Plasmodium parasites, aerobic glycolysis by activated immune cells, and an increase in anaerobic glycolysis in hypoxic cells and tissues as a consequence of parasite sequestration and anemia. Impaired hepatic and renal lactate clearance, caused by underlying liver and kidney disease, might further aggravate hyperlactatemia. Multiple factors thus participate in the etiology of lactic acidosis in malaria, and further investigations are required to fully understand their relative contributions and the consequences of this major metabolic disturbance.Lactic acidosis and hyperlactatemia are common metabolic disturbances in patients with severe malaria. Lactic acidosis causes physiological adverse effects, which can aggravate the outcome of malaria. Despite its clear association with mortality in malaria patients, the etiology of lactic acidosis is not completely understood. In this review, the possible contributors to lactic acidosis and hyperlactatemia in patients with malaria are discussed. Both increased lactate production and impaired lactate clearance may play a role in the pathogenesis of lactic acidosis. The increased lactate production is caused by several factors, including the metabolism of intraerythrocytic  Plasmodium species, which are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, and Plasmodium malariae. It affects more than 200 million people each year, with over 400,000 deaths [P. falciparum and P. vivax with the more pathogenic P. falciparum responsible for the majority of the malaria deaths globally. Severe malaria is a heterogenous disease with several complications that are the main cause of death and that include cerebral malaria (CM), severe malarial anemia (SMA), malaria-associated acute respiratory distress syndrome (MA-ARDS), placental malaria (PM), acute kidney injury (AKI), and metabolic disturbances. CM is a severe neurological complication responsible for many malaria-related deaths globally and is characterized by sequestration of infected red blood cells (iRBCs) in the brain, local inflammation, blood–brain barrier damage, edema, and hemorrhages [Malaria is a hazardous disease caused by 1 of the 5 human orrhages . MA-ARDSorrhages . AKI patBesides these organ-specific complications, metabolic disturbances like hypoglycemia and hyperlactatemia also occur in patients with malaria. Hyperlactatemia and lactic acidosis are important prognostic factors in patients with severe malaria ,8. LactaThe etiology of lactic acidosis in severe malaria is incompletely understood, despite its severity and strong association with mortality. In this review, we discuss possible causes for hyperlactatemia and lactic acidosis in malaria as done in previous reviews, and we will further supplement this with the most recent findings and new hypotheses ,15. We tLactic acidosis is also common in severe non-malarial sepsis and septic shock, in which it is also associated with poor prognosis . Similar+. In the last step, pyruvate shuttles into the tricarboxylic acid (TCA) cycle, is used as a substrate for amino acid synthesis or is converted to lactate by lactate dehydrogenase (LDH). The conversion of pyruvate to lactate provides NAD+, which is needed to assist glycolysis. The LDH enzyme is a tetramer comprised of LDHA and/or LDHB subunits. This results in 5 isoforms of LDH in the human body, which are expressed in different organs. The LDHA subunit has a higher affinity for pyruvate and preferentially converts pyruvate into lactate, compared to the LDHB subunit which catalyzes the opposite reaction [Lactic acid and its anion lactate are mainly formed by the glycolysis pathway. Because the pKa of lactate is 3.8, the anion lactate is the predominant form in the human body . During reaction . Glycolyreaction . Lactatereaction . This inreaction ,21.+) and with the onset of lactic acidosis  and is associated with an increase in hydrogen ions  concentrations in the plasma. The estimated number of sequestered parasites was calculated as the difference between the estimated total parasite biomass and the total circulating parasite biomass, the latter of which was calculated from the observed peripheral blood parasitemia [In a study with Gambian children infected with ass only . Therebyasitemia . This stasitemia ,38. ThisP. falciparum is the only species of the 5 human-infecting Plasmodia with a clear capability to sequester massively. Sequestration is mediated by the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins and enables the parasite to escape clearance by the spleen. This results in the localized release of inflammatory molecules, but is also a unique cause of microvascular obstruction and subsequent hypoxia in the surrounding cells and tissues [P. falciparum malaria, whereas bacteria or fungi in the blood during non-malarial sepsis do not specifically attach to endothelial cells [P. vivax, a Plasmodium species which is unable to sequester, hyperlactatemia occurs less frequently than in patients with P. falciparum malaria [Parasite sequestration is the cytoadherence of iRBCs to a variety of receptors on endothelial cells, mainly in capillaries and postcapillary venules.  tissues . Local hal cells ,40–42. Wal cells . However malaria .P. falciparum malaria [P. falciparum malaria using orthogonal polarized spectroscopy [P. falciparum found an association between lactate levels and the expression of several parasite genes important for cytoadherence [Furthermore, several studies have reported an association between the sequestered parasite biomass and lactate levels. Dondorp and colleagues observed a positive correlation between the sequestered parasite biomass and the plasma lactate concentration in patients with  malaria . No asso malaria . In addi malaria . Hanson troscopy . The amodherence . These sP. falciparum [In 1 study, no correlation was observed between parasite sequestered biomass and blood lactate levels in children infected with lciparum . The autIn malaria, both the destruction of uninfected RBCs and iRBCs after schizont rupture play an important role in the development of anemia . In anemDevelopment of anemia normally leads to the induction of erythropoiesis in the bone marrow and release of newly formed reticulocytes in the circulation. The metabolism of erythroid progenitors, which display a high proliferation rate, depends on aerobic glycolysis leading to high lactate production under aerobic conditions, called the Warburg effect . ErythroAlthough rarely as severe as in SMA, anemia is also a common feature in non-malarial septic patients, and this might thus contribute to lactic acidosis to some extent. In non-malarial sepsis, anemia may be caused by iatrogenic blood loss, depression of serum iron levels and erythropoietin production, intravenous fluid administration, disseminated intravascular coagulation (DIC), and a decreased life span of erythrocytes –58. ThesHypovolemia, hypotension, and shock in malaria patients may result in tissue hypoperfusion and hypoxia-induced hyperlactatemia. However, Hanson and colleagues found that lactic acidosis was worse in less hypovolemic malaria patients . HypotenLeukocyte activation and proliferation require several metabolic processes, which are determined by the leukocyte subtype and activation stimulus . In quiePlasmodium-derived components through their pattern recognition receptors. This is essential for the clearance of malaria infection, but excessive activation of these cells also leads to immunopathology [Antimalarial immunity involves both innate and adaptive immunity, comprises extensive phagocytic activity, T lymphocyte activation, and antibody production, and is regulated by the production of several cytokines and chemokines. Dendritic cells, monocytes, and macrophages sense athology . Both moathology . In partathology . The glyathology . The actTh1 immune reactions with interleukin (IL)-12 and interferon gamma (IFN-γ) as the main driver cytokines are essential for antimalarial immunity . However9 blood cells/day [It is currently not clear to what extent the Th1 reaction in malaria patients quantitatively contributes to lactic acidosis. However, it should be considered that in the erythrocytic stage of malaria, the activation of leukocytes is massive and systemic. IFN-γ produced during acute malaria substantially modulates hematopoiesis in the bone marrow, an organ larger than the liver comprising approximately 5% of the total body weight and producing approximately 500.10ells/day . Inflammells/day . The splells/day . The splells/day ,77. Alsoells/day . Overallells/day .Such Th1 and/or M1 immune activation is obviously not unique to malaria and may also occur in other infections. In non-malarial sepsis, the systemic exposure of the host to pathogens initiates an excessive inflammatory response . This eaGluconeogenesis in the liver occurs with alanine, lactate, and/or glycerol as precursors and is responsible for up to 70% of the lactate removal in the human body. The tight link between glucose metabolism and lactate levels is reflected by the common co-occurrence of lactic acidosis and hypoglycemia and the fact that children with metabolic acidosis and respiratory distress are most at risk of hypoglycemia ,83.In healthy subjects, gluconeogenesis and glycogenolysis contribute evenly to the glucose production . In normP. falciparum [P. falciparum–infected patients, histological evidence of liver injury is minimal. Although in some patients with severe malaria, inflammatory infiltrates, hepatocyte swelling, hemozoin deposition, loss of microvilli at the sinusoidal pole, and centrizonal necrosis have been observed in liver tissue [In malaria, jaundice occurs in up to 5% of the malaria cases in endemic regions and between 10% and 60% in areas of low malaria transmission . Jaundiclciparum ,90. In mfalciparum malaria was reported, and the majority (>95%) of P. falciparum– or P. vivax–infected patients with hyperbilirubinemia had increased serum lactate concentrations [Impaired clearance of lactate by the liver may directly influence the blood lactate concentration . A posittrations ,92. Moretrations . These sAlthough gluconeogenic alanine use is adequate in patients with uncomplicated malaria, alanine consumption is lower if the disease is more severe, possibly explained by decreased liver perfusion or impaired hepatic clearance . A decreThe contribution of liver dysfunction to lactic acidosis in malaria is comparable as in other conditions. In non-malarial septic patients, liver dysfunction has been associated with hyperlactatemia in hemodynamically stable patients as the result of impaired lactate clearance rather than increased production ,99. In cAltogether, these observations indicate that in a subset of malaria patients, impaired hepatic lactate clearance because of liver dysfunction, decreased blood flow, and/or suppressed gluconeogenesis may presumably play a significant role in the development of lactic acidosis.Next to the liver, the kidney is the second organ for lactate clearance via gluconeogenesis and urine excretion. In healthy persons, up to 30% of the lactate is removed via gluconeogenesis in the proximal tubules of the renal cortex ,101. UriP. falciparum or P. vivax, patients with AKI had increased plasma lactate levels compared to patients without AKI [AKI can occur in up to 40% of the adult patients with severe malaria . The maihout AKI ,107,108.AKI may also be an unrecognized contributor to lactic acidosis in non-malarial sepsis, because 1 in 3 patients with non-malarial sepsis develop AKI . HoweverThe most important gap in the current knowledge about lactic acidosis in malaria is the quantitative contribution of the various etiologies, which remains unclear despite approximative calculations . This isA better understanding of the quantitative contribution of the various etiologies in the different subsets of severe malaria is required to develop more targeted and adequate treatments. Correction of acidic blood pH with bicarbonate or other neutralizing agents is useful to avoid the detrimental effects of acidosis, but does not remove the underlying cause. Blood transfusion might be beneficial to reduce lactate in patients with SMA, because there is already evidence that anemia is an important contributor to hyperlactatemia in this subset of patients . Rapid kLactic acidosis is frequently observed in patients with severe malaria and is associated with a poor prognosis. Parasite sequestration is often proposed as the main contributor to lactic acidosis. However, this review supports a more elaborate view on the etiology of hyperlactatemia and lactic acidosis, including both increased production and impaired clearance of lactate. There are both similarities and differences between the etiologies of lactic acidosis in malaria and non-malarial sepsis. The quantitative contribution of the various etiologies in the different subsets of severe malaria should be further studied in the future. The better understanding of the distinct etiologies of lactic acidosis in severe malaria is required to develop more adequate treatments.S1 Appendix(DOCX)Click here for additional data file.


