Plasmodium malariae, Plasmodium ovale spp, and mixed-Plasmodium infections is scarce. In this study, we aimed to analyse the efficacy of pyronaridine–artesunate for the treatment of non-falciparum and mixed-species Plasmodium infections from a large phase 3b/4 clinical trial in central Africa.High-quality evidence for the therapeutic efficacy and effectiveness of antimalarials for infections caused by Plasmodium infections or non-falciparum mono-infections. Day 28 (or unforeseen visit) samples positive for non-falciparum malaria were re-assessed by microscopy to identify microscopic versus submicroscopic infections. Analyses were done on two sample sets: a per-protocol set and an intention-to-treat set.This post-hoc analysis was done in a random subset of samples from two sites (in the Democratic Republic of the Congo and in Gabon) of the CANTAM-Pyramax trial assessing pyronaridine-artesunate therapy. We randomly selected paired dried blood spot samples from day 0 and day 28 (or unforeseen visit) and analysed them by quantitative PCR for mixed Plasmodium infections or non-falciparum mono-infections. We did not detect P vivax in the samples. For both the per-protocol and intention-to-treat sets, the overall day 28 cure rates for P malariae, P ovale curtisi, and P ovale wallikeri were 96·3% or higher (95% CIs from 81·0–99·9 to 95·7–100). Cure rates were consistently high in P malariae  and P ovale spp  infections.Among 1502 randomly selected samples, 192 (12·8%) showed mixed-P malariae, P ovale curtisi, or P ovale wallikeri and mixed-Plasmodium infections in a real-world setting.This post-hoc analysis provides important evidence supporting the high efficacy of pyronaridine-artesunate against mono-infections with Medicines for Malaria Venture. Plasmodium falciparum and, to a lesser extent, caused by Plasmodium vivax.Plasmodium malariae, Plasmodium ovale curtisi, or Plasmodium ovale wallikeri and mixed Plasmodium infections have mostly been excluded from these clinical trials, and data analyses and evidence showing the efficacy of recommended treatments in these infections are thus extremely scarce.Plasmodium species were traditionally regarded as rarely occurring species and of minor importance, the availability of diagnostic methods with increased sensitivity, changes in malaria transmission patterns, and improved control of falciparum malaria have led to an increased importance of these neglected forms of malaria.5Over the past few decades, many clinical trials have shown the efficacy, tolerability, and safety of antimalarial therapies for the treatment of malaria caused by P falciparum, as shown in previous WHO World Malaria Reports.Plasmodium species are finding their way into these reports too. Over the past decade, non-falciparum and non-vivax malaria has moved in the focus of the scientific community, and publications reporting non-falciparum or mixed Plasmodium infections have increased, not only from falciparum-endemic countries, but also in travel returnees.P falciparum is declining in Africa, the transmission of P ovale spp and P malariae persists, with an overall increase in odds of infection.9For years, malaria cases reported from African countries were almost exclusively classified as P ovale spp and P malariae has also been reported in Asia, where asymptomatic infections have the potential to challenge progress of malaria control programmes.Plasmodium infections is important to reduce morbidity and, especially in malaria-elimination settings, to interrupt transmission.11Persistence of Evidence before this studyPlasmodium ovale spp and Plasmodium malariae. We searched WHO's International Clinical Trials Registry Platform on Oct 22, 2021, with the terms “Plasmodium ovale”, “Plasmodium malariae”, “non-falciparum”, “mixed Plasmodium”, and “mixed infection”, with no language restrictions, which resulted in the identification of two small prospective trials for the treatment of acute malaria with artemether–lumefantrine. With the same search terms and on the same date, we also searched the European Medicines Agency webpage and the US Food and Drug Administration webpage (using the tool FDALabel) for registered drugs, and we found no artemisinin-based combination therapies registered for the treatment of P ovale spp, P malariae, or mixed Plasmodium malaria.Two previous systematic reviews have shown a scarcity of evidence for treatment recommendations for malaria caused by Added value of this studyPlasmodium infections have been reported with increasing frequency in malaria endemic countries, as well as in travellers from non-endemic countries. At the same time, evidence-based efficacious antimalarial treatments, which can be widely applied, are needed. However, antimalarial treatments for all Plasmodium species and mixed Plasmodium infections are indispensable for malaria control and elimination. We did a post-hoc analysis focused on non-falciparum and mixed Plasmodium infections on a large subset of participants who took part in a large, prospective, clinical phase 3b/4, cohort event monitoring study designed to evaluate, in a real-life setting, the safety, tolerability, and efficacy of pyronaridine–artesunate therapy for uncomplicated malaria.Over the past decade, non-falciparum and mixed Implications of all the available evidencePlasmodium species reported in this study. This can simplify the future disease management of patients with malaria given the challenges of routine diagnostics of non-falciparum and mixed-species malaria by rapid diagnostic tests and microscopy. The nature of the analysed data highlights not only the efficacy, but also the effectiveness of pyronaridine–artesunate in a real-world setting.The results of this study provide high-quality evidence that support the use of pyronaridine–artesunate for the treatment of all African P falciparum and blood-stage P vivax malaria. For malaria control and malaria elimination programmes, efficacious antimalarial treatments for all Plasmodium species and mixed infection are indispensable. Generating more evidence for the treatment of infections with P malariae and P ovale spp is thus a much-needed undertaking.A fixed dose combination of pyronaridine and artesunate  is the only artemisinin-based combination therapy with positive assessments from stringent regulatory authorities for both the treatment of P malariae and P ovale spp.For this post-hoc analysis, we analysed blood samples from the CANTAM-Pyramax real-world study, in which pyronaridine–artesunate was administered to patients with acute uncomplicated malaria, to provide a patient pool of considerable size from endemic countries with a known prevalence of Plasmodium infections and corresponding efficacy of pyronaridine–artesunate for infections with Plasmodium species and mixed infections to provide urgently needed evidence based on systematically collected data.We aimed to assess the proportions of non-falciparum, non-vivax monoinfections and mixed NCT03201770), which has been published in more detail elsewhere.We did a post-hoc analysis assessing samples collected during the CANTAM-Pyramax trial , and are both known regions of relevant prevalence of non-falciparum malaria.We did a retrospective real-time quantitative PCR (qPCR) analysis using dried blood spot (DBS) samples from the two CANTAM-Pyramax African sites: the Centre Hospitalier du Mont-Amba in Kinshasa (Democratic Republic of the Congo [DR Congo]), representing an urban setting in a large metropolitan region of central Africa, and the Centre de Recherches Médicales de Lambaréné (CERMEL) in Lambaréné (Gabon),For this post-hoc analysis, a random subset of malaria episodes for whom paired samples from day 0 and day 28 (or day 0 and unforeseen visit between day 4 and day 28) were available was identified by generation of a random list by an independent statistician, with the aim of including 750 cases per study site.P falciparum, P malariae, P ovale curtisi, P ovale wallikeri, and P vivax with previously published methods.P falciparum , P vivax , P malariae , and P ovale spp . Samples positive for P ovale spp were further analysed for P ovale curtisi and P ovale wallikeri.Plasmodium species other than P falciparum. For samples in which qPCR identified mixed Plasmodium or non-falciparum infection on day 28 (or unforeseen visit), the corresponding thick smear was reassessed for microscopic presence of non-falciparum malaria, to ascertain microscopic versus submicroscopic parasitaemia and to confirm asexual parasitaemia as opposed to assay positivity triggered by gametocytaemia alone. Microscopic reassessment was done at the Laboratoire Clinique at CERMEL, by use of the Lambaréné method for malaria microscopy.We analysed DBS samples by qPCR at the Department of Biomedical Sciences, Institute of Tropical Medicine , to detect and differentiate Statistical analyses were descriptive and done with SAS (version 9.4) in a Linux environment by Datamap . Two analysis sets were defined for this post-hoc analysis. The post-hoc per-protocol set comprised of all malaria episodes excluding those with major protocol deviations, whereas the post-hoc intention-to-treat analysis set included all malaria episodes of the random sample subset. Clinical patient data were extracted from the CANTAM-Pyramax clinical database and provided for all malaria episodes included in the random sample subset.Demographic data and patient characteristics for the post-hoc analysis set were summarised, with number and percentages for categorical variables, and mean (SD), median (IQR), and range for continuous variables. Results are provided for all non-falciparum and mixed-species infections overall, by study site, and by species present on day 0.Plasmodium species was detected by qPCR in the day 0 DBS sample. To assess efficacy, we determined crude cure rates for all non-falciparum species. We defined the day 28 cure rate on the basis of the post-hoc qPCR analysis, separately for each Plasmodium species other than P falciparum . A patient was considered cured for the species of interest if the species was detected in the day 0 DBS and not detected in the day 28 DBS, irrespective of any other species being present in the day 28 DBS. Patients were considered not cured if the same species of interest was detected in a DBS taken on day 0, as well as in DBSs taken between day 4 and day 28.Effectiveness was depicted for malaria episodes in which any non-falciparum species alone or in combination with any other P falciparum cure rates were not determined because no further qPCR analyses of pure P falciparum samples at day 28 were done.When a mixed infection was present at day 0, the patient was considered for inclusion in the analysis of each species that was present in the day 0 sample, meaning that a patient could be allocated multiple times. Each cure rate is presented as the number and percentage of patients with exact Clopper Pearson 95% CIs overall, by species, and separately for each site. An additional summary of cure rates for the post-hoc per-protocol set is also provided.The funder was involved in the conceptualisation, funding acquisition, methods, project administration, resources, supervision, validation, and review and editing of the manuscript.We included day 0 samples of 1502 malaria episodes in 1413 patients in this analysis; 750 malaria episodes in 715 patients from the DR Congo site and 752 malaria episodes in 698 patients from the Gabon site. Of these, 64 (9·0%) patients at the DR Congo site and 96 (13·8%) at the Gabon site  had multiple episodes of malaria. On day 28, three samples were qPCR-positive for non-falciparum malaria at the DR Congo site and none at the Gabon site. There were no non-falciparum-positive samples from an unscheduled visit between days 0 and 28 on either site.