for two days. In the emergency department, she was found to have a fever of 101.3 °F and tachycardia, with normal blood pressure and respiratory status. She reported no other symptoms. Her last visit to Liberia was four years ago. She denied any complications after her travel, including fever, fatigue, and anemia. The patient reported chemoprophylaxis but was unable to remember the regimen. She reported no travel outside of the New York area since then, including airports. She denied any blood transfusions or any exposure to wooded areas or tick bites. She worked as a home health aide; she denied smoking, alcohol, or illicit drug use.P. falciparum on Giemsa stain with a parasitemia of 1.86%  yielded positive results. A diagnosis of malaria with Plasmodium falciparum was made.Her laboratory tests revealed thrombocytopenia with a platelet count of 32,000 per microliter , leukopenia with a white blood cell count of 3,200 per cubic milliliter , and anemia with a hemoglobin of 9.2 g/dL . Lactate dehydrogenase was 310 IU/L  and bilirubin 3.8 mg/dL , mostly indirect. Labs revealed elevated liver function enzymes with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) being 57 and 67 U/L, respectively . Ultrasound of the liver was normal. Peripheral blood smear showed ImagingChest X-ray was performed and was negative for any focal parenchymal opacities, pleural effusions, or pneumothorax. No splenomegaly or hepatomegaly was evident on computed tomography (CT) abdomen/ pelvis or abdominal ultrasound.Treatment and managementThe patient was treated with Malarone (atovaquone 1000 mg/proguanil 400 mg) once daily for 4 consecutive days. A decline in parasitemia was confirmed as the patient responded to treatment; the last smear showed <1% parasitemia. Thrombocytes, lymphocytes, and lactate dehydrogenase were closely monitored until they normalized. The patient was discharged home with outpatient follow-up with an infectious disease specialist.P. vivax, P. falciparum, P. malariae, P. ovale, and Plasmodium knowlesi. P. vivax and P. falciparum are the two most common species found in Africa. P. falciparum is the most lethal of all. The life cycle of malaria parasites begins when an infected female Anopheles mosquito bites an uninfected individual. Parasites stick to red blood cells and clump deformed erythrocytes in deep capillary beds, predisposing to organ ischemia and shock, as well as tissue hypoxia and lactic acidosis.There are five pathogenic species of Plasmodium causing malaria in humans: Pl.falciparum transmission with 350 to 500 million clinical episodes and 1 million deaths annually [P. vivax and P. ovale. The normal incubation period for malaria is 7-40 days. It is shorter for falciparum species compared to non-falciparum species such as P. vivax and P. ovale [P. falciparum was eight years after visiting an endemic area [P. falciparum malaria can present even after four years in a patient with a remote history of travel to an endemic region, in spite of receiving chemoprophylaxis.A total of 2.4 billion people are at risk of stable or unstable P. falciparum with reported infected United States marines in Liberia in 2003 [Repeated parasite exposure can lead to clinical immunity initially protecting from severe consequences. However, as transmission rates decrease, the risk of infection also increases the rates of severe disease due to the lack of functional immunity . Outbrea in 2003 . In obtaP. falciparum malaria that she acquired in Liberia four years prior to her clinical presentation. As P. falciparum malaria is a fatal disease, it is crucial to have a high index of suspicion and investigate for disease in patients presenting with fever, malaise, and thrombocytopenia, even with a remote history of travel to an endemic region. Early appropriate management in these patients can prevent fatal complications and even death.From this case report, we conclude that our patient had


Plasmodium spp. Accordingly, chemical strategies and knowledge of host immunity to Plasmodium spp. have served as the basis. Evidence is obtained after being tested in experimental rodent models for malaria infection and recognized for human sera from malaria-endemic regions. This encourages us to propose such an immune-potentiating strategy to be further considered in the search for new vaccine candidates.Malaria is a deadly disease that takes the lives of more than 420,000 people a year and is responsible for more than 229 million clinical cases globally. In 2019, 95% of malaria morbidity occurred in African countries. The development of a highly protective vaccine is an urgent task that remains to be solved. Many vaccine candidates have been developed, from the use of the entire attenuated and irradiated pre-erythrocytic parasite forms (or recombinantly expressed antigens thereof) to synthetic candidates formulated in a variety of adjuvants and delivery systems, however these have unfortunately proven a limited efficacy. At present, some vaccine candidates are finishing safety and protective efficacy trials, such as the PfSPZ and the RTS,S/AS01 which are being introduced in Africa. We propose a strategy for introducing non-natural elements into target antigens representing key epitopes of  P. vivax) and 409 thousand deaths in 2019 ..106].Plasmodium infection can result in the development of naturally acquired immunity [P. falciparum merozoite to invade erythrocytes depends on specific interactions between the parasite and red blood cell receptors. Several proteins expressed on the merozoite’s surface which are actively involved in the invasive process have been identified. Among those, the 10 members of the Merozoite Surface Protein (MSP) family have been extensively studied. This group of molecules is not distributed homogeneously in the merozoite membrane, nor processed in a similar way, but are characterized by their structural diversity which includes domains involved in a variety of processes such as protein–protein interactions, essential for the initial formation of the merozoite as well as for contacting the red blood cells (RBCs) membrane , constitutes a family of antigenic proteins of children  and is tnstrated .GLURP (Glutamate rich protein) has a predicted molecular mass of 145 kDa and is also expressed on the surface of the merozoite, as well as in pre-erythrocytic stages. It has conserved, tandem repetitive regions that are capable of stimulating T and B lymphocytes . It has P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var2csa gene has been regarded as the principal binding ligand associated with placental sequestration and binds to Chondroitin Sulfate A (CSA). As it has been recently reported, in malaria-endemic areas, pregnant women commonly suffer from placental malaria wherein P. falciparum-infected erythrocytes (IE) sequester in the placenta and often elicit an inflammatory infiltrate. PRIMVAC is a VAR2CSA-derived placental malaria vaccine candidate aiming to prevent serious clinical outcomes of Plasmodium falciparum infection during pregnancy. Thus, expectations regarding trial progress will be useful for malaria prevention [Interestingly the so-named VAR2CSA antigen is an important vaccine target to prevent placental malaria. VAR2CSA is the principal antigen ligand for chondroitin sulfate A in two allogeneic isolates of evention .Plasmodium. It is expressed in both the merozoite and sporozoite stages. It is stored in micronemes and then translocated to the merozoite membrane, mediating the merozoite reorientation towards the RBCs and stimulating a macromolecular complex formation with a protein group located in the neck region of robe-shaped merozoites [Plasmodium sporozoites to liver cells [AMA-1  is an integral membrane protein of approximately 80 kDa, conserved in the genus rozoites . AMA-1 her cells , this prThe EBA-175 (erythrocyte-binding antigen) is a transmembrane protein also present in micronemes and characterized by revealing two conserved cysteine rich domains. It is expressed on the surface of the merozoite and involved in the invasion of the merozoite to RBCs by binding to its receptors via sialic acid. Assays with antibodies from rabbits inoculated with some of its fragments indicate invasion blocking activity . NaturalP. knowlesi, evidenced some immunity with partial protection [P. falciparum [Studies in primates immunized with AMA-1 purified from