Malaria is major public health concerns which continues to claim the lives of more than 435,000 people each year. The challenges with anti-malarial drug resistance and detection of low parasitaemia forms an immediate barrier to achieve the fast-approaching United Nations Sustainable Development Goals of ending malaria epidemics by 2030. In this Opinion article, focusing on the recent published technologies, in particularly the nuclear magnetic resonance (NMR)-based diagnostic technologies, the authors offer their perspectives and highlight ways to bring these point-of-care technologies towards personalized medicine. To this end, they advocate an open sourcing initiative to rapidly close the gap between technological innovations and field implementation. Malaria continues to claim the lives of more than 435,000 people each year. The challenges with anti-malarial drug resistance and detection of low parasitaemia form immediate barriers to achieve the fast-approaching United Nations Sustainable Development Goals of ending malaria epidemics by 2030 .Critical for the control and elimination of malaria is an accurate diagnosis of the disease. Malaria infection often goes asymptomatic due to undetectable low parasitaemia is in fact inevitably underestimating the number of malaria carriers. Detecting and treating these infections are of utmost importance to ensure patient health and to block disease transmission, since they are a reservoir for future infections. Light microscopy (LM) remains the standard of practice for malaria diagnosis in febrile patients at health facilities, nevertheless this method requires well-trained personnel and does not adequately detect low parasitaemia (detection limit: ~ 50 parasite/μL of blood) . AlternaNonetheless, RDTs are not more sensitive than LM and, similarly, cannot detect the low-level blood-stage malaria infections that can otherwise be identified by genetic methods . NucleicA rapid malarial screening has been recently reported based on point-of-care NMR system , 10, 12.As shown in mice studies, the micro NMR system has ‘hypersensitivity’ with a detection limit of less than 10 parasite/µL of blood . This coSchistosoma), favoring their difference in disease manifestation symptoms to distinguish them. One far more relevant problem would be to distinguish Plasmodium species once treatment is usually administered based on the Plasmodium species detected. There are five Plasmodium species that can infect humans, being Plasmodium falciparum responsible for the highest mortality rate [Plasmodium vivax responsible for more than 2.5 billion people at risk of infection [Plasmodium species, right from the starting of parasites invasion in the intraerythrocytic cycle leads to hemozoin crystals formation, but their shape and condensation differs [While good NMR sensitivity (‘true positive rate’) has been reported, the issue of specificity  remains elusive , 8. Hemoity rate  and Plasnfection , 14. The differs .The process of hemozoin formation is mediated heavily by the transfer of electron. Sienkiewicz had shown using multi-frequency high field electron paramagnetic resonance (EPR) that the spectra obtained for hemozoin and synthetic β-haematin can only correspond to a high spin Fe-III (S = 5/2) . The premolecular fingerprint’ is the first step towards personalized malaria medicine, which has vast implication to the malaria elimination programme due the increasingly rate of phenotypic variations in the field [Taking NMR technology beyond parasite detection, this technology has high potential to address another important factor that hampers the control and elimination of malaria i.e., the rapid parasite drug resistance acquisition. Scaling up to higher dimension , NMR spectroscopy may provide the capability of detecting parasite ‘phenotypic variation’. Multi-dimensional representation would essentially means obtaining multifactorial markers (a panel of associated biomarkers) in a single snapshot which in return increases the accuracy of diagnostic , 21. ObtIn good time, multi-dimensional NMR techniques are well developed within the NMR community. With the development of fast and highly efficient multidimensional Inverse Laplace decomposition algorithm –24, and P. falciparum k13 gene does not explain some cases of phenotypic variations, such as parasite clearance time upon artemisinin-based combination therapy (ACT) treatment within the same gene [Anti-malarial drug resistance has been correlated with specific genetic mutations in the parasite, offering a tool for the development of diagnostic tests to monitor resistance. But the analysis of the genetic maker, usually through nucleic acid amplification and detection systems, demands technical handling expertise and generally above 1-h turnaround time, precluding, up to today, the development of a device/tool that could provide this information at the point-of-care settings. On top of this, there are anti-malarial drugs such as the artemisinin, for which mechanism of action and resistance is yet to be fully disclosed owning to the possible epigenetic involved. As an example, mutations at the ame gene , 28. Theame gene . Therefoame gene , 30, 31 As an outcome of the above mentioned, the anti-malarial drug resistance could hypothetically be captured by multidimensional spectroscopy without a priori knowledge (hypothesis free) on the genetic markers or even independent of other -omics details. Anti-malarial target different features of the parasite’s biology leading to changes in metabolic responses. We hypothesized that the alteration of protein chemistry in the infected RBCs with different anti-malarial susceptible parasites could lead to a distinctive spectrum due to the sensitivity of water-protein interactions in the time-domain multidimensional NMR.Parasites were first detected under the microscope in 1884 by Alphonse Laveran, a military doctor in France’s Health Service of the Armed Forces. It has passed now almost a century and a half since its discovery and imposingly, microscopy continues to be the recommended method for malaria diagnosis in the field.Spectroscopic techniques ), and ultralow cost microfluidic chip , 32–35 apoor-man disease’. In order to accelerate this cause, we need researchers to open-source code  and open-source the ideas/solutions, so that it will continue to spark innovation in the place where the technologies are needed most. With ubiquitous access to internet as meeting place , and low-cost fabrication of hardware , more practical solution can be brought to doorstep. The OPENCORE NMR project [Far too often, we found ideas and proposal being thrown off beyond publications in this tight funding environment, and unwillingness of investors to put in their money in what often dubbed as the ‘ project  by K. Ta project , 40, 41 There is a silver lining in this: having low-cost instrumentation with ultra-low-cost assays for malaria diagnostic (and monitoring), will then immediately translate the know-how  for other infectious diseases or even other chronic diseases aka the ‘rich-man diseases’ (e.g., diabetes mellitus , and canAccurate diagnosis of malaria and the resilient capacity that the malaria parasite has in acquiring resistance to anti-malarial drugs form immediate barriers to the control and elimination of this disease. Researchers in the field are beginning to see more cases of phenotypic variations. These phenotypic variations could be based on geographical location or differential transmission setting or even the different ring developmental stage , 30. In