·0%] events in male participants and 345 [46·0%] in female participants in DR Congo, and 399 [53·1%] in male participants and 353 [46·9%] in female participants in Gabon). The overall median age in this randomly selected population was 10·0 years (SD 14·8) and was also similar at the DR Congo and Gabon sites (9·0 years [13·9] at the DR Congo site and 11·0 years [15·6] at the Gabon site). The majority of infections overall and at each of the two sites occurred in participants aged 5–12 years . The mean body-mass index was similar overall and at each of the two sites at the DR Congo and Gabon sites .Overall, slightly more randomly assigned malaria events occurred in male participants than in female participants , which wP falciparum mono-infections, six (0·4%) were P malariae mono-infections, three (0·2%) were P ovale spp mono-infections , 183 (12·2%) were mixed Plasmodium spp infections, and 73 (4·9%) were negative in qPCR. No patient was reported with a P vivax infection.Of the 1502 malaria episodes on day 0, 1237 [82·4%] were P falciparum infections, with one mixed infection not including this species  and P ovale spp (122 [95·3%]) infections occurred as part of mixed infections. Mixed Plasmodium infections were equally balanced between the site in DR Congo (97 [13·1%]) and the site in Gabon  for P malariae, 272 per μL (712) for P ovale curtisi, and 1808 per μL (8536) for P ovale wallikeri (182 (12·1%) episodes in the post-hoc subset were mixed allikeri .Table 2PP falciparum, P malariae, P ovale curtisi, or P ovale wallikeri mono-infection all had headache and rigours or chills as the two most commonly reported malaria symptoms on day 0. By contrast with patients with a P falciparum mono-infection, patients with a P malariae, P ovale curtisi, or P ovale wallikeri mono-infection did not show any events of sweating, jaundice, and haepatomegaly. Patients with mixed Plasmodium infections commonly reported symptoms of headache (124 [67·8%] of 183 episodes), rigours or chills (92 [50·3%]), cough (67 [36·6%]), and loss of appetite or anorexia (61 [33·3%]). Mean body temperatures at day 0 were similar overall and for patients infected with a P falciparum mono-infection, P malariae mono-infection, P ovale curtisi mono-infection, P ovale wallikeri mono-infection, or mixed-Plasmodium infection, ranging from 36·7°C to 37·2°C (SD 0·35 to 0·98). Detailed listings of signs and symptoms are shown in the Patients with a P falciparum  and the other slide was negative .Overall, two samples on day 28 (both from DR Congo) had the same non-falciparum infection by qPCR as on day 0 and were further assessed by microscopy. In the microscopic reassessment, one slide was positive for Plasmodium species other than P falciparum. The day 28 cure rates in the post-hoc intention-to-treat analysis set for P malariae, P ovale curtisi, and P ovale wallikeri were 96·3% or higher (95% CIs from 81·0–99·9 to 95·7–100). We observed slightly higher point estimates of cure rates in patients with P malariae infection than in those infected with P ovale curtisi or P ovale wallikeri, but the 95% CIs crossed in all subanalyses  such infections in this analysis, which were equally distributed between the urban site in DR Congo and the rural site in Gabon. 183 (12·2%) of all episodes were mixed infections. This underlines the epidemiological importance of non-falciparum and mixed Plasmodium malaria in central Africa and the associated need for antimalarials with confirmed effectiveness for the treatment of these neglected Plasmodium infections.The distributions of baseline characteristics in this post-hoc subset were similar between both study sites, which suggests an unbiased randomisation process for the selection of samples. The underlying expectation that approximately 10% of the 1502 randomly selected malaria episodes would be non-falciparum mono-infections or mixed Plasmodium infections can be challenging. Information about the epidemiology of P malariae and P ovale spp is scarce, which can make it difficult to identify suitable geographical settings where sufficiently large sample sizes can be obtained for clinical trials in this field. Additionally, the diagnosis of non-falciparum malaria requires microscopists to be well trained in identifying potential study participants. In case of reappearance of parasites during the observational period, no established methods are available to reliably distinguish recrudescence from reinfection and relapse, if applicable, which might lead to overestimation or underestimation of failure rates. In this post-hoc analysis, we chose the more conservative approach to consider reappearance of the same Plasmodium species within the follow-up period as failure.Determining antimalarial treatment efficacy for non-falciparum and mixed P falciparum infection alone and that microscopic identification of low-level parasitaemia of non-falciparum malaria is challenging, the observed prevalence in this study might underestimate somewhat the true prevalence of non-falciparum malaria in this setting.To reflect a real-world setting, patients with malaria were recruited in the CANTAM-Pyramax trial on the basis of either a positive RDT result  or malaria microscopy—depending on what was routine procedure at the respective sites. On the basis of the fact that some RDTs were targeting Fewer than 5% of samples of patients recruited on the basis of RDT tests had a negative result in qPCR testing. This discrepancy of results might be explained by the test characteristics of RDTs. Positive predictive values (PPVs) and negative predictive values of RDTs for clinical malaria have been modelled previously,P falciparum and one microscopically negative slide, suggesting there were submicroscopic non-falciparum parasitaemia in both samples. The effect of antimalarial treatment regimens with and without 8-aminoquinolines on P falciparum gametocytes have been described previously, and a 2022 publication has detailed the specific clinical effects of pyronaridine–artesunate on the sexual stages of this species.Plasmodium malariae and Plasmodium ovale spp, and the characteristics of those stages, have not been described conclusively so far.The microscopic reassessment of qPCR-positive day 28 samples showed one slide being microscopically positive for P falciparum and P vivax (>95%).P ovale wallikeri at the DR Congo site. However, this is based on one observation alone, which had such a big effect because of the low sample size in this group (one of 16 observations). Given that the clinical trial was an effectiveness trial with unsupervised treatment intake on days 2 and 3, these high cure rates further support the use of pyronaridine–artesunate in clinical routine. Therefore, our results give confidence that pyronaridine–artesunate can be used to effectively treat blood-stage infections of all African Plasmodium spp reported in this study. This finding can simplify the future management of patients with malaria, given the challenges of routine diagnostics of non-falciparum and mixed-species malaria by RDT and microscopy.Overall cure rates on day 28 in this post-hoc analysis were very good for all non-falciparum species and similar to the effectiveness of pyronaridine–artesunate in P malariae was more prevalent than P ovale spp, which reflects what is reported from other endemic regions.P ovale curtisi than P ovale wallikeri episodes; this differs from data from returning travellers, but it is in line with previous prospective research done in Gabon.vs short-time traveller from an endemic region diagnosed in a non-endemic region).Plasmodium knowlesi have only been reported from southeast Asia.In this sample subset, P vivax infection was thought to be limited to individuals expressing the Duffy antigen–chemokine receptor gene, which served as explanation for the absence of vivax infections in large parts of Africa. However, over the past years, more and more confirmed vivax cases were reported not only from the African continent, but also at the same time from Duffy-negative individuals.P vivax infections have also been previously reported in DR Congo.For a long time, Plasmodium species were determined no matter whether they occurred as part of a mono-infection or mixed infection. We are not aware of any data showing reduced effectiveness of antimalarials in mono-infections compared with mixed Plasmodium infections, and thus we are confident that these data provide reliable estimates for both mono-infections and mixed-species infections with these Plasmodium species. Additionally, it might not be ruled out that some non-falciparum infections were below the threshold of detection at initial assessment due to quantitative suppression by P falciparum or due to primer competition. However, as this study aimed to assess the therapeutic effectiveness of pyronaridine–artesunate, the analysis was restricted to only those patients with a positive PCR at recruitment.A key limitation of this post-hoc analysis is that we observed only nine non-falciparum mono-infections in this large subset of malaria episodes. For this analysis, outcomes for each P ovale, P malariae, and mixed-species malaria.In summary, this post-hoc analysis provides high-quality evidence for the high efficacy and clinical usefulness of pyronaridine–artesunate combination therapy for the treatment of https://www.mmv.org) on reasonable request.All relevant data are presented in this Article and the IB-F and SD are full-time employees of Medicines for Malaria Venture (MMV). JS is a full-time employee of Shin-Poong Pharmaceutical. RM and SA-B are consultants paid by Shin-Poong Pharmaceutical. All other authors declare no competing interests.