otection . Howeverlciparum ,140.P. falciparum known as dense granules, the antigens RESA (ring-infected erythrocyte surface antigen), a 155 kDa protein and RIMA, (ring membrane antigen), among others have been characterized [Also, in the ring-shaped sites of other organelles of cterized . Studiescterized . The REScterized . This prPlasmodium merozoites, and there is not yet a blood-stage vaccine that has reached a phase III clinical trial [In summary, the most representative blood phase antigens regarded as targets for human vaccine candidates that have reached clinical trials are MSP1, MSP2, MSP3, AMA1, EBA175, GLURP, PfEMP1 (VAR2CSA), and SERA. Generally, these have been presented as recombinantly expressed antigens and co-administered with different adjuvants and delivery systems as seen in al trial .Plasmodium’s life cycle, which seems to use quite elaborated evasion mechanisms to escape the host immunity, which probably includes its own antigens’ structure modulation which could be governed by so far unknown strategies and precisely employed by Plasmodium to avoid being targeted by a poorly stimulated immune response.Finding a safe and highly effective malaria vaccine remains a challenge to be faced and solved especially considering the complexity of the P. falciparum, and apparent molecular weight estimated by SDS-PAGE. Orthologous genes for these proteins have been found in other Plasmodium spp., including P. vivax (Pvs25), P. berghei (Pbs25), and P. yoelii (Pys25) [A third approach for the development of vaccines against malaria consists of transmission blockers, that is, they are directed to antigens from pre-fertilization stages (gametes) or antigens expressed once fertilization occurs, thus acting to prevent infection . In prec (Pys25) . Compara (Pys25) .P. falciparum administered with Montanide ISA 51 as adjuvant was evaluated in a controlled phase I study, finding levels of antibodies significantly different from the control in volunteers who completed the vaccination scheme, which could be related to the blockage of the infection, however systemic adverse events were also reported—including erythema nodosum—and were directly associated with the formulation, suggesting extended further studies [Pichia pastoris and conjugated with exoprotein A (EPA), called Pfs25M-EPA/Alhydrogel®, was subsequently proposed. The results of the phase I clinical trial carried out in the United States for this vaccine demonstrated the relationship between antibody titers and transmission blocking activity, however, the highest level of antibody titers was achieved at the highest dose after administration of the fourth immunization and decreased over time, considering that their functional activity was short-lived [The Pfs25 is one of the best described and characterized transmission blocking antigenic proteins. A vaccine of recombinant origin of this antigen for  studies . To imprrt-lived .P. falciparum (strain 3D7) [The Pfs230 is a 230 kDa protein belonging to a structural family characterized by partially conserved cysteine motifs. The gene coding for this protein was originally cloned from ain 3D7) . It is eain 3D7) . This isain 3D7) .Differential expression of the Pfs25 and Pfs230 antigens during the parasite’s sexual development may be useful to improve the efficacy of some vaccines when carrying out the co-administration of these antigens, significantly increasing the antibody response and therefore its functional activity. By its side, the Pfs48/45 participates in gamete–gamete recognition in blood-like tissues within the mosquito, this is expressed on the surface of gametocytes within the human host. The natural development of antibodies against this protein has been reported in individuals living in malaria endemic areas . VaccinePlasmodium falciparum antigen named Pfs47 is a strategic vaccine target. This is a member of the 6-cysteine family of proteins and is expressed on the surface of female gametocytes, gametes, zygotes, and ookinetes; hence it has been regarded as an important target for transmission-blocking vaccines. However, a recombinantly expressed protein rPfs47 did not show transmission-blocking activity (TBA), while its stimulated antibodies were effective tools of TBA. Therefore, the role of this Plasmodium target for eliciting functional antibodies became evident for developing malaria vaccine candidates [Interestingly a ndidates .Those limitations and disadvantages already mentioned for the development of biological and recombinant vaccines against malaria, as well as the knowledge accumulated to date in this field, can be regarded as input information towards the development of new approaches for more potent vaccine candidates where synthetic epitope-antigens gain relevance. Peptide as vaccine candidates could be useful for immunization processes with single or multiple combinations of diverse molecules sizes and origins, designed with the purpose of reaching an optimal specificity to bind major histocompatibility complex (MHC) molecules, which once formed can then be recognized by T cell receptors and initiate a directed immune response by suppressing some natural requirements for antigen processing and presentation of protein epitopes .Peptide-based vaccines represent a potential strategy for the prevention and treatment of pathogenic diseases, their low cost, synthetic feasibility, and inherent safety are all attractive features, however, they have remained largely unsuccessful due to the native peptides’ low immunogenicity, which comes mainly from the reduction of bioavailability due to proteolytic in vivo degradation ,159.Some genetically conserved proteins from different stages of the malaria parasite which could be immunologically relevant are usually silent to the host immune system and so escape their recognition.Plasmodium apical sushi protein were neither toxic nor hemolytic being also highly antigenic and malaria protective when administered in infected rodents, establishing deep differences regarding other previously studied vaccine candidates [Hence, to reveal its immunogenic nature, this non-recognizing phenotype must be overcome by incorporating modulating structure elements of non-natural origin. In consequence modified versions of such low immunogenic antigens now would potentiate their molecular recognition and functionality thus becoming useful for malaria prevention . This prndidates ,165.Plasmodium falciparum proteins were selected and analyzed by employing a bioinformatics scheme to propose some immunogens representing possible B and T epitopes [To start the experimental design, six blood-stage epitopes ,167,168.P. falciparum blood-stage proteins, which were then obtained by solid-phase synthesis and extensively characterized. Introduction of non-natural elements into given amino-acid pairs was then proposed and obtained by liquid and solid-phase synthesis strategies as previously reported [4 infected RBCs with P. berghei ANKA and P. yoelii 17XL. A subsequent 20-day follow-up after being challenged lead us to detect those animals which better controlled the parasitemia percentage and even survived the experiment, characterized by the recognition of P. falciparum 3D7 and FCB-2 strains lysate proteins by western blot ; 72 samples from the Tierralta, village, Cordoba province ; 68 samples from Quibdó city in the Choco province , and 8 samples from the Tumaco village from the Nariño province . A control group of 13 samples was collected from a non-endemic malaria area of Colombia, being the Bogotá city . For geographical location see P. falciparum 3D7 and FCB-2 strains lysate proteins as mentioned above .The results revealed some interesting clues. First, human sera from people from Tierralta, Quibdó, and Tumaco revealed a comparable reactivity by western blot regarding the groups of mice vaccinated with non-natural analogs derived from MSP1, AMA1, and EBA175. Only two samples from San Juan de Nepomuceno displayed some similarity with its rodent counterpart, this fact could be due to this village being the only one in this set that is not located in a high malaria transmission area. Secondly, a remarkable finding is represented by the human sera from Tierralta, Quibdó, and Tumaco  which show a differential high recognition for most non-natural analogs from selected malaria proteins. Interestingly, the EBA175(E515-H516) analog was the most immunogenic for all groups by displaying the highest OD values as observed A–D, follPlasmodium parasite life-cycle, the limited selection criteria for potential targets as immunity inducers, and the multiple immune evasion mechanisms employed by the Plasmodium spp. Employing classical approaches for a vaccination with immunogens resembling natural Plasmodium antigens presented either as recombinant fused proteins or attenuated forms of parasite stages has proven to be useless for a potent host immune system stimulation. Therefore, specific structure modifications of antigens appear to be relevant for recognition in malaria exposed people, and long-lived antibodies directed especially to EBA175, AMA-1, and N-terminus and central portions of the MSP1 seem to play an important role in humoral immunity to malaria. Plasmodium seems to modulate its protein structure at convenience. A lot of work remains to be conducted to analyze the amount of other potential vaccine targets and contribute to overcoming this important public health problem.Difficulties for developing effective vaccines against malaria could arise from our poor understanding of the