The advantages of photodynamic diagnostic technology using 5-aminolevulinic acid (ALA-PDD) have been established. The aim of this prospective cohort study was to evaluate the usefulness of ALA-PDD to diagnose upper tract urothelial carcinoma (UT-UC) using the Olympus VISERA ELITE video system.We carried out a prospective, interventional, non-randomized, non-contrast and open label cohort pilot study that involved patients who underwent ureterorenoscopy (URS) to detect UT-UC. 5-aminolevulinic acid hydrochloride was orally administered before URS. The observational results and pathological diagnosis with ALA-PDD and traditional white light methods were compared, and the proportion of positive subjects and specimens were calculated.A total of 20 patients were enrolled and one patient who had multiple bladder tumors did not undergo URS. Fifteen of 19 patients were pathologically diagnosed with UT-UC and of these 11 (73.3%) were ALA-PDD positive. Fourteen of 19 patients were ALA-PDD positive and of these 11 were pathologically diagnosed with UC. For the 92 biopsy specimens that were malignant or benign, the sensitivity for both traditional white light observation and ALA-PDD was the same at 62.5%, whereas the specificities were 73.1% and 67.3%, respectively. Of the 38 specimens that were randomly biopsied without any abnormality under examination by both white light and ALA-PDD, 11 specimens (28.9%) from 5 patients were diagnosed with high grade UC. In contrast, four specimens from 4 patients, which were negative in traditional white light observation but positive in ALA-PDD, were diagnosed with carcinoma in situ (CIS).Our results suggest that ALA-PDD using VISERA ELITE is not sufficiently applicable for UT-UC. Nevertheless, it might be better particularly for CIS than white light and superior results would be obtained using VISERA ELITE II video system.Trial registration: The present clinical study was approved by the Okayama University Institutional Review Board prior to study initiation (Application no.: RIN 1803–002) and was registered with the UMIN Clinical Trials Registry (UMIN-CTR), Japan (Accession no.: UMIN000031205). Photodynamic diagnostic (PDD) technology using 5-aminolevulinic acid (ALA-PDD) is currently being used for the diagnosis of brain tumors in Japan. For patients with bladder cancer, the sensitivity and specificity of ALA-PDD using scopes and video systems produced by Karl Storz SE & Co. KG  can be as high as 75.8% and 68.2%, respectively . Thus, iUpper tract urothelial carcinoma (UT-UC) includes renal pelvic and ureteral cancer, which are the same as bladder cancer from a pathological perspective. Although standard treatment for UT-UC is total nephroureterectomy and partial cystectomy, techniques involving endoscopic diagnosis and resection with URS have been developed , 4. HoweThe aim of this prospective cohort study was to evaluate the usefulness of ALA-PDD for diagnosis of UT-UC using the VISERA ELITE video system produced by Olympus Co., Ltd.All methods were carried out in accordance with relevant guidelines and regulations.We carried out a pilot, prospective interventional, non-randomized, non-contrast and open label cohort study. Enrollment began following approval by the review board. Patients who were suspected of having UT-UC and were undergoing ureterorenoscopy (URS) were enrolled. Since this study was a pilot study, 20 cases were considered based on the annual number of cases treated at our institution. The primary endpoint was the proportion of positive subjects and secondary endpoints were sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and adverse events for ALA-PDD.Patients that were enrolled in the study were adults who were suspected of having UT-UC and were undergoing URS. All patients were aware of the conditions associated with the disease, and provided informed consent to participate. All enrolled patients agreed to use contraception until 1 month after the administration of 5-ALA. Exclusion criteria were patients with porphyria, allergy to porphyrin or 5-ALA, hepatic dysfunction, use of drugs that induced photosensitivity, pregnancy, or severe underlying diseases such as cardiovascular diseases or infection.On the day of the URS, 20 mg/kg of 5-aminolevulinic acid hydrochloride (5-ALA HCl) was orally administered over 180 min (range: 120–240 min) before the scheduled insertion time for the ureteroscope. After oral administration of 5-ALA HCl, patients drank 250 mL of an L-arginine-intensive drink and 500 mL of fruit juice to maintain blood pressure. At the time of examination, normal observations using white light and ALA-PDD using visible blue light were carried out using a two-color LED light source . All endoscopic images were recorded.After the procedures, patients had proper shading to prevent photosensitivity for up to 48 h after ingesting 5-ALA HCl and light exposure of less than 500 lx was maintained. Hematologic and biochemical examinations were performed on postoperative days (POD) 1 and 3 and the occurrence of adverse events was noted.5-ALA HCl was supplied as a white powder similar to that for ALAGLIO by SBI Pharmaceuticals . The chemical compound was stored according to strict quality control guidelines and detailed usage records were maintained by the Division of Clinical Research of New Drugs and Therapeutics in the Center for Innovative Clinical Medicine, Okayama University Hospital.The endourological techniques and URS procedures performed at our institution for observation of the upper urinary tract and ablation of tumors were previously described , 12. AllThe observational results and pathological diagnoses, the proportion of positive subjects, positive specimens, sensitivity, and specificity were calculated. Adverse events were also investigated and severity was categorized using the Clavien-Dindo classification.Characteristics of the 20 patients enrolled in this study are shown in Table The median (IQR) time from oral administration of 5-ALA HCl to biopsy was 246 (227–269) minutes. One patient had multiple tumors in the bladder and URS was aborted. Among the 19 remaining patients, biopsy was not performed because of absence of any abnormality in one patient, for whom URS was performed as follow-up. Fifteen patients were pathologically diagnosed with UT-UC and of these, 11 (73.3%) were ALA-PDD positive. Fourteen of 19 patients were ALA-PDD positive and of these, 11 were pathologically diagnosed with UC. Among the 50 specimens that are ALA-PDD positive, 25 (50%) were pathologically diagnosed with UC, 17 (34%) were not malignant, and the status for 8 (16%) could not be determined because the specimens were too small. Among the 7 patients that were ALA-PDD positive and white light negative  in 6 patients and hypotension requiring vasopressors (Grade II) in one patient. No allergic episodes, cardiovascular complications or photosensitivity were observed in our study.In this prospective cohort study to evaluate the effectiveness of ALA-PDD for UT-UC diagnosis using the VISERA ELITE video system, the sensitivity and specificity were 62.5% and 67.3%, respectively. Of the 38 specimens that were randomly biopsied without any abnormality under examination by both white light and ALA-PDD, 11 specimens (28.9%) from 5 patients were diagnosed with high grade UC. On the other hand, four biopsy specimens that had abnormal findings with ALA-PDD but not with normal white light observation were diagnosed with CIS.ALA-PDD was approved only for bladder cancer by Japanese national health insurance in 2017. This approach is expected to have value not only for bladder cancer diagnosis but also for treatment, namely complete resection at TURBT –16. On tInterestingly, 11 of the 38 (28.9%) specimens that exhibited no abnormalities in either white light or ALA-PDD examinations were pathologically diagnosed with high grade UC. The reasons for this result are unclear, but some abovementioned factors may have contributed to this outcome. To our knowledge there are no studies that examined random biopsy samples taken from the upper urinary tract, and additional investigations are needed.According to European guidelines , diagnosAdverse events of ALA-PDD have been reported as liver toxicity, allergic episode, cardiovascular complications, photosensitivity, hypotension, nausea and vomiting , 25. In The present study has several limitations. Although this pilot study was prospective, it was performed at a single center, was non-randomized and included small number of patients (n = 20). Second, there were 2 patients who underwent protocol URS after endoscopic laser ablation and cognitive bias could be associated with the previous procedure. Third, we evaluated the usefulness of ALA-PDD only in the diagnostic process and not the treatment process. Fourth, we did not compare endoscopic devices including semirigid/flexible ureteroscopes, light cables, and video systems, and did not evaluate their compatibility with ALA-PDD. Despite these limitation, the ureteroscopy with ALA-PDD might be more useful for diagnosis in patients with UT-UC, particularly with CIS.Our results of this pilot study suggest that ALA-PDD using VISERA ELITE is not sufficiently applicable for UT-UC. Nevertheless, it might be better particularly for CIS than white light and superior results would be obtained using VISERA ELITE II video system. Further studies on patient selection, endoscopic equipment and image processing methods are needed for the broad application of ALA-PDD for UT-UC diagnosis.