Assessment of target organ damage (TOD) is an important part of the diagnosis and evaluation of hypertension. Carotid-femoral pulse wave velocity (cf-PWV) is considered to be the gold-standard for noninvasive arterial stiffness assessment. This study aims to analyze the risk of TOD in people with different phenotypes of peripheral blood pressure and cf-PWV.The study cohort was recruited from December 2017 to September 2021 at Ruijin Hospital in Shanghai. It was divided into 4 groups according to peripheral blood pressure (pBP) and cf-PWV. TOD was assessed as carotid intima-media thickness (CIMT), chronic kidney disease (CKD), urinary albumin-creatinine ratio (ACR), estimated glomerular filtration rate (eGFR) and left ventricular mass index (LVMI).P < 0.01). eGFR, ACR, LVMI and CIMT were significantly different among different groups (P < 0.01). The risk of ACR abnormality was significantly higher in the group with elevated pBP , while left ventricular hypertrophy (LVH) was significantly higher in the group with elevated cf-PWV .A total of 1,257 subjects  was recruited. Age, body mass index (BMI) and fasting blood glucose (FBG), as well as peripheral systolic blood pressure (pSBP), peripheral diastolic blood pressure (pDBP), peripheral pulse pressure (pPP) were significantly different in the four groups (Different profiles based on the status of PBP and cf-PWV associated with different TOD. Individuals with higher pBP have an increased risk of ACR abnormality, while individuals with only cf-PWV elevated have a higher risk of LVH. The Chinese Hypertension Survey 2012–2015) found that the crude prevalence rate of hypertension among Chinese adults (≥18) was 27.9% (weighted rate was 23.2%). The prevalence of hypertension in young people (18–34 years old) is 5.2%, and that of those ≥75 years of age is 59.8%. It is estimated that the number of adult hypertensive patients over 18 years of age in China is 245 million  is still uncertain. For example, the Finn-Home study  suggesteThe aim of this study was to analyze the risk of TOD in people with different BP and cf-PWV. This is to promote patient management in an individualized manner, with precise diagnosis and treatment. This approach will avoid considering only a single marker , which can lead to being over-treated or under-treated.A total of 1,257 participants (age ≥18 years) who received a routine physical examination at Ruijin Hospital, Shanghai, China, between December 2017 and September 2020 was recruited. Subjects with any history of occlusive arterial disease history such as myocardial infarction or acute coronary syndrome, transient ischemic attack or stroke were excluded from the study. 1,300 subjects were invited, of whom 1,257 were enrolled (43 lacked cf-PWV data). The protocol received Ruijin Hospital ethics approval and all participants provided written informed consent.All patients had their medical history taken, underwent physical examination and collection of anthropometric data. Height was mea-sured with a wall-mounted stadiometer to the nearest 0.5 cm with-out shoes and body weight was measured on a balance calibrated to the nearest 0.1 kg with minimum clothing and without shoes. Measurements were made with an up-stretched tape meter and recorded to the nearest 0.1 cm. Body height and weight were measured without shoes. Anthropometric measures such as body mass index (BMI) were calculated using these anthropometric data. Status of current smoking was defined as having smoked the last cigarette within 1 week of when BP measurements were taken.Blood samples were obtained after 12–14 h of overnight fasting. Serum Total Cholesterol (TC), Low-density Lipoprotein Cholesterol (LDL-C), High-density Lipoprotein Cholesterol (LDL-C), Triglycerides (TG), Fasting Blood Glucose (FBG), and serum Creatinine (Cr) were obtained from patient medical records. The Modification of Diet in Renal Disease (MDRD) formula was used to calculate the esti-mated Glomerular Filtration Rate (eGFR) . Urinaryr wave of the electrocardiogram.Seated BP measurements were obtained in 12-h fasting individuals in the morning (7–9 am) with a standard manual brachial cuff sphygmomanometer after a 10-min rest and using the average of two readings in both arms. Pulse pressure (PP) was calculated as the difference between systolic BP (SBP) and diastolic BP (DBP) and mean arterial pressure (MAP) was calculated as (DBP) + (PP)/3. Measurement of cf-PWV was performed using the SphygmoCor CVMS system  as per the manufacturer's protocol in a supine position. Radial artery pressure waveforms were recorded using the high-fidelity tonometer for at least 10 s, until a stable radial tonometric pressure trace was obtained. cf-PWV was calculated as the ratio of the direct distance between the carotid and femoral sites of measurements and pulse transit time calculated as a direct delay between the two waves in relation to the Carotid IMT (CIMT) was examined bilaterally using high-resolution echocardiography Doppler ultrasound  with a broad-band linear array transducer (multiple frequency: 4–12 MHz). IMT was measured on both the left and right common carotid artery starting ~1.5 cm proximal to the carotid artery bulb. Three recordings were taken, and the mean value was calculated for each side. “Plaque = 0” referred to the absence of plaque, while “plaque = 1” referred to the presence of plaque.3 – (LVEDd)3]} +0.6 and LVMI (g/m2) = LVM/BSA , interventricular septal diameter (IVSd), posterior wall thickness at end-diastole diameter (PWTd) and left ventricular end-systolic diameter (LVESd) from the parasternal view , and the LVM/BSA . The abo LVM/BSA .2  or LVMI of 95 g/m2   = urinary albumin/urinary creatinine. eGFR < 60 ml/min per 1.73 m2 was defined as CKD and ACR >30 mg/g was defined as microalbuminuria. Arterial TOD was defined as increased CIMT (CIMT > 900 um) or as presence of arterial plaque.Asymptomatic hypertensive TOD include cardiac, arterial, and renal TOD. Left ventricular hyper-trophy (LVH) was defined as LVMI of 115 g/m . Renal TThe included subjects were grouped according to peripheral BP and cf-PWV. Group(control): normal BP and cf-PWV, with peripheral BP <140/90 mmHg and cf-PWV < 10 m/s; Group (PBP): only elevated BP, normal cf-PWV, with peripheral BP ≥ 140/90 mmHg and cf-PWV <10 m/s; Group(cf-PWV): Only with elevated cf-PWV with peripheral BP < 140/90 mmHg and cf-PWV ≥ 10 m/s. Group (PBP/cf-PWV): abnormal peripheral BP and cf-PWV; peripheral BP ≥ 140/90 mmHg and cf-PWV ≥ 10 m/s.t-test and Chi-square test respectively for males and females. To compare continuous and categorical variables among the four groups, Analysis of Variance (ANOVA) and Chi-square test respectively were used. Logistic regression analysis was used to calculate the Odds Ratios (ORs) adjusted for cardiovascular risk factors including age, sex, BMI, smoking, hypertension, Antihypertensive drugs (yes or no), HDL, LDL, fasting blood glucose, peripheral mean arterial pressure, heart rate. Analyses were performed with SPSS 26.0 for Windows . A two-sided p < 0.05 was considered statistically significant.Continuous variables are expressed as mean ± SD, and frequencies (percentage) are reported for categorical variables. Continuous and categorical variables were compared using P < 0.01), while females had higher levels of total cholesterol, LDL and HDL (P < 0.01). There were no significant differences for peripheral SBP (pSBP) and heart rate (HR) between males and females. Males had a higher level of pDBP, while pPP was significantly higher in females. However, in terms of medical history, the prevalence of hypertension and the rate of antihypertensive treatment were significantly higher in men, as well as cf-PWV (P < 0.05). In the case of TOD, eGFR, ACR and LVMI were significantly higher in male than in female.A total of 1,300 subjects was enrolled in this study, 43 of which were excluded due to data abnormalities, and finally 1,257 subjects  were recruited in our study . ResultsP < 0.01). There was no statistical difference in hypoglycemic treatment  and pPP . ACR was positively correlated with pSBP . lgACR was positively correlated with pSBP , pDBP , pMAP  and pPP . LVMI was positively correlated with pSBP , pDBP , pMAP  and pPP . CIMT was positively correlated with pSBP , pMAP  and pPP . cf-PWV was negatively correlated with eGFR , and positively correlated with ACR , lgACR , LVMI  and CIMT  (EGFR was negatively correlated with pSBP (= 0.292) .P = 0.005, OR 2.264, 95%CI 1.277–4.016; p = 0.003, OR 1.482, 95%CI 1.144–1.920), while the risk of LVH was significantly increased in Group(cf-PWV)  after adjusting age, sex, BMI, smoking history, hypertension, antihypertensive drugs (yes or no), HDL, LDL, fasting blood glucose, pMAP, HR  as the reference group, Group(PBP) and Group(PBP/cf-PWV) had higher risks of ACR abnormality  .The main finding of our study is that the increase in cf-PWV or increase in peripheral SBP alone, have different effect on specific target organs. Elevated peripheral SBP was associated with an increase of ACR, depending on whether cf-PWV was increased, the OR was 2.264 and 1.482, respectively. When peripheral SBP increased, regardless of the cf-PWV level, all participants had a certain risk of kidney and heart damage. Elevated cf-PWV alone was likely to cause LVH (OR 1.868).Previous studies suggested that central and peripheral BP are correlated with urinary albumin excretion and ACR , 15, sugA Chinese study  suggesteOur study also showed that cf-PWV is more closely related to LVH, but no correlation between increased blood pressure and increased risk of LVH has been observed. Theoretically, aortic stiffness increases the effects of wave reflection of the pressure waveform, leading to various unfavorable hemodynamic consequences, such as increased left ventricular afterload, which can lead to damage of the heart muscle , and thiBP elevation is generally the most important risk factor resulting in increased Left ventricular mass (LVM) and leading to Left ventricular hypertrophy (LVH). PWV were greatly dependent on age and blood pressure. A recent study by Yun M et al. confirmed that elevated BP precedes the development of LVH and hemodynamic properties are responsible for the development of cardiac enlargement, during the young-to-midlife adult age period . In our Carotid IMT formation is a valid measure of subclinical TOD which is related to future cardiovascular events. IMT was evaluated with manual measurement while echo-tracking semi-automated system exist , they weAt present, the correlation between BP, cf-PWV and TOD remains to be further studied. The African-PREDICT study  suggesteThe heterogeneity of these research conclusions may be related to many factors including age, personal characteristics, comorbid diseases, race, treatment, PP amplification and other factors. The results of our study showed that antihypertensive therapy is related to lgACR, LVMI, and cf-PWV, and hypoglycemic therapy is related to ACR. Age, gender, and peripheral SBP were independent risk factors for LVMI. Blood glucose, triglycerides, cholesterol, low-density lipoprotein, peripheral SBP, and heart rate were independent risk factors for cf-PWV. This also suggested the joint effect of multiple risk factors in target organ damage and the mutual influence of these risk factors.There are some shortcomings and limitations in this study. This is a cross-sectional study with a small sample size and large differences among the four groups of grouped samples, which may lead to bias in the results. At the same time, due to the small sample size, this study did not further conduct male and female grouping and age stratification analysis. There was a large proportion of people taking antihypertensive drugs in the study, the time on antihypertensive and different types of antihypertensive drugs may have differing effects on the target organs of hypertension. The cuff sphygmomanometer is cylindrical rather than conical, which may be more appropriate for obese participants with large upper arms.In conclusion, we found that BP and cf-PWV have different effects on TOD. This suggest that TOD is affected by many factors, and it is not comprehensive to consider only BP or PWV. Factors such as age, comorbid diseases, treatment plan should be considered. For example, studies have suggested that for untreated non-diabetic hypertensive patients, there was a correlation between proteinuria, cf-PWV and LVMI, suggesting that TOD was progressing in parallel . This caThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Ruijin Hospital. The patients/participants provided their written informed consent to participate in this study.HC, YH, and JZ designed the research study, wrote the manuscript, analyzed the data, drafted, and revised the manuscript. XD, QW, and YB performed the research. YH, JZ, and XD analyzed and interpreted data. JZ and AA contributed to the thorough reading of the manuscript and several revisions. All authors contributed to the article and approved the submitted version.This project was supported by the National Natural Science Foundation of China (Grant No. 81500190), Clinical Science and Shanghai Municipal Hospital New Frontier Technology Joint Project (SHDC12019X20), and Shanghai Municipal Commission of Health and Family Planning (Grant No. 2020YJZX0124).