Plasmodium falciparum causes the majority of malaria cases worldwide and children in sub-Saharan Africa are the most vulnerable group affected. Non-sterile clinical immunity that protects from symptoms develops slowly and is relatively short-lived. Moreover, current malaria vaccine candidates fail to induce durable high-level protection in endemic settings, possibly due to the immunomodulatory effects of the malaria parasite itself. Because dendritic cells play a crucial role in initiating immune responses, the aim of this study was to better understand the impact of cumulative malaria exposure as well as concurrent P. falciparum infection on dendritic cell phenotype and function.P. falciparum (n = 8) was assessed. Additionally, plasma cytokine and chemokine levels were measured in these adults and in Malian children (n = 19) with acute symptomatic malaria.In this cross-sectional study, the phenotype and function of dendritic cells freshly isolated from peripheral blood samples of Malian adults with a lifelong history of malaria exposure who were either uninfected (n = 27) or asymptomatically infected with P. falciparum blood-stage parasites by up-regulating the costimulatory molecules HLA-DR, CD80, CD86 and CD40 and secreting IL-10, CXCL9 and CXCL10. In contrast, myeloid dendritic cells of asymptomatically infected Malian adults exhibited no significant responses above the uninfected red blood cell control. IL-10 and CXCL9 plasma levels were elevated in both asymptomatic adults and children with acute malaria.With the exception of lower plasmacytoid dendritic cell frequencies in asymptomatically infected Malian adults, peripheral blood dendritic cell subset frequencies and HLA-DR surface expression did not differ by infection status. Peripheral blood myeloid dendritic cells of uninfected Malian adults responded to in vitro stimulation with P. falciparum infections in malaria-endemic areas.The findings of this study indicate that myeloid dendritic cells of uninfected adults with a lifelong history of malaria exposure are able to up-regulate co-stimulatory molecules and produce cytokines. Whether mDCs of malaria-exposed individuals are efficient antigen-presenting cells capable of mounting an appropriate immune response remains to be determined. The data also highlights IL-10 and CXCL9 as important factors in both asymptomatic and acute malaria and add to the understanding of asymptomatic  Plasmodium falciparum remains a major public health threat. An estimated 3.4 billion people in 92 countries are at risk of malaria. In 2018, P. falciparum caused approximately 228 million malaria cases and 405,000 deaths worldwide, the majority among children in sub-Saharan Africa [Malaria caused by P. falciparum and the human immune system [P. falciparum infections [Ultimately, a key tool for the control, elimination, and eventual eradication of malaria is an effective vaccine, yet the leading vaccine candidate RTS,S confers only partial, short-lived protection in African children . A consie system , particufections –12.P. falciparum infections lead to high parasitemia and a strong pro-inflammatory cytokine and chemokine response, referred to as “cytokine storm”, causing fever and other malaria symptoms [P. falciparum infection, but only after years of repeated exposures [In malaria-naïve individuals, symptoms , 14. Howsymptoms –17. Althxposures . Once acxposures .A crucial part of this process are dendritic cells (DCs). As sentinels of the immune system, DCs are not only important for early cytokine responses but are also essential for bridging and regulating the innate and adaptive immune responses to pathogenic infections and vaccines . DCs resThe two major human DC subsets, plasmacytoid dendritic cells (pDCs) and myeloid DCs (mDCs), have different roles in initiating and coordinating the immune response. While pDCs secrete high levels of IFNα and TNF upon activation, mDCs up-regulate costimulatory molecules and are highly efficient antigen presenting cells .Plasmodium, especially in human malaria infections. Studies both in humans and mouse models have been contradictory [P. falciparum suggested that DC maturation was blocked by the parasite leading to dysfunction and suboptimal allogenic T cell activation [. showing the blocking effect was parasite dose-dependent [Despite their essential role in initiating adaptive immune responses to natural infections as well as vaccines, little is known about the role DCs play in the immune response to adictory . The semtivation . Conclusependent . The expP. falciparum malaria from an endemic area in Papua, Indonesia [Very few studies have addressed DC function using purified primary DCs. One study analysed primary DC functions ex vivo from individuals with acute ndonesia . The autndonesia . Recentlndonesia . During ndonesia .These previous studies provide valuable insight into the effect of highly inflammatory conditions during malaria on circulating DCs and early processes during primary DC activation by the parasite in malaria-naïve individuals. However, they do not reveal how isolated DCs from malaria exposed or asymptomatically infected individuals in malaria-endemic areas respond to the parasite. Addressing this question will help us understand whether initial immunological responses to vaccines or natural infections are affected by regulatory networks in individuals living in endemic areas.P. falciparum-infections in malaria-endemic Mali have an effect on DC frequency, viability and response to the parasite.Therefore, this study aimed to address whether intense, lifelong malaria exposure or concurrent asymptomatic P. falciparum transmission from July through December each year [P. falciparum exposure was unlikely.Both study sites, Kambila  and Kalifabougou  in Mali, have been described in detail before . Both arP. falciparum infections in these individuals were detected by PCR analysis of blood spots at the NIH after in vitro assays had been conducted in Mali. Detailed methods for the detection of P. falciparum blood-stage infection by PCR have been described before [The cross-sectional study in Kambila included 35 adults Table , who exhd before . Plasma Peripheral blood was collected by venipuncture into 8 mL sodium citrate-containing cell preparation tubes  and transported to the laboratory in Bamako where PBMCs were isolated according to the manufacturer’s instructions. The blood volume was 48 mL for adults and 8 mL for children. PBMCs and dendritic cells were analyzed or isolated immediately. Plasma samples were cryopreserved at -80 °C and later shipped to the NIH. The same sample collection and processing was conducted for the US donors.P. falciparum strain 3D7 were maintained at 5% haematocrit in RPMI 1640, 25 mM HEPES supplemented with 10 μg/mL gentamicin, 250 μM hypoxanthine, 25 mM sodium bicarbonate, and 0.5% Albumax II under atmospheric conditions of 5% oxygen, 5% carbon dioxide, and 90% nitrogen. Late-stage P. falciparum-iRBCs (trophozoites and schizonts) were isolated using MACS Cell Separation LS Columns (Miltenyi Biotec). Plasmodium falciparum-iRBCs were washed, resuspended at 1 × 106/µL and lysed by three consecutive freeze/thaw cycles. Uninfected RBC lysates were prepared accordingly