Accumulating evidence suggest that compromised CYP2D6 enzyme activity caused by gene mutation could contribute to primaquine failure for the radical cure of vivax malaria. The current study aims to preliminarily reveal the association between the recurrence of vivax malaria in Yunnan Province and CYP2D6 gene mutation by analysing polymorphisms in the entire coding region of human CYP2D6 gene.Blood samples were collected from patients with vivax malaria, who received "chloroquine and 8-day course of primaquine therapy" in Yunnan Province. The suspected relapsed cases were determined by epidemiological approaches and gene sequence alignment. PCR was conducted to amplify the CYP2D6 gene in the human genome, and the amplified products were then sequenced to compare with the non-mutation “reference” sequence, so as to ensure correct sequencing results and to determine 9 exon regions. Subsequently, the DNA sequences of 9 exons were spliced into the coding DNA sequence (CDS), which, by default, is known as maternal CDS. The paternal CDS was obtained by adjusting the bases according to the sequencing peaks. The mutation loci, haplotypes , genotypes and odds ratios (OR) of all the CDSs were analysed.Χ2 = 16.177, P < 0.05). Of the defined 24 genotypes, 8 genotypes, including *4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10, were detected only in the SR group.Of the119 maternal CDS chains in total with 1491 bp in length, 12 mutation sites in the 238 maternal and paternal CDS chains were detected. The c.408G > C mutation was most frequently detected in both suspected relapsed group (SR) and non-relapsed group (NR), reaching 85.2% (75/88) and 76.0% (114/150), respectively. The c.886C > T mutation was most closely related to the recurrence of vivax malaria . Among the 23 haplotypes (Hap_1 ~ Hap_23), Hap_3 was non-mutant, and the sequence structure of Hap_9 was the most complicated one. Five star alleles, including *1, *2, *4, *10 and *39, were confirmed by comparison, and CYP2D6*10 allele accounted for the largest percentage . The frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (Mutation of CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutations of c. 886C > T and CYP2D6*2 allele, which correspond to impaired PQ metabolizer phenotype, are most closely related to the relapse of vivax malaria. In addition, the genotype *4/*4 with null CYP2D6 enzyme function was only detected in the SR group. These results reveal the risk of defected CYP2D6 enzyme activity that diminishes the therapeutic effect of primaquine on vivax malaria.The online version contains supplementary material available at 10.1186/s12936-021-03685-3. Plasmodium infection [Plasmodium [Plasmodium vivax [P. vivax.In recent years, the global malaria epidemic has gradually scaled down, yet the death tolls remain high and the number of malaria cases in certain areas has increased . Meanwhinfection , the expasmodium , 4, and asmodium , pose daasmodium , China sasmodium . From 20um vivax . TherefoP. vivax via 5-hydroxy-primaquine (5-hydroxy-primaquine) formed by the enzyme CYP2D6  in human liver cells [Tafenoquine and primaquine are recommended by the World Health Organization (WHO) for the treatment of relapsed malaria owing to its efficiency of eradicating the hypnozoites of er cells –12. CYP2er cells , and thaer cells , 14, 15.er cells , 17.CYP2D6 enzyme activity is measured mainly through phenotypic observation and genotypic analysis. Compared with phenotypic characterization, genotypic prediction manifests the advantages of greater stability, more convenience and being less affected by environmental or physiological factors –20. CYP2Primaquine is commonly used in the malaria endemic areas of Yunnan Province, yet the systematic survey on the genetic polymorphisms of CYP2D6 has been not conducted. In this study, Whole Genome Sequencing (WGS) was conducted to investigate the polymorphisms of CYP2D6 gene in the vivax malaria cases, thereby revealing the association between human CYP2D6 gene polymorphisms and the relapsed cases of vivax malaria.P. vivax by both microscopy examination and Plasmodium 18S rRNA gene detection (PCR) in Yunnan Province Malaria Diagnosis Referent Laboratory (YPMDRL)  were enrolled in the study. The subjects were diagnosed of mono-infection of P. vivax infection within the interval of 28–180 days after the initial diagnosis; (2) epidemiological investigation confirmed that the patients were not re-exposed to malaria endemic areas between the two malaria episodes; and (3) the comparative sequencing of CSP (Circumsporozoite Surface Protein) and MSP-1 (Merozoite Surface Protein 1) of P. vivax with those of the previously infected strains found 100% similarity and 0 variable sites.The subjects diagnosed from 2014 to 2018 were subdivided into suspected relapse of vivax malaria (SR group) and non-relapse of vivax malaria (NR group). The inclusion criteria of the SR group are: (1) the patients were re-diagnosed as A simple random sampling method was conducted to selected 75 NR cases from the 143 patients confirmed in 2018. The subjects in the NR group had no history of relapsed vivax malaria after 1 year of follow-up. The source of vivax malaria infection was determined through epidemiological investigations. The subjects had no travelling history to malaria endemic areas within 30 days before the onset of malaria were regarded as local cases, otherwise they were regarded as imported cases. The study was approved by Yunnan Institute of Parasitic Diseases and Ethical Committee document No. 201904 . Genotyping was performed by using the stored blood samples obtained from the suspected patients who experienced fever and chills. Informed consent was obtained from all the patients. The blood samples of the subjects were provided by the Centers for Disease Control and Prevention in Yunnan Province, according to the described protocol . Dried bThree dried blood spots (diameter = 5 mm) were made, and genomic DNA was extracted according to the manufacturer’s instructions of the QIAgen Mini Kit , according to the manufacture’s instruction. The extracted DNA was stored at − 20 ℃.https://www.ncbi.nlm.nih.gov/gene/), reference sequence (ID: NC_000022.11) was used as template, following the reaction condition in previous studies [2O. PCR reaction conditions were clarified as follows: 92 to 94 °C for 2–5 min; 92 to 94 °C for 10–30 s, 50 to 56 °C for 15–30 s, 68 to 72 °C for 2 to 3 min, 35 cycles; 68 to 72 °C for 7 min. The triplicated parallel repetition was adopted for each PCR reaction. The amplified products were observed using 1.5% agarose gel electrophoresis. The positive amplification products were sent to Shanghai Meiji Biomedical Technology Co, Ltd. for sequencing by using the Sanger method. Only the sequences that showed identical results in at least two tests were used for subsequent analysis.The primers of CYP2D6 for polymerase chain reaction (PCR) amplification were designed by using GenBank  on the NCBI platform. The sequences having a homology of 100% and query cover of above 99% with the non-mutated “reference” sequence (ID: NC_000022.11) were considered as the human CYP2D6 gene sequence.The sequencing reads were aligned by using DNAStar and BioEdit software packages. The obtained DNA sequences were assessed by using the Basic Local Alignment Search Tool  of CYP2D6 gene in the order from exon 1 to exon 9. The paternal CDS was obtained by replacing the undisplayed diploid bases on the maternal CDS chain , 32, andP. vivax. The significance level was set as P < 0.05; OR < 1 indicates that the haplotype is a protective factor, while OR < 1 indicates a risk factor.The haplotype  of CDSs was determined according to the criteria of Human Cytochrome P450 Allele Nomenclature Committee (NM_000106.6)  and prevA total of 45 SR vivax malaria cases were confirmed by epidemiological investigation and gene sequence alignment  were obtained. Base substitutions at 12 loci, including c.31 and c.100, on the 238 maternal and paternal CDS, were determined (Table The comparative results of 12 single nucleotide polymorphisms (SNPs) between the two groups were listed in Table A total of 23 haplotypes Hap_1 ~ Hap_23) were identified amongst the 238 CDSs of CYP2D6 gene. Of them, 9 haplotypes were only observed in the sequences of SR group , while 6 haplotypes were only found in the NR group (π = 0.0014 and He = 0.760). 8 haplotypes, including Hap_2, Hap_3, Hap_4, Hap_5, Hap_6, Hap_7, Hap_13 and Hap_14, were found in both SR and NR group. The compositions of every haplotype were shown in Fig.  ~ Hap_23x2 = 2.560, P > 0.05). The frequency of alleles *1 in the NR group , and the frequency of alleles *2 in SR group was significantly higher .Among the 23 haplotypes, 11 sub-alleles  that could be accurately termed belonged to 5 star (*) alleles, including *1, *2, *4, *10 and *39. Moreover, 12 sub-alleles (*m-*x) had no referencing information. The most common alleles included *10 , *1  and *39   consists of 8–9 exons and 7–8 introns. Its CDS is 1338–1491 bp in length and encodes 446–497 amino acids. The heterogeneity of CYP2D6 monooxygenase activity varies from complete dysfunction to ultra-rapid enzyme metabolism, depending on the various genotypes of CYP2D6 , 33, 36.P < 0.05). In the findings of Wang et al. [. [The current study is the first one that attempts to analyse CYP2D6 polymorphisms in the entire coding region sequence in the blood specimen of relapsed vivax malaria patients in Yunnan. The current study reveals the association between CYP2D6 polymorphisms and thwarted CYP2D6 enzyme activity, which is accountable to the failed treatment of using primaquine. This study found that mutation at c. 886 locus of CYP2D6 gene elevated the risk of vivax malaria relapse risk by 2.167-fold . Consistently, previous studies found the highest frequency of CYP2D6*10 alleles mutation in the Chinese Han population and in Asian subjects , 39–41. . [The current study found that CYP2D*2 allele was more frequently determined in the relapsed patients. This result was not in agreement with that of Brasil et al. , which r. –44.In this study, all the alleles were identified based on the mutations in the exon regions of CYP2D6, which is different from allelic recognition that covers intron mutations , 45. TheEight mutant allelic types were found only in the SR group, yet the association between these different CYP2D6 genotypes and the relapse of vivax malaria could not be established. Given that the *10/*10 genotype still accounted for the largest proportion (30.3%) in the overall study cohort and the no-function *4/*4 genotype was found in the SR group , the risP. vivax was ruled out from the SR group, the influences of primaquine doses and primaquine resistance were not evaluated. Finally, polymorphisms in the entire coding region of CYP2D6 gene were investigated yetthe possible mutation in intron region and the copy number of CYP2D6 gene, which has been considered to disturb the activity of CYP2D6 enzyme, were not analysed. Thusly, the activity of CYP2D6 cannot be indirectly predicted based on the results of CYP2D6 genotyping in the present study. In view of such shortcoming, the sampling size should be expanded to conduct molecular epidemiological investigations on CYP2D6 genotype.The limitations of the current study should be noted. Firstly, the sample size of suspected recurrence of vivax malaria is not large enough, and the alleles  of CYP2D6 genes with low frequency of distribution might not be quantitatively analyzed  and other advanced methods will be applied to optimize the accurate identification of human CYP2D6 genotypes, so as to swiftly predict CYP2D6 enzyme activity of individual subjects. These findings will contribute to the efficacy of primaquine in the treatment of vivax malaria in Yunnan province.This pilot study reveals the polymorphisms of CYP2D6 gene coding region and its association with the recurrence of vivax malaria in the population of Yunnan Province, suggesting that the radical cure of vivax malaria by using primaquine could be hampered due to the impaired CYP2D6 enzyme activity in the population of Yunnan Province. The study revealed that (1) CYP2D6*10 allele accounts for the highest proportion in the vivax malaria cases of Yunnan Province; (2) mutation at c. 886 locus is most closely related to the relapse of vivax malaria and CYP2D6*2 allele containing this mutation locus was significantly higher in SR; and (3) The *4/*4 genotype that indicates null CYP2D6 enzyme function (poor metabolizers) was only found in the SR cases.Additional file 1. The details of nested PCR testing for differentiating between various Plasmodium species.Additional file 2. Sequencing Peak Map of CYP2D6 Gene Polymorphic loci.Additional file 3. Epidemiological information and alignment results of the genes of suspected recurrent cases of vivax malaria.Additional file 4. Electrophoretic map of PCR products in the coding region of CYP2D6 gene.Additional file 5. Analysis the CYP2D6 genotypes in SR group and NR group.


Somalia is affected by a civil war and a protracted humanitarian crisis for more than two decades. The international community has put in place nutrition surveillance systems to monitor the situation and inform decisions. However, the indicators commonly used to identify acute malnutrition, weight-for-height Z-score (WHZ) and mid upper arm circumference (MUAC), do not always converge in their estimations of acute malnutrition, creating challenges for decision making. Furthermore, the divergences are not consistent across livelihood populations within the country. We explored the MUAC-for-age Z-score (MUACAZ) as an alternative indicator in Somalia to minimize the discrepancy.We analyzed data from nineteen cross-sectional surveys conducted in Somalia between 2007 and 2016. We compared the acute malnutrition prevalence estimates by each of the indicators and the degree of overlap in the individual diagnosis of acute malnutrition between the WHZ and the MUAC-based indicators. We performed multivariate regression analysis with sex, age and stunting as independent variables and acute malnutrition as the dependent outcome, defined by WHZ, MUAC or MUACAZ. We performed all the analysis in the population overall and in each of the livelihood populations separately.A total 255,623 measurements of children 6–59 months of age were analyzed. The overall prevalence of global acute malnutrition by MUACAZ (15.8%) was similar to the one obtained using WHZ (16%), whereas prevalence based on MUAC was much lower (7.8%). These patterns of divergence were sustained throughout the nineteen surveys and the livelihoods studied, with only few exceptions. However, the proportion of overlap in the individual diagnosis of children as acutely malnourished was low between WHZ and absolute MUAC diagnosis (18.1%) and also between WHZ and MUACAZ (28.3%). Results show that age, sex and stunting status of the child affected the likelihood of being diagnosed as acutely malnourished to varying degrees, depending on the indicator used.The MUAC-for-age (MUACZ) indicator yielded acute malnutrition prevalence estimates convergent with those obtained by WHZ indicator. However, the degree of overlap between these two indicators for individual diagnosis of acute malnutrition is low. Further studies of MUACAZ as an alternative indicator for nutrition surveillance are needed.The online version of this article (10.1186/s40795-018-0213-3) contains supplementary material, which is available to authorized users. Somalia has been affected by a civil war since 1991, with the food security situation further aggravated by recurrent and prolonged droughts. Located in the Horn of Africa, the combination of conflict and natural disasters has eroded its livelihoods, caused structural food insecurity, population displacements and extreme poverty . As partOn-going surveillance of anthropometric indicators is crucial to detect the deterioration of the nutritional status of a population. It can provide information on trends, allowing for comparison overtime and against baseline years results, and it permits for geographical and contextual comparisons which can inform the prioritization of actions and the allocation of resources . For thiIn two recent reviews of nutrition surveillance systems in humanitarian settings and low income countries , 4, the The recommended anthropometric indices and measures for acute malnutrition surveillance are weight-for-height/length Z-score (WHZ), absolute value of Mid-Upper Arm Circumference (MUAC) and MUAC-for-age Z-score (MUACAZ), with the cut-off points detailed in Box 1, being WHZ and MUAC the most widely used.However, and although the absolute MUAC cut-off points were selected from statistical analysis to best match the WHZ cut-off points, so that the same prevalence of acute malnutrition would be found with each criterion , it has For this reason, in nutrition surveillance systems aimed at identifying children with severe acute malnutrition (SAM) for admission in feeding programs, the World Health Organization (WHO) and United Nations International Children’s Emergency Funds (UNICEF) recommend the use of WHZ < − 3 and also the use of absolute MUAC below 115 mm, as the latter indicator presents operational advantages in terms of time and cost of data collection, and is also believed to identify the children at higher risk of death . AlthougHowever, if the aim of the surveillance is to assess the severity of the situation in the population and the targeting of humanitarian actions, WHO has recommended the use of WHZ or the use of MUAC adjusted by age and sex, that is the MUACAZ, but not the use of absolute MUAC . The maiNonetheless, in recent times, the use of the MUAC measurement (or absolute MUAC) below 125 mm has been advocated as a proxy for global acute malnutrition for population estimates, grounded on the operational advantages of the indicator described before . This isThe reasons for these discrepancies are not fully understood, but an important part of this variation must be linked to the age and sex dependency of the absolute MUAC measurement . That isFor this purpose we compare the nutrition outcomes yielded by absolute MUAC and MUACAZ with those obtained using WHZ in different populations of Somalia over the 2007–2016 period. We analyse also the effect of factors, namely sex, age and stunting status, expected to contribute to the discrepancy observed between malnutrition rates derived from the three indicators.The data