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plasmodium knowlesi (Pk). A 5-plex chemiluminescent assay simultaneously measures pan-Plasmodium lactate dehydrogenase (pLDH), P. falciparum (Pf)-LDH, P. vivax (Pv)-LDH, Pf-histidine-rich protein-2 (HRP2), and C-reactive protein. We assessed its diagnostic performance on whole blood (WB) samples from 102 healthy controls and 306 PCR-confirmed clinical cases of Pf, Pv, Pk, P. malariae (Pm) and P. ovale (Po) mono-infections from Southeast-Asia. We confirm its excellent HRP2-based detection of Pf. Cross-reactivity of Pf-LDH with all non-falciparum species tested was observed (specificity 57.3%). Pv-LDH performance was suboptimal for Pv (93.9% sensitivity and 73.9% specificity). Poor specificity was driven by strong Pk cross-reactivity, with Pv-LDH detecting 93.9% of Pk infections. The pan-LDH-to-Pf-LDH ratio was capable of discerning Pv from Pk, and robustly differentiated Pf from Pm or Po infection, useful in regions with hrp2/3 deletions. We tested the platform’s performance in plasma for the first time, with WB outperforming plasma for all analytes except Pv-LDH for Pk. The platform is a promising tool for WB malaria diagnosis, although further development is warranted to improve its utility in regions co-endemic for multiple non-falciparum species.Commercial point-of-care tests remain insufficient for accurately detecting and differentiating low-level malaria infections in regions co-endemic with multiple non-falciparum species, including zoonotic  Ongoing challenges in malaria elimination include the lack of adequate tools to detect the low parasite densities seen in asymptomatic infection, with this group serving as a significant reservoir for transmission3. Light microscopy and rapid diagnostic tests (RDTs), the most commonly used point-of-care malaria diagnostics in endemic areas, lack sensitivity in low-level infection, and often demonstrate poor accuracy for differentiating non-falciparum species5. The development of highly sensitive and specific malaria diagnostic tools is essential both for effective treatment and for prevention of onward transmission6.Malaria remains a major global problem, with 241 million cases and 627,000 deaths reported in 2020Plasmodium falciparum lactate dehydrogenase (Pf-LDH), P.vivax-LDH (Pv-LDH), pan-Plasmodium-LDH (pan-LDH) and C-reactive protein (CRP), a host inflammatory marker7. The performance of 4-plex8 and 5-plex7 assays for antigen-based malaria screening has been tested on whole blood from asymptomatic and symptomatic infections with P. falciparum, P. vivax, P. malariae and P. ovale and on matching dry blood-spots9, reporting high sensitivity for P. falciparum, including for those with hrp2/3 deletions. Apart from P. cynomolgi10, genus-specific pLDH has enabled detection of all human Plasmodium species in whole blood with varying sensitivity. However, because of the high level of conservation of the Plasmodium protein, anti-pLDH antibodies designed to detect species-specific epitopes in the protein have demonstrated cross-reactivity with other species, resulting in loss of specificity10.A 5-plex chemiluminescent enzyme-linked immunosorbent assay (ELISA) (Quansys Biosciences) simultaneously detects and quantifies histidine-rich protein-2 (HRP2), Plasmodium knowlesi causes zoonotic malaria across most of Southeast Asia, including severe and fatal disease12. Knowlesi malaria is co-endemic with P. falciparum, P. vivax, P. malariae and P. ovale across much of its geographical range. Because of high levels of conservation in antigenic targets between P. knowlesi and other non-falciparum species, especially P. vivax, there is significant cross-reactivity between species16. RDTs evaluated to date have had low sensitivity and poor specificity for diagnosis of P. knowlesi19. As a zoonosis, its epidemiology and strategies for control differ significantly from other species causing malaria, making accurate diagnosis important. The 5-plex Quansys platform has significant cross-reactivity between Pv-LDH and P. knowlesi generated from in-vitro cultures10. Detection of low parasitemia with high specificity and sensitivity for all Plasmodium species causing human malaria in co-endemic areas is essential.Plasmodium species, and provide novel data for clinical P. knowlesi infections.Here, we evaluated the diagnostic performance of the 5-plex platform in whole blood and for the first time in plasma from malaria patients in regions co-endemic for the five major human Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae or P. ovale spp. as part of prospective clinical studies in Sabah, Malaysia21. Venous blood was also concurrently collected from healthy asymptomatic adult controls negative for malaria on microscopy and subsequent polymerase chain reaction (PCR) testing22. At the time of sampling Sabah had co-endemic transmission of the five aforementioned Plasmodium species. Additional samples from a small number of patients with P. ovale infection were obtained from a clinical trial in Timika23, a lowland forest region of Indonesian Papua with very low prevalence of this species24. Malaria patients in these regions were enrolled according to inclusion and exclusion criteria as previously described25. Clinical and laboratory features to define severe malaria were based on WHO 2014 research criteria26. Whole blood and plasma samples were stored at − 80 °C within 30 min and thawed prior to antigen evaluations. Plasmodium species mono-infections were confirmed for all malaria cases by PCR using validated protocols described elsewhere28. Parasite counts per 200 white cells (thick smear) or 1,000 red cells (thin smear) were determined by experienced research microscopists, then converted to parasites per microlitre blood using automated cell counts.Clinical malaria cases from two endemic regions in Southeast Asia were included in the study. Peripheral venous blood was collected from malaria patients presenting to health facilities with fever and infected with Plasmodium antigens and CRP in ethylenediaminetetraacetic acid-anticoagulated whole blood and heparinised plasma. The assay was performed according to manufacturer’s instructions and serially diluted protein standards from the kit were used to construct standard curves. Chemiluminescent imaging of each plate was captured on a Q-View™ Imager LS and analysed using Q-View™ software (Quansys Biosciences). Samples were run neat and diluted up to 100-fold until results were obtained in a reliable area of the standard curves. Several samples needing further dilution for HRP2 and CRP were assigned a value of the upper limit of quantification on their standard curve. Samples run neat that had antigen(s) below the detection limit were conservatively assigned a value corresponding to the assay’s lower limit of detection for that antigen(s).The commercially-available Q-Plex™ Human Malaria assay  was used to detect and quantify four Plasmodium species result was used as the gold-standard reference. For the Q-Plex™ Human Malaria assay, the mean plus 3 × standard deviation of the control group was then used as the threshold cut-off to determine positivity for each antigen. Two other standard methods to determine threshold cut-offs were used as additional comparators – values giving > 99% specificity on receiver-operating-characteristic (ROC) curves versus (a) controls and (b) non-cognate Plasmodium species 7. Sensitivity was calculated as true positives divided by the sum of true positives and false negatives. Specificity was calculated as true negatives divided by the sum of true negatives and false positives. These values were expressed as percentages and reported with 95% confidence intervals (CI) determined using the binomial exact method. The McNemar paired chi-squared test with Yates correction was used for the comparison of assay sensitivities between paired plasma and whole blood.Statistical analyses were performed using Graphpad Prism 9.2.0 . The Mann–Whitney test was used for the comparison of two patient groups, and the Kruskal–Wallis test with Dunn’s multiple comparison was used for comparisons with > 2 groups. To evaluate diagnostic sensitivity and specificity, the PCR Studies were approved by the Human Research Ethics Committees of the Ministry of Health, Malaysia; the National Institute of Health Research and Development, Ministry of Health, Indonesia; and Menzies School of Health Research, Northern Territory, Australia. Written informed consent was obtained from all participants or their legal guardian. All methods were performed in accordance with relevant guidelines and regulations.P. falciparum, 78 P. vivax, 126 P. knowlesi, 26 P. malariae and 10 P. ovale spp. . Median parasitemia was significantly higher in P. falciparum patients compared to other infecting species (Table P < 0.0001). Plasma and a subset of corresponding whole blood samples .A total of 102 healthy controls and 306 malaria patients were studied, comprising the following PCR-confirmed clinical cases: 66 Plasmodium antigen concentrations were consistently higher in whole blood and generally showed less variability in concentration compared to plasma. For both sample types, HRP2 concentrations were significantly higher in falciparum malaria compared to controls or other malaria patients . The measured level of Pf-LDH was higher in samples from subjects infected with all Plasmodium species compared to controls, but was highest in patients with P. falciparum infection . For Pv-LDH, whole blood and plasma concentrations were markedly higher in vivax and knowlesi malaria (P < 0.0001), with whole blood concentrations also elevated in infections by other species compared to controls . Concentrations of pan-LDH and CRP were significantly elevated in malaria patients compared to controls for both sample types .The Q-Plex™ Human Malaria assay measured whole blood and plasma concentrations of HRP2, pan-LDH, Pf-LDH, Pv-LDH and CRP. Overall, Plasmodium species . The diagnostic