and used as negative control. Lysates were stored at − 80 °C until use.Asexual blood stage cultures of the 2. Enriched mDCs were seeded at 1.5 × 105 per well in 96-well tissue culture-treated plates and cultured with P. falciparum-iRBC lysates at a ratio of 1:3 (DC/iRBC) for 24 h. Uninfected RBC lysates were used as a negative control. mDCs were then harvested for FACS analysis and supernatants were stored at − 80 °C until shipment to the NIH and cytokine/chemokine analysis.Myeloid Dendritic Cell Isolation Kits (Miltenyi Biotec) were used to negatively enrich primary peripheral blood mDCs from PBMCs, following the manufacturer’s instructions. Primary mDCs were cultured with RPMI 1640 supplemented with 10% heat-inactivated human AB serum  at 37 °C and 5% COP. falciparum-iRBC lysate was analysed using the following antibodies: HLA-DR (L243), CD80 (2D10), CD86 (IT2.2) and CD40 (5C3). All antibodies, Annexin V and 7-AAD were purchased from BioLegend or BD Biosciences.Flow cytometry was performed using a BD LSR II cytometer (BD Biosciences), and data were analysed with FlowJo 10 (Tree Star). Primary DC subsets were identified using the following labelled monoclonal antibodies: a lineage cocktail , HLA-DR (L243), CD303 (201A), CD1c (L161), CD141 (M80) and CD16 (3G8). DC apoptosis was quantified by staining a separate PBMC aliquot with monoclonal antibodies against lineage, HLA-DR (L243) and Annexin V as well as 7-AAD. To normalize to microlitres of blood, the total number of PBMCs was determined using a counting chamber after isolation, prior to further analysis and dendritic cell isolation. Assuming this number was close to the number of PBMCs in whole blood, FACS analysis percentages were used to determine cell #/µl of blood. mDC maturation after culture with To analyse cytokine and chemokine levels in plasma and culture supernatants, the following kits were used: LEGENDplex Human Inflammation Panel 1 (13-plex) (Biolegend), Cytometric Bead Array Human Inflammatory Cytokine Kit (BD Biosciences) and Cytometric Bead Array Human Chemokine Kit (BD Biosciences).Statistical analyses were performed using Prism 8 (GraphPad Software) and JMP (SAS Institute). Depending on the experimental design, a paired or unpaired t-test with Welch’s correction was performed. For data in Fig. P. falciparum transmission or concurrent asymptomatic P. falciparum-infections lead to altered frequency or function of DCs, freshly isolated, primary myeloid DC activation by P. falciparum-iRBC lysate in vitro was analysed in a malaria-endemic setting in a cross-sectional study. In the rural village of Kambila, Mali, where malaria transmission is intense and seasonal [P. falciparum blood-stage infection . Due to naturally acquired clinical immunity to the parasite, adults in this cohort rarely develop symptomatic infections [P. falciparum-infections along with asymptomatic infections, plasma samples from 19 children between the ages of 6 and 9 years were added to the study  negative and HLA-DR positive cells [+ and CD141+ DCs, and a third subset characterized by the expression of CD16 , during  and after  an acute symptomatic malaria episode. Plasma obtained from US adults was used as a control. As expected, most inflammatory cytokine and chemokine levels tested increased significantly during acute malaria and decreased to baseline levels at the convalescent time point 7 days post treatment. Similar to asymptomatically infected adults Fig. a, plasmaP. falciparum in malaria-endemic settings.Most plasma cytokine and chemokine levels returned to baseline by the convalescent timepoint, with the exception of IL-18, CCL5 and CXCL9 Fig. . Taken tP. falciparum in particular remains very limited . In ligiewed in , it is oP. falciparum infections compared to uninfected individuals. Several other studies have reported lower numbers of circulating DCs in acute malaria patients in both children and adults [P. falciparum infections in children and adults in Papua, Indonesia, DC numbers were found to be unchanged, including pDC frequencies [The findings suggest that mDC numbers in circulation remain stable during asymptomatic malaria in Malian adults. pDC frequencies, however, were lower in individuals with asymptomatic d adults , 45–47. quencies . This diquencies , 49. Indquencies –52, lowequencies , or migr+ mDCs in children with acute malaria [+ mDCs through Flt3 ligand.A few studies have reported an increase in CD141 malaria , 39, 53. malaria , no suchP. falciparum-iRBC lysate. Whether mDCs from malaria-naïve US individuals are also capable of responding to the parasite with IL-10 secretion will have to be addressed in future experiments. The absence of a significant increase in the previous study could be due to a lower sample size and does not permit further conclusions at this time. Comparable to the response exhibited by DCs from US donors to the parasite, DCs from Malians secreted the Th1-associated chemokines CXCL9 and CXCL10 as well as CCL2 but no CCL5. Except for IL-10 secretion, in vitro DC responses to the parasite in malaria-exposed uninfected Malians seem, at least qualitatively, comparable to malaria-naïve US DCs. Future studies will have to address whether up-regulation of costimulatory molecules and chemokines in these DCs translates into effective T cell priming in malaria-endemic areas.As observed previously with a subset of the data , DCs isoP. falciparum infections, contributes to pathology [A number of studies have assessed cytokines in the plasma of acute malaria patients, concluding that the so called “cytokine storm”, characteristic for athology . Here, aathology . TNF levP. falciparum-iRBC lysates, CXCL9 and CXCL10 have been reported to exhibit increased expression in murine splenic DCs during Plasmodium chabaudi infection, with no significant increase in inflammatory cytokine gene expression [P. falciparum infections. Similar to the “cytokine storm”, inflammatory chemokines like CXCL9 and CXCL10 are likely important to mount an effective immune response to the parasite early during the infection, but may be detrimental under certain circumstances.Plasma chemokine levels during human malaria are less well documented. Similar to other studies in Africa , 57, thipression . Comparapression , suggestPlasmodium infection.In addition to CXCL9 and CXCL10, we observed that low levels of IL-10 were secreted by mDCs in Malian adults upon stimulation with the parasite. That IL-10 was detectable in the plasma of asymptomatically infected adults and at high levels in children with acute malaria is consistent with an important role for this regulatory cytokine in malaria. Indeed, Il-10 has been extensively described in murine malaria models and human studies as an important regulatory factor (reviewed in ). AlthouP. falciparum-infected Malian adults. While children with asymptomatic malaria have increased IFNγ, TNF and IL-4 levels [Studies that assess plasma cytokine or chemokine levels in asymptomatic malaria infections are scarce. In this study, IL-10 and CXCL9 were significantly elevated in asymptomatic 4 levels , IL-10 a4 levels –66. ThisLimitations of this study include the low number of infected individuals enrolled, the cross-sectional design and the lack of data on DC-T cell interactions. Future studies should include a larger sample size, and longitudinal analysis of DC responses and functionality that include T cell activation. The Malian study site is particularly well suited for such studies due to the sharply demarcated and predictable malaria transmission season which enables the analysis of DCs from uninfected individuals before the malaria season and the same individuals as they become infected during the ensuing malaria season. Despite these limitations, the present study provides valuable insight into baselines DC frequencies and responses as well as in vivo cytokine and chemokine responses to the parasite in a malaria-endemic setting.P. falciparum-iRBCs. However, whether these responses translate into efficient T cell activation remains to be determined. This report also suggests that IL-10 and CXCL9 are upregulated during asymptomatic malaria, which may be of interest given the high prevalence of asymptomatic infection in endemic areas [Although far from complete, together with the report on peripheral blood DC frequencies in asymptomatic malaria in Papua, Indonesia , this stic areas , 68.