used for this study were obtained from 19 surveys undertaken by FSNAU and partners working in Somalia, in a biannual basis, in the 2007–2016 time period.There are three main livelihoods in the country: the pastoralists; the agro-pastoralists; and the riverines, that are mainly agrarian . BecauseA two-stage cluster sampling with probability proportional to size design was employed where sample sizes for the surveys (number of households and number of children) were calculated using Standardized Methodology for Survey in Relief and Transition (SMART) methods. Previous estimations of global acute malnutrition measured by WHZ and crude mortality rates for the surveyed areas were used for the calculations. An additional 10% was added to the sample size to allow for dropout or refusal to participate.n = 650, 0.25% of the total sample). Weight-for-height and MUAC-for-age Z-scores were calculated using WHO Anthro  and macros using WHO 2006 growth standards. Extreme biologically implausible values were excluded based on WHO standards with recommended flag limits of WHZ and MUAC Z-scores − 5, + 5 [The data were cleaned by deleting the records of individual children with any of the following criteria: age < 6 months, age > 59 months, and age, sex, weight, height, oedema or MUAC not recorded. Cases of oedematous malnutrition were excluded from analysis because the WHZ indicator is not valid as proxy of acute malnutrition in such cases  and severe acute malnutrition (SAM) using the indexes and cut offs described in Table We calculated the prevalence of GAM according to each of the three indicators in a sample pooling the data of the nineteen surveys, stratified by livelihood system, and then, for each of the surveys separately.WHZ− 2only or WHZ− 3only when the child had WHZ below − 2 or WHZ below − 3 but MUAC over 125 mm or 115 mm respectively, and MUAC125only or MUAC115only when MUAC was below 125 or below 115 but WHZ was equal or greater than − 2, or above − 3, respectively. When comparing WHZ and MUACAZ the WHZ− 2only or the WHZ− 3only refer to children identified as GAM or SAM by WHZ but not by MUACAZ, and the MUACAZ− 2only and the MUACAZ− 3only to children with MUACAZ below − 2 or − 3 but above these same values for WHZ.For comparing the diagnosis of acute malnutrition using WHZ and absolute MUAC, we defined − 2, MUAC125 or MUACAZ− 2 for GAM and WHZ− 3, MUAC115 or MUACAZ− 3 for SAM including all covariates  in the overall sample and then in each of the livelihood samples separately, to control for potential confounding and mediating effects.We performed multivariate logistic regressions on GAM and SAM prevalence defined by WHZ, absolute MUAC and MUACAZ. The independent variables used were sex , young age (age < 24 months vs. age ≥ 24 months), and stunting (height for age Z score < − 2 vs height for age Z score > − 2), as they are the hypothetical contributors to the discrepant malnutrition diagnosis between WHZ and absolute MUAC . We testStata 14  was used for statistical analysis.A total of 255,623 measurements of children aged 6 to 59 months from 19 surveys were examined. There was a nearly equal proportion of girls and boys (49% girls), and the mean age of the children was 31.6 (±0.03) months, with a distribution of 66% children with 24 months or older and 33% children between 6 months and 24 months of age. By livelihood system, 16.5%, 35.2% and 10.4% of children were from areas of agro-pastoral, pastoral and riverine livelihoods, respectively, while 38% lived in IDPs camps.In Table − 2 and by MUACAZ− 2 (16% vs. 15.8%), and half of it when calculated by MUAC125 (7.8%). In pastoralists, the difference was even greater, as the prevalence of GAM by MUAC125 was less than half the GAM prevalence yielded by the other two indicators. The SAM estimates showed similar patterns to GAM  using MUAC125.The discrepancies between the estimations of GAM by each of the three indicators were sustained throughout the 19 surveys  was higher than the overlap between WHZ− 2 and MUAC125 (28.3 to 18.1%). When the analysis was stratified by livelihood the highest overlap between WHZ− 2 and MUAC125 was found among agro-pastoralists (20.5%), and the lowest among pastoralists (14.4%)  than by WHZ− 3 and MUAC115 (9.6%) for the overall population. The largest discrepancy was found for SAM between WHZ and absolute MUAC in pastoralists children.The differences for SAM were more pronounced, and the degree of overlap was significantly lower than for GAM, being higher for children identified by WHZ115 was the sole criteria to be used, and 8276 if the children were to be diagnosed by WHZ− 3 alone. This difference was more marked among the pastoralists population .When comparing the SAM diagnosis by WHZ and by MUACAZ the differences were less pronounced, as out of the 15,052 children diagnosed as SAM by either of these two indicators, 7375 children, would have been diagnosed as severely acutely malnourished if WHZIn Table Female children were less likely than boys to be diagnosed as acutely malnourished by WHZ or MUACAZ, whereas they were identified as such more than boys (around 40% more) with absolute MUAC. On the other hand, stunted children were identified as acutely malnourished about three times more than non-stunted children when MUACAZ and absolute MUAC were used, compared to 1.3 times, at most, when WHZ was the indicator utilized. Among pastoralists children the impact of stunting on the acute malnutrition outcomes was higher than in the other livelihood groups.Regarding age, there was a change in the direction of the association when the outcome was GAM or SAM, as older children were more likely to be diagnosed as GAM, and less likely to be diagnosed as SAM when measured by WHZ or by MUACAZ in the overall population. Among agropastoralists and pastoralists populations, the children older than 2 years were also more likely to be diagnosed as SAM when using MUACAZ but not when using WHZ  [In 1995 the WHO proposed a set of thresholds to classify the severity of malnutrition in emergency situations according to the prevalence of GAM, measured by WHZritical) . Up to d125 is above 10.7% [− 2 and only “Serious” by FSNAU threshold using MUAC125, thus triggering different responses.In response to this limitation, FSNAU developed absolute MUAC emergency thresholds adapted to the Somalia context and based on the calculation of the quintiles of the distribution of absolute MUAC. According to those thresholds, the situation is considered “Critical” if the prevalence of GAM as measured by MUAC− 2 the population estimates are similar to those obtained by WHZ− 2, and so they are the resulting classifications according to WHO thresholds in sixteen out of the nineteen surveys analysed.According to our results if the GAM is measured by MUACAZOne of the main arguments for not using MUACAZ it is the age dependency of the indicator, as it is reported that in certain contexts with a high burden of acute malnutrition, age is difficult to ascertain accurately . This maWe recommend to revise the age dependency limitation in view of the importance of bringing age in the estimation of acute malnutrition when using the MUAC measurement. As other authors have already suggested, it could also be possible to construct tally sheets for MUAC divided by age groups in order to minimise the constraints regarding age ascertainment and its accuracy .In relation to the individual screening of acutely malnourished children the results obtained by either indicator did not converge. The discrepancy observed between absolute MUAC and by WHZ in children screening had already been studied , 18 and The results obtained by Grellety and Golden in their recent review of 47 countries . HoweverMoreover, our results disagree with the hypothesis of pastoralist populations showing systematic higher estimates for acute malnutrition as compared to other populations in the same context  as they Finally, our results show that age, sex and stunting status of the children also play a role in their likelihood of being diagnosed as acutely malnourished depending on the indicator to be used. Female, younger, and stunted children were more likely to be identified as acutely malnourished if the indicator to be used was absolute MUAC, whereas boys older than two years and stunted were more likely to be diagnosed as GAM by WHZ. Our results confirm those of other studies conducted in countries as Philippines, Chad, South Sudan and Bangladesh , Kenya  or SouthThe sample characteristics in terms of size and number of surveys allowed for a high precision in the analysis, and for the stratification by livelihood system. The so called IDPs livelihood was over-represented in the overall population as recent data indicated that the proportion of IDPs in Somalia population was around 14% , thus thThe data were collected in field conditions which may have an impact on the accuracy of measurements, although the FSNAU survey enumerators long time experience and routinely training may have minimized this limitation.Another limitation of our study is that the analysis was performed on a single country and results cannot be generalizable for other countries.The estimates of acute malnutrition by MUACAZ are convergent with the estimations