sensitivity of the HRP2 assay was less for plasma samples (92.4%), but specificity remained high (98%). The overall specificity of the HRP2 assay for P. falciparum compared against other Plasmodium species was 93% in whole blood and 94.6% in plasma.The assay’s diagnostic performance was evaluated against clinical samples with PCR-confirmed mono-infection of five different es Table . ResultsP. falciparum infection in whole blood (100%) but again was less sensitive in plasma . In fact, the Pf-LDH assay was more sensitive for detection of P. malariae than P. falciparum in plasma samples (96.2%) and detected 100% of P. malariae cases in whole blood. The specificity of Pf-LDH for detection of P. falciparum was similarly low compared to P. vivax (10.6%) and P. knowlesi (47.0%) infections in whole blood, improving to 61.0% and 69.8% in plasma, respectively. The sensitivity of Pf-LDH in detecting P. vivax and P. knowlesi infections was 89.4% and 53.0% in whole blood, and 39.0% and 30.2% in plasma, respectively. In the two cases of P. ovale where whole blood was available, there were strong positive signals for Pf-LDH  and pan-LDH , while measures of other Plasmodium antigens were below threshold. Therefore, while Pf-LDH is highly sensitive in detecting P. falciparum infections in whole blood, our results indicate that Pf-LDH alone displays limited specificity and requires an alternate analyte such as HRP2 for specific diagnosis of P. falciparum infection in co-endemic regions. Given that this may be problematic in cases where hrp2/3 gene deletions are prevalent, we calculated the pan-LDH-to-Pf-LDH ratio in whole blood and found that 97% of P. falciparum cases had ratios < 1.5 (median 0.75 [IQR:0.69–0.91]) while 92% of P. malariae and P. ovale cases had ratios > 1.5 . Thus, the pan-LDH-to-Pf-LDH ratio may offer a strategy to diagnose P. falciparum infections in the absence of a positive HRP2 antigen result.Like HRP2, Pf-LDH showed excellent sensitivity for P. vivax infections . The specificity of the Pv-LDH assay for P. vivax and P. knowlesi infection in whole blood was high , but specificity was lower in infection with other species in plasma samples (80–88.5%). The pan-LDH-to-Pv-LDH ratio did not distinguish P. vivax from P. knowlesi infection. However, the pan-LDH-to-Pf-LDH ratio in whole blood was above a threshold of 3,000 in 88% of P. vivax cases , while 83% of P. knowlesi cases had ratios < 3,000 . Likewise, the diagnostic sensitivity of Pv-LDH at detecting P. vivax cases was significantly higher in whole blood (93.9%) than in plasma , while its sensitivity for P. knowlesi infections was similar in the two sample matrices . There was no significant difference in Pf-LDH sensitivity for P. malariae infections between whole blood (100%) and plasma . Collectively, the assay showed superior diagnostic performance using the recommended sample type of whole blood, and adequate performance in plasma. In contrast, the host-response protein, CRP, was elevated in significantly more malaria cases when using plasma (89.6%) compared to whole blood (80%).The performance of the assay in plasma samples was compared to matched whole blood samples Table . In wholPlasmodium species, resulted in lower sensitivities for all species except for P. vivax and P. malariae detection using whole blood Pv-LDH and Pf-LDH, respectively  did knowlesi malaria samples test negative using the Pv-LDH antibody in whole blood, which were irrelevant when applying the lower recommended WHO cut-off of 200 parasites/µL29. Pf-LDH antibodies detected all P. malariae infections, including parasitemias as low as 71 parasites/µL.We evaluated the range of peripheral blood parasite concentrations in samples where parasite antigen levels were below the detection threshold in whole blood and plasma . The inclusion of Pv-LDH in the assay panel allowed the detection of P. vivax cases with 94% sensitivity. However, the Pv-LDH target could result in misdiagnosis of P. knowlesi infections in co-endemic areas due to very high cross-reactivity with this species, as demonstrated by the excellent reported sensitivity for P. knowlesi. This is not surprising as there are only five amino acid polymorphisms between P. vivax and P. knowlesi in the 316 residues of the LDH protein. Examination of whole blood demonstrated superior diagnostic performance compared to plasma for the majority of antigens. Overall, our study further supports the Quansys 5-plex assay as a promising platform for malaria diagnostic research and development but, in its current form and price per specimen , may be limited to high-throughput usage in well-funded reference laboratories in selected malaria-endemic settings.Our study evaluated the diagnostic performance of the Q-Plex™ Human Malaria assay against a set of 306 PCR-confirmed cases of malaria mono-infection with five different 8. Here, we opted to set a different threshold (mean plus 3 × SD of controls) and found this did not significantly alter the diagnostic performance of the assay. In a clinical or surveillance setting, the expected profile for malaria diagnostics includes a minimum sensitivity and specificity of 95%, and for optimum performance a sensitivity of 98% and specificity of 99%30. The Quansys assay met these criteria for the detection of P. falciparum infections due to the excellent performance of the HRP2-detecting component. HRP2 positivity in a small proportion of non-falciparum species was possibly a reflection of recent P. falciparum infection and the longer persistence of HRP2 after treatment and parasite clearance31. Importantly, we found that the assay requires further development for precise identification of non-falciparum species. The high level conservation of the LDH protein among the different Plasmodium species, with resultant cross-reacting antibody specificity, means that the Pf-LDH cannot distinguish P. falciparum from P. malariae7 and P. ovale, and is consistent with previous findings10. Despite this, it was possible to differentiate P. malariae and P. ovale from P. falciparum by using the lack of HRP2 positivity. In regions with high rates of hrp2/3 gene deletions where P. falciparum and P. malariae/ovale may co-exist33, using the whole blood pan-LDH-to-Pf-LDH ratio would allow distinction of P. falciparum infections (ratio < 1.5) from the two cross-reacting species (ratio > 1.5).A different approach to the determination of threshold cut-offs for positivity  was used in previous evaluationsP. knowlesi cross-reactivity with Pf-LDH19, the platform’s diagnostic performance for P. knowlesi revealed strong cross reactivity with the P. vivax marker (Pv-LDH) as previously reported with both cultured A1H1 strain samples10 and using other diagnostic methods35. The superior sensitivity of RDTs targeting Pv-LDH in detecting P. knowlesi over P. vivax has previously been reported35 and was seen in our plasma samples. We were unable to explain the 4 false negative P. vivax cases with relatively high parasitemia. Genetic diversity of the protein 36 or sample damage in these cases are unlikely, and testing of these cases at higher whole blood dilutions did not improve detection, excluding any inhibitory effects as a cause. The pan-LDH-to-Pf-LDH ratio may be a strategy to differentiate P. vivax from P. knowlesi infection but with some uncertainty. While the sensitivity and specificity of Pv-LDH to detect these two species in whole blood (~ 94%) fell short of the expected diagnostic profiles (≥ 95%), it may be useful in areas endemic for P. knowlesi without or with very low P. vivax prevalence such as Malaysia, or in areas endemic for P. vivax with very low P. knowlesi prevalence. The development of diagnostic tools to robustly distinguish P. vivax from P. knowlesi infection is urgently needed to support clinical management and public health responses. In co-endemic areas, misdiagnosing knowlesi malaria as vivax infection may result in suboptimal oral treatment of potentially life-threatening knowlesi infection which may require immediate parenteral artesunate therapy38. Conversely, it may result in unnecessary primaquine therapy in knowlesi malaria and complicate public health responses for elimination of human-only Plasmodium infections, while misdiagnosis of vivax malaria as P. knowlesi infection would result in failure to administer primaquine to prevent relapse.In addition to demonstrating known P. falciparum from P. malariae/ovale in the absence of HRP2. Inclusion of CRP has been argued to be of prognostic value40. We were unable to assess the utility of CRP in predicting severe malaria in our study due to low numbers of severe malaria cases with whole blood samples available (n = 5). Further, without additional testing to exclude potential concomitant bacterial infections that may contribute to raised CRP41, it was not possible to the infer the utility of this biomarker for assessment of malaria-only severity. Moreover, as assay dilutions beyond 100-fold did not allow identification of exact concentrations in samples with very high CRP levels, we were unable to quantitatively assess for this relationship. A limitation of this assay platform, where a multiplex approach is used, is that the optimal dilution for each analyte may not be achieved, and rerunning samples at multiple dilutions may not be possible, for example where sample volumes are limited. Other diagnostic limitations of the assay include duration of the assay (~ 4 h – similar length as PCR), high cost, and the need for an imager and power, overall restricting the current Quansys platform for use in larger facilities.Pan-LDH was a useful additional marker in the assay. It has utility for calculation of the pan-LDH-to-Pf-LDH ratio to discriminate Plasmodium antigens in whole blood compared to plasma samples  was expected given greater antigen quantity released from lysed red cells than that present in plasma. The greater variation in Plasmodium antigen concentrations seen in plasma versus whole blood, together with recent observations comparing native versus recombinant species-specific pLDH using this platform10, also point towards differences in antibody-binding to target pLDH epitopes. Conformational changes of the target protein in plasma and/or a higher proportion of immune-complexed protein in plasma from humoral immunity42, may also contribute to greater sensitivity in whole blood. As a diagnostic tool, whole blood is a preferred analyte, given the extra work and instrumentation needed to separate plasma. Scenarios where the latter may be necessary include serological surveillance surveys, retrospective studies where whole blood is not available, or analytical studies evaluating protein secretion and biomarkers of parasite biomass44.Greater diagnostic sensitivity of Plasmodium species8. In its current configuration as an ELISA assay with imager, suitable applications include a reference assay for RDT quality assurance, research and development and potentially at reference laboratories where simultaneous testing of large numbers of samples is required. Further optimisation of the assay platform will be required to improve specificity for diagnosis of non-falciparum species if it is to be considered a platform for evaluating biomarkers for expansion into next-generation RDTs. Moreover, the study reiterates the limitations of current reagents developed to detect pLDH and perhaps the suitability of LDH as a target analyte to selectively discriminate non-falciparum malaria species.In summary, our findings support the Q-Plex™ Human Malaria assay as a sensitive tool for the diagnosis of symptomatic malaria from all five major Supplementary Information.