Plasmodium knowlesi is a potential cause of severe and fatal malaria, but comprehensive studies of its pooled prevalence and risk factors are lacking. This study aimed to explore the prevalence and risk factors related to severe P. knowlesi infection.P. knowlesi available in Web of Science (ISI), Scopus, and PubMed (MEDLINE). Titles, abstracts, and full-text articles were screened, and any irrelevant studies were excluded. The random-effects model was used to compute the pooled prevalence estimate of severe P. knowlesi infection by a metaprop command provided in STATA software. Differences in demographic characteristics, clinical characteristics, and laboratory data were analysed using Review Manager Version 5.3 software for patients in the following groups: 1) patients with severe and non-severe P. knowlesi infection and 2) patients with severe P. knowlesi and severe P. falciparum infection.A systematic review was conducted by retrieving all published articles on severe P. knowlesi infections were eligible to be included in this systematic review and meta-analysis. The pooled prevalence estimate of severe P. knowlesi infection was 19% . Severe acute kidney injuries (AKI) , jaundice , and hyperparasitaemia  were the common clinical manifestations found among patients with severe complications. In comparison to non-severe P. knowlesi infections, patients with severe P. knowlesi infections had significantly higher age, leucocyte count, and parasitaemia levels (P < 0.05). In comparison to patients with severe P. falciparum infections, patients with severe P. knowlesi infections had significantly higher age, neutrophil count, and creatinine levels (P < 0.05).Out of the 2382 studies retrieved from the three databases, seven studies with a total enrolment of 1124 patients with P. knowlesi infections. Patients with severe P. knowlesi infections had higher age, leucocyte count, and parasitaemia levels than those with non-severe P. knowlesi infections. In addition, patients with severe P. knowlesi infections had higher age, neutrophil count, and creatinine levels than those with severe P. falciparum infections.This systematic review and meta-analysis demonstrated a high proportion of severe  Plasmodium knowlesi is a naturally occurring malarial parasite in monkeys [P. knowlesi are the long-tailed macaque (Macaca fascicularis) and the pig-tailed macaque  [P. knowlesi is found only in Southeast Asian countries because its natural hosts and the female Anopheles mosquito vectors are very limited and specifically exist in these areas [Leucosphyrus Complex transmitted P. knowlesi in southern Asia, while the Dirus Complex transmitted P. knowlesi in Vietnam, Myanmar, Laos, Cambodia and Thailand [P. knowlesi is transmitted from human to human but rather through mosquito bites [P. knowlesi occur through quotidian (24-hour schizogonic cycles) malarial parasites and lead to severe and lethal manifestations. monkeys  and is a monkeys . The natleonine) –6. The gse areas . A previThailand . At presto bites . InfectiP. knowlesi infection in humans was reported by Singh et al. in Sarawak, Malaysia, in patients previously misdiagnosed with P. malariae infections [P. knowlesi is similar to that of P. malariae, a quartan malarial parasite causing non-severe human malaria [P. knowlesi confirmed by polymerase chain reaction (PCR) were miss-detected as P. malariae by microscopic methods (76%) [P. knowlesi, but they were insensitive in cases of low parasitaemia and were unable to distinguish P. knowlesi from P. vivax [P. knowlesi requires a molecular technique, such as the nested PCR assay. A previous study demonstrated that P. knowlesi misdiagnosis as P. malariae caused severe malaria and resulted in death because of inappropriate treatment [P. vivax and to prevent the fatal outcome of patients by administering intravenous therapy with artesunate or quinine as the first drug of choice for severe malaria, especially caused by P. knowlesi. Although P. knowlesi is a potential cause of severe and fatal malaria, comprehensive studies of its pooled prevalence and risk factors are lacking. In addition, a meta-analysis determining the difference in the prevalence estimate of severe P. knowlesi infection among studies conducted on P. knowlesi is needed. Therefore, this study aimed to estimate the prevalence and risk factors related to severe P. knowlesi malaria. In addition, the comparison of demographic characteristics, clinical data, and laboratory data between severe P. knowlesi and severe P. falciparum infection was evaluated to predict disease severity in co-endemic primary care settings of P. knowlesi.The first case of fections . The mor malaria . This sids (76%) . The rapP. vivax , 12. Henreatment . It is vPlasmodium knowlesi”) OR knowlesi)”  guidelines (PRISMA 2009 Checklist S” Table S. This seTwo reviewers (MK and KUK) selected and extracted the data from the included studies independently. We used the Endnote software X7  to manage the references. First, we removed any duplicate papers retrieved from the three databases. Then, we screened the titles and abstracts according to the inclusion criteria. Finally, we assessed all full-text articles for detailed assessments for further data extraction. The data extraction was conducted using the Microsoft Excel 2010  and included data on the primary author, year of publication, study area, study design, demographic characteristics of patients (age and sex), and clinical characteristics . Laboratory profiles including parasite count, haemoglobin, leucocyte count, platelet count, creatinine, sodium, bilirubin, glucose, albumin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were additionally collected. The malaria endemicity of the years when the included studies were conducted by their authors was demonstrated by the slide positivity rate as reported by the World Malaria Report 2010–2014 . DisagreP. knowlesi malaria were defined by the World Health Organization (WHO) [The criteria of severe on (WHO) .A quality assessment was performed following the quality assessment tool developed by the Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomized studies in meta-analysis . It provP. knowlesi infection. The data analysis for the pooled prevalence began with entry of the extracted data into the Excel sheet and to the STATA Statistical Software version 15.0 . The number of patients with severe P. knowlesi and total cases of P. knowlesi were imported and analysed using a command “metaprop case population, random/fixed” provided in the STATA software. A metaprop command allowed Freeman-Tukey double arcsine transformation to stabilize the variances. The random-effects or fixed-effect model could be used to compute the pooled estimate, depending on the heterogeneity among the included studies [CI).The primary outcome of the present study was the pooled prevalence of severe  studies . The resP. knowlesi malaria using the demographic characteristics, clinical characteristics, and laboratory profiles of patients with severe and non-severe P. knowlesi malaria. The third outcome of the present study was to compare the demographic characteristics, clinical characteristics, and laboratory profiles between patients with severe P. knowlesi and severe P. falciparum malaria. The results of the second and third outcomes were presented as the pooled mean difference (MD) with 95% CI for continuous outcomes and odds ratio along with 95% CI for categorical outcomes. The data heterogeneity was assessed using Cochrane Q (chi-square) and Moran’s I2 (inconsistency). The I2 was quantified as low, moderate, and high, with upper limits of 25, 50, and 75% for I2, respectively. If moderate or high heterogeneity was presented by Moran’s I2, the random-effects model was used to calculate