reported by WHZ, as opposed to the high discrepancies found in relation to the absolute MUAC estimates, in our study population. We recommend further research on the MUACAZ indicator in the estimations of GAM in other contexts in which WHZ cannot be collected, as well as the reporting of this indicator in nutrition surveillance reports in Somalia whenever possible.We encourage the inclusion of MUACAZ in the array of outcomes to be calculated by the available software for nutrition surveillance estimates like the Emergency Nutrition Assessment (ENA) software.In relation to screening for program admission, we recommend adherence to the current guidelines of using both absolute MUAC and weight-for-height for the screening of severely and moderately malnourished children, and promote research on the role that MUACAZ or alternative indicators can play in this type of surveillance.Additional file 1:Table S1. Comparison of acute malnutrition diagnosis by WHZ and/or absolute MUAC in four Somalian livelihoods. Proportion of children with acute malnutrition (GAM or SAM) as defined by WHZ and/or absolute MUAC (see Methods section for definitions) that are diagnosed as malnourished by WHZ only, by absolute MUAC only, or by both criteria simultaneously. Table S2. Comparison of acute malnutrition diagnosis by WHZ and/or MUACAZ in four Somalian livelihoods. Proportion of children with acute malnutrition (GAM or SAM) as defined by WHZ and/or absolute MUACAZ (see Methods section for definitions) that are diagnosed as malnourished by WHZ only, by MUACAZ only, or by both criteria simultaneously. Table S3. Factors associated with GAM diagnosis based on WHZ, absolute MUAC and MUACAZ by livelihood. Results of the multivariate regression on GAM as measured by either three of the indicators  conducted in each of the four livelihood populations. Table S4. Factors associated with SAM diagnosis bases on WHZ, MUAC and MUACAZ by livelihood. Results of the multivariate regression on SAM as measured by either three of the indicators  conducted in each of the four livelihood populations. (PDF 211 kb)


Plasmodium and despite concerted and at times valiant efforts, the underlying pathophysiological processes leading to severe disease are poorly understood. Here we describe zoonotic malaria caused by Plasmodium knowlesi and the utility of this parasite as a model system for severe malaria. We present a method to generate long-read third-generation Plasmodium genome sequence data from archived clinical samples using the MinION platform. The method and technology are accessible, affordable and data is generated in real-time. We propose that by widely adopting this methodology important information on clinically relevant parasite diversity, including multiple gene family members, from geographically distinct study sites will emerge. Our goal, over time, is to exploit the duality of P. knowlesi as a well-used laboratory model and human pathogen to develop a representative translational model system for severe malaria that is informed by clinically relevant parasite diversity.Malaria is responsible for unacceptably high morbidity and mortality, especially in Sub-Saharan African Nations. Malaria is caused by member species’ of the genus  Plasmodium, have a highly complex lifecycle, intimately dependant on an invertebrate mosquito host for the diploid sexual stage of reproduction and equally dependant on specific vertebrate hosts for asexual replication and transmission. Lifecycle complexity, including adaptation to specific vertebrate hosts, invertebrate host restriction to particular Anopheline vector species with spatial and ecological niche requirement may augur unfavourably for Plasmodium spp. survival in a dynamic world. Yet, despite sustained efforts, human malaria persists to the extent that the altruistic World Health Organization (WHO) malaria eradication goal of the 1950s, was downgraded to country and at times species-specific elimination https://www.who.int/malaria/areas/elimination/en/. Even so, eradication is not a forgotten dream and may well be achievable within a new 30-year time-frame  and an area that lags behind available technologies . No diffModelling malaria is especially difficult because malaria parasites have co-evolved with their vertebrate hosts each exerting selective forces on the other in a dynamic dance for survival . That dyP. falciparum gene clusters involved in erythrocyte invasion  of the Plasmodium knowlesi normocyte binding protein (Pknbp)xa on chromosome 14 and continuous data on markers of disease severity  frozen whole blood. Undeterred and as proof of concept, we developed a method to deplete human DNA and concentrate parasite DNA in the samples. We produced P. knowlesi genome sequences from six clinical isolates using massively parallel Illumina short-read sequencing platforms  produces a disease more representative of human malaria and it is surprising that this opportunity to model severe malaria has not been taken forward  and identify virulence gene candidates. We show how short and long-read Plasmodium genome sequence data can be generated from fresh or archived frozen samples held in the many malaria research centres worldwide. Plasmodium genome sequence outputs over time and space will facilitate the construction of a substantial genetic reference resource, based on diverse wild-type parasites isolated from patients, that will inform model systems  by centrifugation: 2,000 x g; 20 minutes; 4°C (pellet 1). The supernatant was transferred to a fresh tube and centrifugation repeated to maximise parasite recovery (pellet 2). The pellets were combined and resuspended in 1.2mL of cold PBS. Surviving host WBCs, a source of human DNA (hDNA) contamination, were removed using magnetic beads coated with antibodies to the ubiquitous WBC surface marker CD45 . Dynabeads were prepared as per manufacturers’ instruction and 100ul bead suspension added to the 1.2mL pellet suspension followed by incubation at 4°C with rotation for 30 minutes. WBC’s bound to the beads were removed by placing in a magnetic field for two minutes.As highlighted in the findings . We havequencing . To overg for 20 min to recover the parasite enriched pellet (PEP). The PEP was suspended in 200ul PBS for DNA extraction . Recovered DNA was eluted in 150ul of Buffer AE. Percent hDNA depletion and Plasmodium DNA recovery were determined using quantitative PCR (qPCR) . P. knowlesi DNA enriched samples (post hDNA depletion) from isolates with a starting parasitaemia of <40,000 per ul had low parasite DNA yield and sequence coverage.The human WBC depleted eluate was carefully removed and transferred to a fresh Eppendorf tube and centrifuged at 2,000 x R (qPCR) . P. knowTwenty-one (21) samples from 15 different patients, median parasitaemia 193,600 parasites/ul  with >90% hDNA depletion were taken forward to PCR-free rapid barcoding library preparation . SQK-RBK004 library preparation is suitable for small yield DNA samples in the region of 400ng and includes a tagmentation step that generates read lengths normally distributed around a mean length of 4,500 kb. Of 21 library preparations 13 (62%) had >10x genome sequence coverage and six of these >30x coverage. Coverage of 100x was achieved especially when >1 sequencing library was prepared per isolate.Plasmodium genome sequence data from small clinical samples from malaria patients. Samples that are archived or collected prospectively can be sequenced in a cost-effective and time-efficient manner anywhere. The importance of this capability is the opportunity to move forward from a necessary dependence on Plasmodium genome sequence generated from experimental lines to genome sequence generated from clinical samples with matched clinical data. The methodology we describe is particularly applicable to P. knowlesi infections that tend to be single genotype and reach relatively high parasitaemia. The methods can be applied to, albeit, relatively uncommon single genotype P. falciparum infections. Although multiple genotype infections present a limiting factor to the methods described here, the potential to generate valuable genome-wide information on even a small number of clinical isolates to inform studies on P. falciparum virulence should not be overlooked.For the first time it is possible to generate long-read pGWAS on long read sequence data from clinical isolates with matched high quality continuous clinical and laboratory data, relative to particular clinical manifestations of severe malaria, will help unravel the contribution of parasite diversity to virulence. In addition to accessibility and field application of low-cost real-time sequencing in-house, Nanopore MinION long-read sequencing can resolve important multiple gene family members, including the P. knowlesi kirs and SICAvars . Genome sequencing was supported by Tenovus Scotland (T16/03).DRO is supported by the Wellcome Trust ISSF award 204821/Z/16/Z. Bioinformatics and computational biology analyses were supported by the University of St Andrews Bioinformatics Unit (AMD3BIOINF), funded by Wellcome Trust ISSF award 105621/Z/14/Z. The sample BioBank was compiled with informed consent (Medial Research Council, 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.