Malaria is an infectious disease considered as one of the biggest causes of mortality in endemic areas. This life-threatening disease needs to be quickly diagnosed and treated. The standard diagnostic tools recommended by the World Health Organization are thick blood smears microscopy and immuno-chromatographic rapid diagnostic tests. However, these methods lack sensitivity especially in cases of low parasitaemia and non-falciparum infections. Therefore, the need for more accurate and reliable diagnostic tools, such as real-time polymerase chain reaction based methods which have proven greater sensitivity particularly in the screening of malaria, is prominent. This study was conducted at the French National Malaria Reference Centre to assess sensitivity and specificity of two commercial malaria qPCR kits and two in-house developed qPCRs compared to LAMP.® Malaria test, the BioEvolution Plasmodium Typage test, the in-house HRM and the in-house TaqMan qPCRs. The specificity and sensitivity of each method and their confidence intervals were determined with the LAMP-based assay Alethia® Malaria as the reference for malaria diagnosis. The accuracy of species diagnosis of the Ampliquick® Malaria test and the two in-house qPCRs was also evaluated using the BioEvolution Plasmodium Typage test as the reference method for species identification.183 blood samples received for expertise at the FNMRC were included in this study and were subjected to four different qPCR methods: the Biosynex Ampliquick® Malaria test (sensitivity at 97.2%) and the BioEvolution Plasmodium Typage (sensitivity at 95.4%). Additionally, with the in-house qPCRs we were able to confirm a Plasmodium falciparum infection in microscopically negative samples that were not detected by commercial qPCR kits. This demonstrates that the var genes of P. falciparum used in these in-house qPCRs are more reliable targets than the 18S sRNA commonly used in most of the developed qPCR methods for malaria diagnosis.The main results showed that when compared to LAMP, a test with excellent diagnostic performances, the two in-house developed qPCRs were the most sensitive (sensitivity at 100% for the in-house TaqMan qPCR and 98.1% for the in-house HRM qPCR), followed by the two commercial kits: the Biosynex AmpliquickOverall, these results accentuate the role molecular methods could play in the screening of malaria. This may represent a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis, which could be essential for the detection and treatment of malaria carriers and even for the eradication of this disease.The online version contains supplementary material available at 10.1186/s12936-022-04219-1. Plasmodium. According to the World Malaria Report of 2021, 241 million malaria cases were estimated in 2020 in 85 malaria endemic countries ). Kappa’s coefficient shows that the qPCRs tested are highly concordant with the LAMP method, in particular the in-house qPCRs , followed by the two commercial kits: the Ampliquick–99.8 resPlasmodium Typage is a routinely used qPCR kit for malaria diagnosis at the FNMRC. It allows the simultaneous identification of P. falciparum, P. ovale, P. vivax, P. malariae and P. knowlesi and was therefore considered as the reference method for the Plasmodium species identification when the sample was positive by this method.On each included sample, microscopy diagnosis and species identification by qPCR were systematically done  after the in-house TaqMan-qPCR analysis, which correlated with the initial LAMP results .Tables Plasmodium Ct of the Ampliquick® Malaria (median Ct = 23.40), the species Ct of the Bio-Evolution Plasmodium typage (median Ct = 27) and the undifferentiated Ct of the in-house HRM-qPCR (median Ct = 22.88) and the in-house TaqMan-qPCR (median Ct = 23.58) were calculated. The data show that the in-house qPCRs and the commercial Ampliquick® Malaria have lower Ct values than the Bio-Evolution Plasmodium Typage  , concordant with a better sensitivity. For the in-house TaqMan-qPCR, Ct of the var genes and the 18S rRNA targets were not different .The median Pan-As shown in Fig. To control malaria, accurate and rapid diagnostic tools are very essential not only for the establishment of an effective treatment but also for surveillance and epidemiological monitoring. The quality and sensitivity of the diagnosis are very important criteria to avoid misdiagnosis which could lead to severe health problems, recrudescence, drug resistance and possibly death .® Malaria and two in-house developed qPCRs were assessed by comparing them with the highly sensitive LAMP method Alethia® Malaria for sensibility evaluation and with the Plasmodium typage (Bio-Evolution) qPCR method for species diagnosis evaluation. Unlike other LAMP methods that can differentiate Plasmodium spp., P. falciparum or P. vivax infections [® Malaria does not allow species diagnosis and requires, for that reason, an equally sensitive species PCR. It is very necessary to differentiate the Plasmodium species to identify P. ovale or P. vivax infections because they require specific treatment with primaquine, but also P. knowlesi infections which can lead to severe illness and are commonly misidentified with conventional methods.The implication of the molecular diagnosis of malaria has been highlighted in many studies. As reported in the literature, PCR and qPCR methods displayed high sensitivity compared to other conventional methods such as microscopy or RDTs , 13, 24.fections , AlethiaPlasmodium Typage was the least sensitive at 95.2% and the in-house TaqMan-qPCR was the most sensitive at 100%. When it comes to commercial kits, it is very clear that Ampliquick® Malaria is better at detecting Plasmodium infections than the Bio-Evolution Plasmodium Typage. While analysing the data, the Bio-Evolution kit results were compared to microscopy results and when the initial microscopy diagnosis is positive for a Plasmodium infection, the subsequent species identification by this method is accurate. Mixed infections were even distinguished in some cases. However, when compared to negative microscopy results it is clear that this qPCR method is not as reliable as the others in detecting false negatives. Indeed, out of the 61 negatives with microscopy, 48 were negative with Plasmodium Typage (Bio-Evolution) while 43 were considered truly negative after the LAMP and the in-house TaqMan qPCR analysis. Of the 48 negatives one was identified as a Plasmodium spp. infection and four were identified as P. falciparum mono-infections. These findings show that the in-house TaqMan qPCR was able to detect P. falciparum infections that were not detected by the commercial kit.The main results of this study showed that when compared to LAMP, the gold standard for positivity throughout this study, the Bio-Evolution var genes for P. falciparum detection, a multigenic family with approximately sixty copies in each P. falciparum genome [Plasmodium genome. This was also highlighted when three samples were only positive for the var genes target after the in-house TaqMan qPCR. These samples were either microscopically negative or had very low parasitaemia implying that the var genes of P. falciparum are more sensitive targets than the 18S rRNA (Pan), despite no statistical difference in Ct values. Moreover, the same DNA extracts were evaluated with the four qPCR methods implying that the different sensitivities are proper to each method and not due to the quality of DNA extraction.The in-house developed qPCRs target the m genome . It may Plasmodium Typage (Bio-Evolution), the major limitation of the other techniques is the absence of species identification. The other downside is, with it being a commercial kit, the Ampliquick® Malaria test is more expensive and thus cannot be systematically used in developing countries for the diagnosis of malaria. However, the TaqMan and HRM in-house qPCRs adapted from Schindler et al. [Compared to r et al.  showed bP. falciparum infections, which is the most common and life-threatening species.This paper represents a comparative study in which four qPCR methods for malaria diagnosis were evaluated to provide information about their sensitivities and accuracy. This could be a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis. Compared to the LAMP assay, the four tested qPCR methods showed varying sensitivities with the in-house TaqMan qPCR being the most sensitive. Although it does not enable the detection of the five common malaria human infecting species, the in-house TaqMan has a comparable sensitivity to that of the LAMP assay with the advantage of identifying Taken together, the results demonstrate the role molecular methods could play in the screening of malaria and infectious diseases in a rapid and effective way by providing critical information for the clinical context and the epidemiological survey.Additional file 1. The different methods used to evaluate the included samples. A flow-chart showing the number of samples included and the different methods we used to analyze these samples.Additional file 2. Results of the microscopic diagnosis and molecular assays for each included sample. A table detailing the results of: microscopic diagnosis (species identification and parasite densities), LAMP Alethia® Malaria and the four different qPCR methods tested in this study for the 183 included samples.


Plasmodium knowlesi is a zoonotic malaria parasite that has gained increasing medical interest over the past two decades. This zoonotic parasitic infection is prevalent in Southeast Asia and causes many cases with fulminant pathology. Despite several biogeographical restrictions that limit its distribution, knowlesi malaria cases have been reported in different parts of the world due to travelling and tourism activities. Here, breakthroughs and key information generated from recent  studies conducted on P. knowlesi were reviewed, and the knowledge gap in various research aspects that need to be filled was discussed. Besides, challenges and strategies required to control and eradicate human malaria with this emerging and potentially fatal zoonosis were described. Plasmodium. Over the past few decades, healthcare professionals and general public were educated that malaria was caused by four species of human malaria parasites, namely Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale [Plasmodium knowlesi, a simian malaria parasite, has been recognized as the fifth medically important Plasmodium [P. knowlesi are described and reviewed here.Malaria is one of the oldest infectious diseases. Despite the significant reduction of global malaria cases decade by decade, malaria remains a significant healthcare and economic burden to many countries, especially the developing nations around the tropical and subtropical regions , 2. Thisum ovale . After easmodium –6. In thasmodium , the stoP. knowlesi discovery was relatively short . The macaques caught from the Kapit division of Sarawak, Malaysia demonstrated the highest P. knowlesi infection rate, with an infection prevalence of 86.6% in the M. fascicularis population and 50% in the pig-tailed macaques (M. nemestrina) population [P. knowlesi-positive’ monkeys [P. knowlesi-positive’ long-tailed macaque from the 44 monkeys examined [P. knowlesi was detected in M. fascicularis from Puerta Princesa Subterranean River National Park, Palawan; but not in macaques from another wildlife centre  in the same province [The infections of lsewhere , 44. Herlsewhere , 46. Plaprimates . Ever siprimates , only stpulation . Indones monkeys , 50, whiexamined . In the province .Table 2SP. knowlesi  [. suggested the Northern pig-tailed macaque (M. leonina) in Shan state of Myanmar as a potential host for P. knowlesi as it is closely related to M. nemestrina [P. knowlesi has yet to be found in M. leonina (Table P. knowlesi was found in a stump-tailed macaque (M. arctoides) in Prachuap Kiri Khan province, Thailand, based on nested PCR method without further backing of evidence by the gold standard microscopy or other molecular tools like sequencing [P. knowlesi was detected in a dusky leaf monkey (Semnopithecus obscurus), as confirmed by sequencing and phylogenetic analysis [As the natural transmission of knowlesi malaria from human to human via mosquito bite remains to be validated, the zoonotic transmission path is still regarded as the main route of knowlesi malaria acquisition in humans. In fact, recent studies have linked close macaque contact by humans with an increased risk of acquiring nfection , 63. Howl hosts’ , 34, 64.alophos) . Moyes ena Table . Meanwhiquencing . In anotanalysis .M. fascicularis in the Port Blair Zoo on Smith Island [P. knowlesi transmission via the human-vector-human route, as demonstrated in experimental infections [P. knowlesi i.e. several members of Anopheles leucosphyrus group are known to be primarily forest-dwelling [P. knowlesi transmission is far from complete, especially in certain