the pooled estimates. If a low heterogeneity (I2 < 25%) was presented, the fixed-effect model was used to calculate the pooled estimates. The subgroup analysis of study sites was performed to demonstrate whether the transmission intensity between Sarawak and Sabah, the area with the highest transmission intensity of all malaria cases and the area with the highest cases of P. knowlesi in Malaysia during 2013–2017, affected the pooled prevalence estimate or affected the risk of severe P. knowlesi infection in the meta-analysis. The analysis of the second and third outcomes was performed using the Review Manager 5.3 . The meta-regression analysis of a covariate, namely, malaria endemicity (slide positive rate), between severe P. knowlesi and non-severe P. knowlesi was performed using STATA Statistical Software version 15.0.The secondary outcome of the present study was to identify factors associated with severe P. knowlesi infection. The evaluation of publication bias among the included studies helped explore the underlying cause of data heterogeneity if it was present in the included studies.The funnel plot was used to demonstrate any symmetrical funnel plot among patients with severe n = 117), Scopus (n = 2139), and Web of Science (n = 126) databases  had severe complications. Most of the severe complications found in patients included severe acute kidney injury (AKI) , jaundice , and hyperparasitaemia . The number of patients with P. falciparum infections included a total of 394 patients in five studies [P. knowlesi and P. falciparum infections are shown in Table The characteristics of the seven studies included in this study are presented in Table  studies , 28, 29  studies , 24, 27, studies , 28, 29, studies , 24, a r studies , and a cP. knowlesi malaria was 19% , calculated by a random-effects model since high heterogeneity (Moran’s I2: 93.7%) was found  [CI: 25–40, I2 = 93.7%) [P. knowlesi malaria was reported in a study by Grigg et al.  [CI: 8–44%, I2 = 96.7%) was higher than that of the studies conducted in Sarawak .The pooled prevalence of severe  28–52%)  and by B= 93.7%) , while tI: 4–8%) . The subI2 analysis of age (I2 = 66%), leucocyte count (I2 = 54%), and parasitaemia level (I2 = 91%) between patients with severe and non-severe P. knowlesi infections demonstrated that medium and high heterogeneities were found among the included studies; therefore, the random-effects model was used to calculate the pooled MD along with 95% CI. In comparison to patients with non-severe P. knowlesi infections, the results demonstrated that patients with severe P. knowlesi infections were significantly older than patients with non-severe P. knowlesi infections   and those studies conducted in Sarawak . The meta-regression analysis using the slide positive rate as a covariate revealed that the slide positive rate was not a confounding factor for the age difference .Cochrane Q and Moran’s 6%) Fig. . The subP. knowlesi infections had a significantly higher leucocyte count than patients with non-severe P. knowlesi infections   but not in the included studies conducted in Sabah . The meta-regression analysis using the slide positive rate as a covariate revealed that the slide positive rate was not a confounding factor for the leucocyte count difference .Patients with severe P. knowlesi infections had a significantly higher parasitaemia level than patients with non-severe P. knowlesi infections  , and the subgroup analysis demonstrated that no significantly higher parasitaemia level was found in the included studies conducted in Sarawak  compared to the included studies conducted in Sabah . The meta-regression analysis using the slide positive rate as a covariate revealed that the slide positive rate was not a confounding factor for the parasitaemia level difference .Patients with severe I2 analysis of platelet counts between patients with severe and non-severe P. knowlesi infections demonstrated a high level of heterogeneity (I2 = 89%); therefore, the random-effects model was used to calculate the pooled MD along with 95% CI. In comparison to patients with non-severe P. knowlesi infections, patients with severe P. knowlesi infections had a significantly lower platelet count  , and a significantly lower platelet count was found in both the included studies conducted in Sabah  and those studies conducted in Sarawak . The meta-regression analysis using the slide positive rate as a covariate revealed that the slide positive rate was not a confounding factor for the platelet count difference .Cochrane Q and Moran’s I2 analysis of age (I2 = 92%), neutrophil count (I2 = 66%), and creatinine level (I2 = 89%) between patients with severe P. knowlesi and severe P. falciparum infections demonstrated that medium and high heterogeneities were found among the included studies; therefore, the random-effects model was used to calculate the pooled MD along with 95% CI. In comparison to patients with severe P. falciparum infections, patients with severe P. knowlesi infections were older    , neutrophil count , or creatinine level .Cochrane Q and Moran’s vel I2 = 9% betweeP. knowlesi was not different from those infected with P. falciparum    was higher than that of the studies conducted in Sarawak (10%). Previous studies reported that P. knowlesi, not P. falciparum or P. vivax, was the most common cause of malaria in Malaysia, particularly in Sabah and Sarawak [P. knowlesi malaria was observed in wild macaques (87%) and long-tailed macaques (50%) living in Sarawak, Malaysian Borneo [P. knowlesi was higher in Sarawak, the highest proportion of severe malaria caused by P. knowlesi was found in another state, Sabah. The result of our study was supported by the average mortality rate per 100 000 patients with P. knowlesi being higher in Sarawak (0.074) than in Sabah (0.064) [This study provided prevalence data and risk factors related to severe  Sarawak , 32. Anon Borneo , 33. Then Borneo . Our res (0.064) .P. knowlesi in wild long-tailed macaques (19%) was also reported from the island of Palawan, the Philippines [P. knowlesi parasites were also found in individuals living close to forested areas in Cambodia (0.59%) [P. malariae should be reported as P. knowlesi to increase recognition and be treated with intravenous artesunate for severe P. knowlesi malaria [P. knowlesi malaria management guidelines recommend an initial treatment with intravenous artesunate for any patients > 50 years of age or with a parasitaemia level > 20 000/μl [In addition to being endemic in Malaysia, the presence of lippines . In huma (0.59%) . A previ malaria . In Mala0 000/μl , 37.P. knowlesi malaria found in the present study were severe AKI, jaundice, and hyperparasitaemia. The results were similar to the largest study of P. knowlesi malaria conducted in three hospitals in Malaysia, where AKI was frequently observed [P. knowlesi in humans are not well understood. However, even a low parasite density from P. knowlesi infections can lead to severe diseases [P. knowlesi malaria, hyperparasitaemia has been defined as a parasite count of 100000 parasites/μl or higher [P. knowlesi in the human host is a 24-hour life cycle in which it rapidly reproduces at the asexual stage at a high parasitaemia level more than P. falciparum [P. knowlesi is still unknown; however, microvascular sequestration similar to that in P. falciparum infections might be the cause that plays a role in kidney injury [The most common complications of severe observed . The mecdiseases . In sever higher , 38. Thelciparum . For AKIy injury .P. knowlesi infections than younger patients, which was related to the low risk of anaemia and high risk of thrombocytopenia [P. knowlesi infected a wide range of ages from young teenagers to mature adults, which was in accordance