locations.However, an issue remains with the Smith Island and Car Nicobar, as there are no known monkey populations that are native to these areas . Other th Island , there afections . Insteaddwelling . There mAnopheles has yet to be validated or disputed, it should be noted that there is abundant evidence demonstrating that P. knowlesi is primarily a zoonosis. Many phylogenetic and haplotype network analyses did not show unique clusters associated with human cases, strongly suggesting that most, if not all of the human cases are originated from macaques [P. knowlesi in the macaque population is much higher than in the human population, implying that the human P. knowlesi infections are mainly the spill-over infections from the macaque population via biting activities by anopheline mosquitoes that bite both humans and monkeys [P. knowlesi parasites derived from M. fascicularis and M. nemestrina in Borneo had distinct microsatellite genotypes, and human cases were associated with either the M. fascicularis- or M. nemestrina-derived parasite subpopulations, indicating that the majority of the clinical knowlesi malaria cases in Borneo were of zoonotic nature [P. knowlesi cases within household members of a known P. knowlesi case, suggesting the presence of peri-domestic transmission. Obviously, the human-to-human transmission of knowlesi malaria could not be ruled out. The feasibility of simian-independent knowlesi transmission among humans will influence the strategies required to control and eradicate malaria in the affected areas. Hence, the feasibility of natural knowlesi transmission from human to human has to be investigated further.Although the human-to-human transmission of knowlesi malaria via macaques , 52. Fur monkeys , 68. Of c nature  and Fornc nature  found a P. knowlesi infection plays a crucial role in disease intervention and prevention. Epidemiology studies in recent years have led to the identification of various factors that influence the disease occurrence, which can be classified as individual and environmental factors . As a result, they are exposed to infective mosquito bites. At peri-domestic and household-level, long grass around the house, and open roof eaves or gaps in house walls are welcoming signs of mosquito invasion, hence the risk factors of knowlesi malaria exposure. Interestingly, having rice paddy fields around the house, as well as residing at areas with higher altitudes are associated with lower infection risk [Ecological variations, both natural and human-induced, are the direct drivers of fections . Loss offections . Usuallyion risk . These aion risk –85. CollP. knowlesi to the list of medically important malaria parasites, the standard operating protocols of malaria diagnosis in knowlesi malaria-endemic areas have to be adjusted to enable accurate detection of all etiological agents of malaria. To date, microscopic examination remains as the gold standard for the diagnosis of malaria. Although limitations have been described, i.e. time consuming and low sensitivity [P. knowlesi parasites with other human malaria species [P. knowlesi resemble the ring forms of P. falciparum. At the late trophozoite stage, P. knowlesi may appear as band forms, which resemble those of P. malariae. Hence, misidentification of knowlesi malaria as infections by P. falciparum or P. malariae is common, especially in areas where the microscopists are not familiar with the parasites [P. knowlesi as P. vivax were reported previously [P. knowlesi patients were given treatment with primaquine, which is a radical cure to clear hypnozoites (dormant liver stage) of P. vivax. This resulted in unnecessary increase of treatment cost and potential health risks to individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency if G6PD testing was not performed prior to the treatment, which could lead to severe acute haemolytic anaemia [Plasmodium, by taking into considerations the parasite’s morphology and patient’s travel history prior to the infection.With the addition of sitivity , this disitivity , 88. The species . Early earasites , 90, 91.arasites . Misideneviously . P. know anaemia . TherefoP. falciparum histidine-rich protein 2 (PfHRP2), plasmodial lactate dehydrogenase (pLDH) and plasmodial aldolase. Antibodies targeting these antigens are used for specific detection of P. falciparum or P. vivax. They are also used in combination with pan-malarial antibodies that target all Plasmodium species [P. knowlesi infection has been evaluated. Foster et al. found that OptiMAL-IT was the most sensitive RDT in detecting P. knowlesi antigen, with 71% sensitivity for fresh samples [P. falciparum test line, suggesting that the P. falciparum lactate dehydyrogenase (LDH) monoclonal antibody used in this kit cross-reacts with the P. knowlesi LDH antigen, which is in agreement with findings from other studies [P. knowlesi remained low [P. knowlesi detection, without compromising its performance of detecting malaria caused by other species of Plasmodium. Of note, Krause and Goldring reported the potential of phosphoethanolamine-N-methyltransferase (PMT) as a biomarker candidate for RDT design due to its presence in erythrocytic stages of the Plasmodium parasites [Plasmodium species orthologues. Hence, the production of a RDT with PMT monoclonal antibodies targeting species-specific epitopes is deemed possible.Malaria rapid diagnostic test (RDT) is a convenient alternative for malaria diagnosis due to its ease of use, low cost and rapid yield of results. The tests are immunochromatographic lateral flow devices that provide qualitative results, which is especially valuable in resource-limited settings and mass screenings. Current malaria RDTs target three proteins, namely  species . The per samples . However studies –99. A syined low . Thus, tarasites . The proPlasmodium antibodies. Most of the tests used P. falciparum and P. vivax recombinant antigens as antigenic targets. A commercial ELISA (EUROIMMUN EIA) which used recombinant antigens from all five medically important Plasmodium species was shown to exhibit high concordance rate to routine screening test (> 94%), with sensitivity and specificity of 85% and 95.2%, respectively [Plasmodium parasites, Müller-Sienerth et al. expressed and evaluated a panel of recombinant proteins to be used as target antigens in ELISA [P. knowlesi merozoite surface protein 10 (PkMSP10), 6-cysteine protein 12 (PkP12), and 6-cysteine protein 38 (PkP38) could be used as antigen panels in serological assays as they accurately determined the patient’s history of exposure to P. knowlesi.Despite of its limited use in diagnosing acute infections, serological assay plays an important role in malaria disease surveillance, screening in blood donation centres, and identification of parasite exposure history . There aectively . Using tin ELISA . The stuP. knowlesi at low parasitaemia [Plasmodium up to species level, with the ability of detecting much lower load of parasites in the samples. The polymerase chain reaction (PCR)-based diagnosis has gained popularity among the researchers. Such methods encompass nested PCR, real-time PCR and multiplex PCR. The PCR-based diagnoses yield much higher sensitivity than microscopy in the detection of Plasmodium spp. [P. knowlesi and P. vivax detection, which yielded superior sensitivity of detection, as compared with the established nested PCR method [P. knowlesi plasmepsin gene as low as 0.01 copies/µL further highlighted its potential in detection of low-density malaria cases [P. falciparum infections than qPCR, and both methods diagnosed an equal number of P. vivax infections [Microscopy and RDT have a detection limit of 100 and 5–50 parasites/μL, respectively . This lesitaemia . Besidesium spp. . Hofmannium spp. . Moreoveium spp. . Since tR method . When coia cases . The finfections . In addiP. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi), with a detection limit of one copy/µL for P. vivax, P. falciparum, and P. malariae; and ten copies/µL for P. knowlesi and P. ovale [P. knowlesi by targeting the 18s rRNA gene, where a positive reaction is indicated by green colour whereas the negative reaction is indicated by orange colour [P. falciparum and P. vivax, which showed a tenfold higher detection limit than nested PCR [Plasmodium species [The PCR-based methods are not without limitations. The major drawbacks of PCR-based assays include the requirement of expensive machines, costly reagents, longer turnaround time and a well-equipped laboratory, which restrict its application for field diagnoses. Furthermore, it is not suitable to be used in countries with low resource settings and unstable power supply. As an alternative, isothermal methods have been pushed forward. Since year 2000, loop-mediated isothermal amplification (LAMP) has been widely used to assist malaria diagnosis in some areas, due to its highly sensitive and rapid performance, in addition to its requirement of cheaper equipment and resources . With anP. ovale . To faciP. ovale –115, fluP. ovale , 117 andP. ovale  can be ie colour . This nested PCR . In 2018 species . Another species , 123. Th species –126.Plasmodium species [In recent years, new generation of molecular diagnostic tools have been developed, one of these is the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay. This is a novel diagnostic approach, where CRISPR technology and RPA assay are combined. SHERLOCK is an ultrasensitive CRISPR-based method that allows the detection of infections from asymptomatic carriers , 128. Th species . This te species .Plasmodium spp. [P. knowlesi was first reported in 1965 [P. knowlesi genetic studies are summarized in Table P. knowlesi can be unravelled more systematically, providing an important foundation to understand the pathobiology of knowlesi malaria.Information on the genetic diversity of a parasite species is crucial for the development of vaccines against malaria. High antigenic variation of proteins expressed by the parasites on the surface of infected cells may compromise the feasibility of generating protective immunity via vaccine and applying neutralizing antibody-mediated therapy on malaria patients , 131. Inium spp. , 133. Th in 1965 . Subsequ in 1965 , 136. ThP. knowlesi protein-coding genes, particularly those related to erythrocyte invasion, are experiencing purifying (negative) selection [P. knowlesi, which could have been mitigated by the parasite population growth and adaptation to the mosquito vectors [P. knowlesi, only a few genes have been found to be under positive selection [P. knowlesi thrombospondin-related adhesive protein (TRAP) gene is one of such genes, which is similar to that of P. falciparum and P. vivax [To date, most of the election , 166–168election . Purifyi vectors , 157. Ho vectors , 164. InP. vivax .P. knowlesi genetic structure, we can understand the lineages of the parasites better. Phylogenetic studies allow us to understand the evolutionary progress of a species and its evolutionary relationship with other related species. For example, P. knowlesi shares a more recent common ancestor with Plasmodium coatneyi, another simian malaria parasite [P. knowlesi were unravelled in Malaysia [P. knowlesi subpopulations, indicative of chromosomal-segment exchanges events between two distinct Malaysian Borneo subpopulations associated with either M. fascicularis or M. nemestrina [M. nemestrina-associated Malaysian Borneo subpopulation [Through intensive studies of parasite . Dimorphparasite , 156. FoMalaysia , 159. MiMalaysia . Studiesmestrina , 164. Onpulation . GeneticP. falciparum, as compared to other human malaria parasites that have yet to be adapted successfully into the in vitro culture condition. Likewise, the rapid advancement in P. knowlesi research relies on the establishment of a reliable in vitro cultivation system for this species. Interestingly, the method used for P. knowlesi cultivation is largely based on the cultivation method established for P. falciparum in the 1970s [P. knowlesi culture [P. knowlesi can adapt to the in vitro conditions directly using human serum and Albumax II [2, 5% CO2). Large amounts of parasites can be obtained consistently, which is important for high throughput studies. Rhesus or long-tailed macaque red blood cells (RBC) are used, where most studies require an adaptation period of about 3 weeks [P. knowlesi infects human RBC naturally, culturing the parasites in human RBC requires an adaptation process that takes around 5–8 months [P. knowlesi to human RBC invasion. The first method uses a mixture of human and macaque RBC (1:4 or 1:9 ratio of macaque RBC to human RBC) [P. knowlesi typically invades younger RBCs. Hence, P. knowlesi can be cultured with human reticulocyte-rich (~ 12%) packed RBC. The parasites are able to adapt to older RBC after a period of cultivation. This trick was utilized by a study group to adapt P. knowlesi parasites to human RBC without the need of macaque RBC. They initiated the culture using human RBC enriched with 16% reticulocytes, followed by gradual reduction of the reticulocyte fraction, over the course of five months [The biological studies of a pathogen benefit tremendously from the establishment of a continuous cultivation system for the pathogen. This is clearly demonstrated in malaria research, where a greater depth of knowledge has been obtained from the large volume of in vitro studies conducted on the culturable he 1970s . The parhe 1970s –174. Ear culture , 175, whbumax II , and evebumax II , without 3 weeks , 176. Al8 months . Two metman RBC) , 177. ThP. falciparum and P. knowlesi. The factors that enable P. knowlesi to adapt to human RBC in vitro using this cultivation protocol remain to be investigated and deciphered. Of note, the normocyte binding protein Xa (NBPXa) gene has been shown to be essential for human RBC invasion, but not for macaque RBC invasion [P. knowlesi line created in their study contained a Duffy binding protein α (DBPα) gene duplication and a Duffy binding protein γ (DBPγ) gene deletion [DBPα gene. Meanwhile, a separate human-adapted P. knowlesi line demonstrated a V943L substitution in DBPα, which could be responsible for the adaptation to human RBC [P. knowlesi to human RBC. Nevertheless, the role of the accompanying deletion of the DBPγ gene in such invasion adaptation is not known and deserves to be investigated as well, to completely decipher the invasion plasticity of P. knowlesi with RBC derived from different hosts. The successful adaptation of P. knowlesi to long term in vitro culture using human RBC has been a key turning point for knowlesi malaria research as it eliminates the need of macaque monkeys and macaque blood [The RPMI 1640-based in vitro cultivation method supports the growth of invasion . In addideletion . Hence, uman RBC . Thus, iue blood .Plasmodium lactate dehydrogenase enzyme [P. falciparum are now applicable to P. knowlesi, with some adaptations and usage of newer, high throughput methods [P. knowlesi to various anti-malarials and novel therapeutic agent candidates have been evaluated using high throughput drug screening, thanks to the established in vitro culture system. Through these assessments, P. knowlesi has been revealed to demonstrate distinct drug susceptibility profiles, as compared to P. falciparum. For example, P. knowlesi has significantly lower susceptibility to a few sodium channel ATP4 inhibitors  [Plasmodium species  demonstrated drug susceptibility profiles that were closer to that of P. knowlesi than P. falciparum [P. knowlesi strains can be recruited for future evaluation of anti-malarials. Besides, attempts to in vitro induce drug resistance in P. knowlesi cultures can be done to investigate the development of drug resistance in this parasite, and predict gene candidates that drive the parasite towards drug resistance.With the established culture system, the efficacy of anti-malarials, new therapeutic compounds or inhibitory antibodies have been assessed using growth inhibition assays (GIA). These assays can be performed with microscopic examination , radiolae enzyme , and floe enzyme . Basical methods . The in  target) –186. Intlciparum . With thP. knowlesi Duffy binding protein α (PkDBPα) and apical membrane antigen 1 (PkAMA1) significantly inhibited parasite growth in a concentration-dependent manner [With the in vitro culture system, the effects of antibodies raised against a potential target can be evaluated, as part of the vaccine candidate screening. Indeed, the antibodies raised against both t manner , supportP. knowlesi line maintained with macaque RBC was demonstrated to retain its ability to form gametocytes that gave rise to successful mosquito infection, as confirmed with the recovery of oocysts and sporozoites, albeit with inconsistency [P. knowlesi are deemed feasible.Most of the adapted in vitro parasite cultures do not produce sexual-stage (gametocyte), possibly due to the loss of gametocytogenesis ability in the prolonged in vitro passage under well-regulated, optimal culture conditions , 189. Hosistency . NeverthPlasmodium knowlesi is unique in that both in vitro and in vivo systems are available for research. Interestingly, the P. knowlesi maintained in in vitro cultures were shown to be capable of readapting to the in vivo conditions via a single blood passage into a macaque [P. knowlesi clones to be selected in vitro, subsequently used to infect a macaque (in vivo) to study the host-parasite interactions such as strain-specific virulence and factors that drive host tolerance upon infection. Notably, the in vitro-adapted P. knowlesi demonstrated clear difference in gene expression profile from the ex vivo parasites , where the SICAvar genes were found to be downregulated in the in vitro cultures [SICAvar is crucial for antigenic switching of P. knowlesi, possibly for the purpose of evading the host’s immune responses. The differences between in vitro and in vivo parasites have been demonstrated in P. falciparum, with differences being reported in gene expression and regulation, drug susceptibility, and cytoadherence characteristics [P. knowlesi research platform that is not available with P. falciparum and other human malaria parasites. macaque . This alcultures . SICAvareristics , 192. ThP. knowlesi strain can be genetically modified with a conventional single or double crossover homologous recombination [P. knowlesi demonstrated 1000-fold higher transfection efficiency than P. falciparum. Due to its shorter erythrocytic cycle, adequate quantity of transgenic parasite lines can be obtained within a week. The ability of the parasites to be cloned by limiting dilution allows transfection studies to be performed without the need of laboratory macaques, making functional studies on P. knowlesi cheaper, more ethical and more accessible to laboratories without monkey facilities. With that, targeted genes can be evaluated to decipher their roles in parasite multiplication/growth, and the specific interactions of ligands with the host receptors can also be investigated. For instance, disruption of pkNBPXa has led to impaired merozoite invasion into human RBC but not the macaque RBC, indicating NBPXa as a key mediator for human RBC invasion by P. knowlesi [The human RBC-adapted bination . Remarkaknowlesi .P. knowlesi has been established [P. knowlesi cysteine rich protective antigen (PkCyRPA) and RH5-interacting protein (PkRIPR) were demonstrated to be essential for the parasite’s survival and RBC invasion via the CRISPR-Cas9 system in combination with dimerisable Cre-recombinase (DiCre) system [P. knowlesi, but also other closely related species that lack a continuous in vitro culture system, such as P. vivax. Various studies on P. vivax have been performed using the orthologue replacement (OR) approach by creating chimeric P. knowlesi lines carrying the P. vivax ortholog genes to evaluate the drug resistance genes and vaccine candidates. For example, several P. vivax drug resistance markers including multidrug resistance protein 1 (Pvmdr1), dihydrofolate reductase (Pvdhfr), dihydropteroate synthase (Pvdhps) were expressed in P. knowlesi model system and their role in antimalarial resistance were assessed [P. vivax Duffy binding protein (PvDBP) has been evaluated by using a stable P. knowlesi PvDBPOR line, and the findings are favorable to support PvDBP as a leading P. vivax blood stage vaccine candidate [P. vivax 6-cysteine protein P12 (Pv12) and P. vivax Asparagine-rich Protein (PvARP) have been identified [P. knowlesi does not share many of the morphological characteristics of P. vivax and does not form hypnozoites. The ability of forming hypnozoites is possessed by another simian malaria parasite, Plasmodium cynomolgi, whose long-term in vitro cultivation has been established recently [P. knowlesi cultivation system is undeniably huge.The CRISPR-Cas9 genome editing of ablished , which w) system . This brassessed . In addiandidate . Using tentified . Howeverrecently . DespitePlasmodium. Each species has unique features such as the parasite carriage duration within the host, ability to form hypnozoites, infectivity to different species of Anopheles, and availability of reservoir hosts in the natural surroundings. In addition, different species of vectors have different characteristics, including different breeding ground requirements. Each of these features possesses different obstacles to the malaria eradication programme [The ultimate goal of battling an infectious disease is the complete eradication of the infection from human population. Unfortunately, smallpox remains the only eradicated human infection in our history . Ironicarogramme , reflectAnopheles that possesses zoo-anthropophilic feeding behaviour that can support the development of P. knowlesi salivary gland sporozoites. Various human activities such as logging industry, harvesting of jungle resources, subsistence cropping, expansion of housing development to the fringe of forests and eco-tourism at forested areas have brought humans closer to the natural hosts (monkeys) and vectors (Anopheles), completing the circuit of knowlesi malaria transmission in humans. Hence, the risk of knowlesi malaria transmission depends on the degree of overlap between the monkey habitats and areas with human activities  can be built between the forest (monkey habitat) and ‘buffer zone’ to hamper monkey intrusion. In addition, these ‘buffer zones’ should not be open to the public. Nevertheless, they can be designed to serve multiple key purposes, such as flood control system, water reservoir, solar panel field, and aquaculture sites.Obviously, the simian reservoirs cannot be culled just to get rid of  malaria , 206–211 outdoor , 213. Be outdoor , 215. Woer depth –221. Nexer depth , 222. Heer depth , 223. Foof 475 m . Hence, P. knowlesi infection by healthcare workers in the travellers’ countries of residence if they came down with the infection after travel.Cooperation and coordination between policy makers, law enforcement officers and public members are crucial to halt the transmission of zoonotic malaria. Local socioeconomic activities should be arranged accordingly to avoid outdoor activities during the feeding period of vectors. At the same time, public education plays an important role. High awareness about this zoonotic infection among the public will give rise to high compliance to measures against knowlesi malaria transmission. For example, public members, especially those involved in tourism, forest resource collecting and logging sector should be educated to plan their activities in parallel with the knowlesi malaria control programme. Farmers that practice subsistence cropping at the fringe of forests should be encouraged to adopt the much more productive farming techniques at relocated farm lands further away from the forests. Certain agricultural practices that employ simian primates such as the coconut harvesting should be replaced with simian-free alternatives. Besides, community relocation from forested areas to non-forested places equipped with better building design that reduces mosquito invasion and better layout of healthcare and sanitary infrastructure should be implemented proactively. All socioeconomic activities should be reviewed and approved by relevant authorities prior to implementation. The itinerary of the activities should minimize, if not completely avoid the risk of being bitten by the vectors. Furthermore, warnings regarding knowlesi malaria transmission and measures to prevent knowlesi malaria transmission should be stated clearly when promoting tourism in knowlesi malaria endemic areas. Foreigners should be informed clearly about this zoonosis before and upon arrival at the destination. Tourists should clearly report their travel history if they fall sick after returning to their countries of residence from the knowlesi malaria endemic region. A health alert card about knowlesi malaria may be given to travellers arriving at knowlesi malaria endemic areas. This may reduce the chance of overlooking P. knowlesi research over the past few years has contributed to our greater understanding on the pathobiology, genomics and evolutionary biology of this parasite, at the same time improved our ability to detect this parasitic infection. The knowledge gap in several aspects about P. knowlesi that deserve more research attention in future has been elaborated. Knowlesi malaria further complicates the malaria eradication program. However, with tailor-made strategies, the transmission of knowlesi malaria in humans may be blocked without severely compromising the welfare of the simian natural hosts, wildlife biodiversity and economic development of knowlesi malaria endemic region.The emergence of knowlesi malaria has definitely changed the dynamics of how we manage and control malaria towards a complete eradication from human population. The exponential increase of