with a study in Sarawak, Malaysia [P. knowlesi infections occurred more frequently among older patients was the strong correlation of increasing age with increasing parasitaemia [P. knowlesi infections [P. falciparum malaria, where endothelial activation increases with age [P. knowlesi infections among these age groups [P. knowlesi over a wide age range might be related to the presence of family clusters [P. knowlesi and P. falciparum infections. This demonstrated that patients who worked as farmers and plantation workers could be infected with either P. knowlesi or P. falciparum at the same proportion. However, a previous study indicated that P. knowlesi parasitaemia was significantly higher in farmers and plantation workers than in other occupations [P. knowlesi malaria but not for non-P. knowlesi malaria [P. knowlesi infection was related to human behaviours, such as higher forest exposure or working in agricultural areas [P knowlesi infection and that the risk of infection by P. knowlesi and non-P. knowlesi was comparable in males [P. knowlesi seropositivity in a study by Fornace et al., 2018, who conducted a study in northern Sabah, Malaysia, and Palawan, the Philippines [P. knowlesi infection were work-related, including Armed Forces and security personnel, boatmen and researchers, Department of Forestry officers, border patrol officers, and non-work-related, including peri-domestic transmission [The meta-analysis demonstrated that older patients had more severe complications from ytopenia , 28, 29.Malaysia . Two preMalaysia , 28. A pfections . This mewith age . In addie groups . A previclusters . The resupations . Moreove malaria . Previoual areas , 28. A pin males . In contlippines . The mossmission .P. knowlesi malaria, the present study demonstrated that patients with severe P. knowlesi malaria had a higher leucocyte count and parasitaemia level than those with non-severe P. knowlesi malaria. The subgroup analysis of study sites demonstrated that a significantly higher leucocyte count was found in the included studies conducted in Sawarak but not in the included studies conducted in Sabah. These results demonstrated that P. knowlesi patients in Sawarak had an immune response that was strongly correlated to P. knowlesi infection, as the leucocyte differential count and neutrophil counts were strongly correlated with P. knowlesi parasitaemia [P. knowlesi malaria had a parasitaemia level greater than 100 000 parasites/μl [P. knowlesi parasitaemia levels, suggesting that severe P. knowlesi malaria can lead to parasite-induced inflammation such as lung injury [P. knowlesi malaria than in those with non-severe P. knowlesi malaria. A previous study reported thrombocytopenia (84.9%) among patients with P. falciparum and P. vivax infections [P. knowlesi malaria demonstrated a decrease in coagulation system markers [P. knowlesi infection demonstrated in Sabah, Malaysia, were female sex, age ≥ 45 years, cardiovascular disease, microscopic misdiagnosis, and delay in administrating intravenous treatment [Comparing patients with severe and non-severe sitaemia . The ressites/μl . They alg injury . The prefections . The thrfections . A previ markers  or an in markers . The risreatment .P. knowlesi malaria were higher than those with P. falciparum malaria. This result supported the conclusion of a previous study in Sabah, Malaysia, which demonstrated severe P. knowlesi malaria at 29% with a greater than 3-fold risk of severity when compared to P. falciparum malaria [P. knowlesi and severe P. falciparum malaria, patients with severe P. knowlesi infections were older than patients with severe P. falciparum malaria. This result was similar to a previous study demonstrating that patients with P. knowlesi malaria were older than those with P. falciparum or P. vivax malaria [P. knowlesi infections and death have not been reported in children, unlike severe P. falciparum infections [The results of the present study demonstrated that severe complications in patients with  malaria . Compari malaria . Previoufections .P. knowlesi malaria had a higher neutrophil count than patients with severe P. falciparum malaria. These results were supported by a previous study that revealed that neutrophil counts strongly correlated with P. knowlesi parasitaemia [P. knowlesi parasitaemia and neutrophils involved the cytokines IL-1ra and IL-10, which were related to anti-inflammatory effects on the patient’s response to infection [P. knowlesi malaria might be due to a consistent correlation between high neutrophil counts and high parasite densities in P. falciparum-infected patients when compared to those with low and moderate parasitaemia groups [P. knowlesi than in non-severe P. knowlesi infections [P. knowlesi malaria had a higher creatinine level than those with severe P. falciparum malaria. These results demonstrated the association between a higher creatinine level and a higher proportion of severe AKI among patients with severe P. knowlesi malaria. As the risk of severity occurred in a high proportion compared to P. falciparum malaria in Malaysia, all patients recognized with severe malaria due to P. knowlesi were given intravenous artesunate as a first-line treatment upon admission to reduce the potential of mortality [The present study demonstrated that patients with severe sitaemia . They sunfection . A previnfection . A high a groups , 49. Prefections , 27. Theortality .P. knowlesi infections could be selected and extracted, so the pooled analysis needs to be carefully interpreted. Second, some clinical and laboratory data were missing among the seven included studies, causing a low statistical power to infer the differences between severe P. knowlesi and severe P. falciparum malaria. Third, subgroup analysis of study sites between Sabah and Sarawak in the meta-analysis of severe P. knowlesi and severe P. falciparum infections could not be performed, as three studies included in the analysis were conducted in Sabah and only one included study was conducted in Sarawak. Fourth, some confounding factors might have affected the haematological analysis of the present study, such as demographic factors, malaria immunity, underlying diseases, and the haemoglobinopathy of patients with malaria. Due to the limitations of the data on haemoglobinopathies and G6PD deficiency of the study populations reported in the included studies, the effect of haemoglobinopathies and G6PD deficiency on the severity or laboratory parameters between patients with severe P. knowlesi and non-severe P. knowlesi infections could not be examined in the present study.The present study had limitations. First, only a few articles on severe P. knowlesi infections. Patients with severe P. knowlesi malaria were older, had a higher leucocyte count, and had a lower platelet count than those with non-severe P. knowlesi malaria. Patients with severe P. knowlesi infections were older, had a higher neutrophil count, and had a higher creatinine level than those with severe P. falciparum infections. Future work must aim to explore the risk factors with a higher number of enrolled studies to aid in the understanding of severe P. knowlesi infections in malaria-endemic settings.This systematic review demonstrated a high rate of severe Additional file 1. PRISMA 2009 Checklist S1.Additional file 2: Table S1. Search term.Additional file 3: Table S2. Characteristic of severe and non-severe complications of Plasmodium knowlesi.Additional file 4: Table S3. Characteristic of severe Plasmodium knowlesi and P. falciparum.Additional file 5: Figure S1. The proportion of farmers infected with Plasmodium knowlesi and P. falciparum.Additional file 6: Figure S2. The proportion of plantation workers infected with Plasmodium knowlesi and P. falciparum.


The increasing incidence of multi-antibiotic-resistant bacterial infections, coupled with the risk of co-infections in malaria-endemic regions, complicates accurate diagnosis and prolongs hospitalization, thereby increasing the total cost of illness. Further, there are challenges in making the correct choice of antibiotic treatment and duration, precipitated by a lack of access to microbial culture facilities in many hospitals in Ghana. The aim of this case report is to highlight the need for blood cultures or alternative rapid tests to be performed routinely in malaria patients, to diagnose co-infections with bacteria, especially when symptoms persist after antimalarial treatment. P. falciparum with a parasitemia of 224 parasites/μl. The patient was treated with Intravenous Artesunate, parental antibiotics (cefuroxime and gentamicin) and oral dispersible zinc tablets in addition to intravenous fluids. Blood culture yielded Acinetobacter baumanii, which was resistant to all of the third-generation antibiotics included in the susceptibility test conducted, but sensitive to ciprofloxacin and gentamicin. After augmenting treatment with intravenous ciprofloxacin, all symptoms resolved.A 6-month old black female child presented to the Agogo Presbyterian Hospital with fever, diarrhea, and a 3-day history of cough. A rapid diagnostic test for malaria and Malaria microscopy was positive forEven though this study cannot confirm whether the bacterial infection was nosocomial or otherwise, the case highlights the necessity to test malaria patients for possible co-infections, especially when fever persists after parasites have been cleared from the bloodstream. Bacterial blood cultures and antimicrobial susceptibility testing should be routinely performed to guide treatment options for febril illnesses in Ghana in order to reduce inappropriate use of broad-spectrum antibiotics and limit the development of antimicrobial resistance. Bloodstream infections (BSI) are major causes of hospitalization and mortality worldwide , 2. GlobOverlapping clinical symptoms of febrile illnesses make it difficult for medical doctors to diagnose bacterial infections. Furthermore, the increasing burden of bacterial multi-resistance both limits the choice of antibiotics and makes empirical use of broad-spectrum antibiotics . Blood cAcinetobacter baumanii, where empirical treatment according to WHO recommendation [The World Health Organization (WHO) therefore recommends the combination of antimalarial and broad-spectrum antibiotics in children with severe malaria . With riendation  was unsuA previously well 6-month-old black female infant weighing 6.5kg, with a history of fever in the past, and with no known underlying conditions were referred to Agogo Presbyterian Hospital (APH) in the Asante Akim North District of the Ashanti region after having presented to a peripheral facility with 3 days of fever, diarrhea, and cough as reported by her mother. The diarrhea was watery and mucoid but non-bloody and was associated with episodes of non-projectile cough and vomiting. The infant was treated with homemade oral herbal preparations without improvement.Upon presentation at the first point of call health facility, the child was managed with parenteral antibiotics, including intravenous cefuroxime, gentamicin and oral dispersible zinc tablets in addition to IV fluids according to the referral note. The child was subsequently referred to APH on account of her deteriorating health condition. Per the referral note, we were unable to ascertain if any laboratory tests or antimalarial treatment was initiated.P. falciparum with a parasitemia of 224 parasites/µL. Based on these findings the admission diagnosis was severe malaria with prostration and severe bronchopneumonia.On examination, the child appeared ill, but was not febrile, with an axillary temperature of 36°C, pale, not jaundiced and adequately hydrated. Further examination focusing on the central nervous system found the child to be lethargic, prostrated (loss of neck control since the onset of illness) and with a Blantyre Coma Score (BCS) of 4/5 . The patient was tachycardic with a heart rate of 146 beats per minute (bpm). The child was in severe respiratory distress with tachypnoea at rest (respiratory rate: 46 cycles per minute), grunting, nasal flaring and severe intercostal recession. Chest auscultation revealed adequate air entry with bilateral bronchial breath sounds. A rapid diagnostic test for malaria  was positive. The full blood count revealed moderate anemia with a hemoglobin concentration of 8.3 g/dL, leukocytosis  and thrombocytosis . Malaria microscopy was positive for The patient was started on parenteral cefotaxime at 300 mg four times daily and 30 mg of gentamicin. Later on, the same day of admission, IV clindamycin at 40 mg four times daily for 4 days was administered. IV Artesunate was also started at 20 mg daily at time 0, at 12 h and 24 h per standard recommended treatment in addition to supportive care, being paracetamol when needed for pyrexia and supplemental oxygen for the respiratory distress. The patient was judged to be at a high risk of aspiration and was kept nil-by-mouth and maintained on IV fluids.By day 2 of admission, the BCS had dropped to 2/5 . Her chest findings had not changed significantly. The same day, a diagnosis of cerebral malaria was included in her records. Treatment was continued and urine output monitored. The patient was started on a minimal enteral feed of 5 mL every 2 h. After 12 h the clinical state improved, with a decreased respiratory rate and Heart rate of 38 cpm and 126 bpm respectively, no signs of respiratory distress and an increasing BCS of 3/5 , which improved further three days into her admission to 5/5 . Her urine output was found to be less than 100 mL over the 24 h but improved following furosemide challenge.On day five following admission, the child continued to experience bouts of fever despite having completed antimalaria treatment and being on broad-spectrum antibiotics. All antibiotics were stopped for 48 h. On day seven, blood was taken for culture, full blood count and further malaria microscopy. The hemoglobin had dropped to 6.1 g/dL from initial 8.3 g/dL on admission, with persistent leukocytosis (24.500/μL) and thrombocytosis (295.000/μL). Malaria microscopy did not reveal any parasites in the thick and thin smear. IV ceftriaxone 500 mg daily for 72 h was administered while awaiting the blood culture results. She was also hemotransfused with 120ml of whole blood over 4 h at 30 mL per hour.et al. [Acinetobacter baumannii by Vitek MS  with a reliability of 99.9%.The blood culture sample was transported in a pediatric aerobic blood culture bottle  to the Kumasi Center for Collaborative Research in Tropical Medicine (KCCR) for analysis. The blood culture was performed as described by Hogan et al. . The bloth day after admission. Based on these results treatment was switched to a combination of parenteral ciprofloxacin (70 mg three times daily) for 72 h and gentamicin (250 mg 8 hourly for 4 days) which is readily available and affordable in addition to 250 mg of ampicillin which was later withdrawn. A second blood culture taken after 7 days of treatment was negative and the child had a consistently normal temperature. She was discharged after a total of 19 days of hospitalization on oral ciprofloxacin, which she was to continue for 5 days after discharge. The course of treatment of the patients has been summarized in Fig. The blood culture result was communicated to the treating physician on the 9Acinetobacter baumannii bacteremia in a 6-month old child. The child presenting to APH with a persistent fever after treatment and clearing of parasite, still harbored Acinetobacter baumannii. Malaria/BSI coinfections are suggested to be a major burden in SSA with the current rise of antibiotic-resistant bacteria [This case reports the existence of concomitant malaria and bacteria . It has bacteria .Plasmodium infection reveal a concomitant invasive BSI [Plasmodium spp. and bacterial pathogens in blood cultures [A study from Ghana showed that 5% of hospitalized children with a sive BSI . Similarcultures , 9. A stcultures , 10.Malaria and bacteremia often present with similar clinical symptoms making differential diagnosis difficult , 11, 12.This case report highlights the necessity of blood culture or alternative effective diagnostics for rural hospital settings in Ghana, that otherwise pose a significant health threat for pediatric patients. It also indicates that the WHO recommendation of broad-spectrum antibiotics and antimalarial treatment  may riskAcinetobacter baumannii strain in this 6-month-old child, hospital or community-acquired, could not be determined. Acinetobacter baumannii is a gram-negative coccobacillus commonly found in soil, water and dry environments [Acinetobacter baumannii is a clinically significant pathogen that can cause serious infections, including respiratory tract infections, bacteremia, and urinary tract infections, in addition to a multi-resistance to antibiotics [The source of the isolated ronments , which hronments . Acinetoibiotics .Acinetobacter baumannii and Plasmodium spp. is important in clinical diagnostic. In children with malaria, undetected bacteria may contribute to morbidity due to untreated bacterial infections. the fact that bacteria was not treated. With the current rise in resistance among important bacterial pathogens, the necessity for blood (and other specimens) cultures for the enrichment of bacteria cannot be ignored. The ability for laboratories to perform bacterial cultures is, therefore, a key to avert the current trend in antibiotic resistance, especially in rural hospital settings. Antimicrobial test results should guide treatment decisions for patients in Ghana to reduce the inappropriate use of broad-spectrum antibiotics. The development of rapid and cost-effective alternatives for the identification of bacterial (co-) infections could be a solution for low-resource settings, where lack of financial means and expertise do not allow blood cultures to be performed.Even though this study cannot confirm whether the bacterial infection was nosocomial or otherwise, the presented case highlights the need to perform blood cultures on malaria positive patients, especially when fever persists after parasites have been cleared. Awareness of possible concomitant infection of