As in most eliminating countries, malaria transmission is highly focal in Haiti. More granular information, including identifying asymptomatic infections, is needed to inform programmatic efforts, monitor intervention effectiveness, and identify remaining foci. Easy access group (EAG) surveys can supplement routine surveillance with more granular information on malaria in a programmatically tractable way. This study assessed how and which type of venue for EAG surveys can improve understanding malaria epidemiology in two regions with different transmission profiles.Plasmodium falciparum by a highly sensitive rapid diagnostic test (hsRDT). Factors associated with hsRDT positivity were assessed by negative binomial random-effects regression models.EAG surveys were conducted within the departments of Artibonite and Grand’Anse (Haiti), in regions with different levels of transmission intensity. Surveys were conducted in three venue types: primary schools, health facilities, and churches. The sampling approach varied accordingly. Individuals present at the venues at the time of the survey were eligible whether they presented malaria symptoms or not. The participants completed a questionnaire and were tested for Overall, 11,029 individuals were sampled across 39 venues in Artibonite and 41 in Grand’Anse. The targeted sample size per venue type (2100 in Artibonite and 2500 in Grand’Anse) was reached except for the churches in Artibonite, where some attendees left the venue before they could be approached or enrolled. Refusal rate and drop-out rate were < 1%. In total, 50/6003 (0.8%) and 355/5026 (7.1%) sampled individuals were hsRDT positive in Artibonite and Grand’Anse, respectively. Over half of all infections in both regions were identified at health facilities. Being male and having a current or reported fever in the previous 2 weeks were consistently identified with increased odds of being hsRDT positive.Surveys in churches were problematic because of logistical and recruitment issues. However, EAG surveys in health facilities and primary schools provided granular information about malaria burden within two departments in Haiti. The EAG surveys were able to identify residual foci of transmission that were missed by recent national surveys. Non-care seeking and/or asymptomatic malaria infections can be identified in this alternative surveillance tool, facilitating data-driven decision-making for improved targeting of interventions. Plasmodium falciparum, and the main malaria vector is Anopheles albimanus, which has a tendency to bite and rest outdoors [P. falciparum, which is still used as a first-line malaria treatment, and the absence of pyrethroid resistance in An. albimanus [Haiti and the Dominican Republic are committed to eliminating malaria –3. The ioutdoors –7. Houseoutdoors , 8. Elimlbimanus –14.As in most eliminating countries, malaria transmission is highly focal in Haiti , 16. In A promising alternative for malaria control programs to supplement passive surveillance is to conduct surveys of populations within easily accessible venues, commonly referred to as easy access group (EAG) surveys –25. LogiThe aim of this study was to assess if, and if so, how and which type of EAG surveys can improve understanding malaria epidemiology, identify residual foci in two regions with different transmission profiles, and inform programmatic decision-making and intervention targeting within Haiti. Different types of EAG venues including churches, primary schools, and health facilities were investigated.www.openstreetmap.org)—a category 5 hurricane (Matthew) hit Grand’Anse in October 2016 and severely impacted the habitat. The two contrasted regions were purposively selected in collaboration with the National Malaria Control Program (PNCM) due to the relatively higher malaria incidence in these communes compared to the rest of Haiti; in 2016, national passive surveillance suggested that the annual incidence rate was 3 per 1000 in the selected Artibonite communes and 27 per 1000 in the Grand’Anse communes. Mean malaria incidence across Haiti is < 2 per 1000. Both regions are characterized by mountainous terrain with intersecting rivers and valleys, with Artibonite being landlocked and Grand’Anse along the Caribbean Sea coast. They encompass rural, semi-rural, and urban populations—the few largest towns in the study region all have < 20,000 inhabitants.This study was conducted in 2017 in rural Haiti. Two sets of EAG surveys were performed. The first took place in May/June in the Artibonite Valley of central Haiti, with the second in October/November in the Grand’Anse department, south-western Haiti  to provide a sampling frame. Information collected included the Global Positioning System (GPS) coordinates of the school, number of registered pupils for the current school year, and contact details. A sample of 21 (Artibonite) or 25 (Grand’Anse) schools with at least 100 enrolled pupils was selected in each region using a stratified random sampling procedure to ensure equal distribution across sub-communes and by remoteness (defined as close or far from main roads according to Euclidean distance). At each school, a maximum of 25 pupils per grade, in grades 2 to 6, were selected to ensure a broad age range, with a maximum of 150 pupils included per school [A census of all primary schools, including public, private, and faith-based institutions, was conducted  was sought from children above age 6 years after having the study and procedures explained. Children who refused to participate were replaced by another child from the same grade.A list of all functioning  public and private health facilities in each study region was obtained from the Ministry of Public Health and Population and checked for completeness and accuracy. All functioning health facilities in the study regions  were included in the survey. All individuals attending the facility during the study period, as well as anyone accompanying them , were eligible for inclusion. Those under 6 months of age, having had previously visited the facility during the study period, attending a scheduled treatment clinic , or requiring urgent care were excluded. At each facility, a maximum of 150 people from each of three age categories  were targeted, following the protocols described in Stresman et al. . Informen = 83). The GPS coordinates, denomination, estimated number of regular attendees, and contact details were collected. Nine churches were selected from the list generated as part of the convenience census using a stratified random sample by sub-commune and denomination. This approach was employed due to logistical constraints: sampling was limited to weekends, with Adventists congregating on Saturdays and Catholics and Protestants on Sundays. All those aged over 6 months who attended the service were eligible for inclusion. There was no limitation on the maximum number of participants included per church. Informed consent and assent procedures followed those described above for health facilities. Churches were no longer included in the study in Grand’Anse due to the logistical challenges in obtaining a sampling frame and the large number of people to sample in a short period.Except for Catholic churches, no comprehensive list of churches was available, so a convenience census was conducted by visiting the area with a local guide and by asking community members to target those likely to have at least 100 regular attendees  on an Android tablet . Questionnaire data were automatically pushed to a secure cloud-based server using the local mobile phone network. Questions included age, sex, how they traveled to the venue, history of fever in the past 2 weeks, treatment-seeking behaviors, travel history, and any vector control methods used in the home. Malaria risk associated with travel was ascertained using the self-reported communal section  of their destination. The malaria burden associated with the reported travel destination was described as high (any cases reported) or low (no cases reported), according to the number of cases reported by the Ministry of Health surveillance system ). Burden within the study site was dichotomized into high and low burden communal sections and was grouped according to highly sensitive RDT (hsRDT) prevalence being greater or lesser than 10%. Reported bednet ownership and use the night before the survey were also collected. Current fever was defined as those with an axillary temperature greater than 37.5 °C. The questionnaire for school children was simplified to ensure it was age appropriate.P. falciparum parasite infection. Malaria parasite infections were detected using a conventional histidine-rich protein 2 (HRP2)-based rapid diagnostic test  and a hsRDT . Individuals found to be positive by the cRDT were provided treatment according to the national guidelines in Haiti, which combines a 3-day course of chloroquine and a single low dose of primaquine. The hsRDT results were the primary outcome of this study, but did not inform treatment as the hsRDTs are only approved for investigational use in Haiti. The intention of performing both tests was to contribute to a multi-site study that aimed to assess the hsRDT performance compared to the cRDT—results have been published elsewhere [All participants were asked to provide a finger-prick capillary blood sample to test for a lsewhere . TreatmeIn schools, children with positive cRDT were traced to their home at the end of the day, where a member of the study team provided the drugs to the parents or caretaker and advised them on how to administer the course to the child. In schools and health facilities, all participants testing positive by either type of RDT and a random selection of 30% of those testing negative by both RDTs were traced to their household. At the household, spatial coordinates were obtained with a handheld GPS device . In primary schools, household location was obtained using a wearable GPS tracker for an additional randomly selected subset of 30% of individuals, irrespective of RDT status or whether they were also selected for GPS tracking, where the spatial location of the household was extracted based on the nighttime location of the participants.All statistical analyses were conducted in open-source R statistical software V3.5.2 . Malaria foci were identified by visual inspection of the hsRDT malaria prevalence by venue and the number of hsRDT positives per household within the subset of individuals for whom coordinates were available. Active foci were therefore conceptualized as circumscribed areas—within a malarious region—that sustain malaria transmission, as per WHO’s definition . Mixed eThe procedures for both study regions were approved by the National Bioethics Committee in Haiti (1516-30), the London School of Hygiene & Tropical Medicine Ethics Committee (103939), and the Tulane Institutional Review Board (795709). All participants provided informed written consent and/or assent, with parental consent for the school surveys using an opt-out process (described above) approved by all ethics committees. Participation in the study was not remunerated.p < 0.0001).Overall, 11,029 individuals were sampled across the two study regions, with 6003 and 5026 participants across 39 and 41 venues in Artibonite and Grand’Anse, respectively  as compared to Artibonite . The majority of infections in both regions were detected in health facilities  with only six hsRDT positives out of 1769 individuals identified in churches in Artibonite. The hsRDT prevalence ranged between and across venues with the highest hsRDT prevalence in Artibonite detected at a health facility (9.5%) whereas in Grand’Anse, the highest burden was observed at a school (44.8%)  and 1.08 km (IQR 0.47–1.78 km) to go to school in Artibonite. In contrast, for the Grand’Anse region, the mean distance traveled to the venue was 1.26 km (IQR 0.68–1.71 km) for health facilities and 1.12 km (IQR 0.33–1.13 km) for schools. The mean distance traveled to the venue of those individuals testing positive for malaria by hsRDT was over 3 km and 1 km in Artibonite and Grand’Anse, respectively  of hsRDT-positive individuals were over 15 years of age. In contrast, 181/355 infections were detected in the 5- to 15-year-old age category in Grand’Anse (Table 9/50 of hn = 29), Saint-Marc , or Verrettes  with only eight participants visiting an area with high malaria risk. Similarly, in Grand’Anse, the vast majority of the participants recruited in health facilities did not report recent travel, both among those with a positive hsRDT (96.3%) and with a negative hsRDT (93.3%). The most common travel destinations of those residing in Grand’Anse included Port-au-Prince (n = 27), Jeremie , or Anse-d’Hainault , with 31 visiting an area with high malaria risk. Both in Artibonite and in Grand’Anse, the two most common reasons for travel were to visit friends and family  and to engage in business .The majority of the participants recruited at health facilities in Artibonite did not report recent travel, both among those testing positive by hsRDT (95.7%) and those testing negative (95.5%). Of those residing in Artibonite, the most common travel destinations were Port-au-Prince  and those with current or recent fever  had significantly increased odds of being hsRDT positive, but there were no clear associations between sampling venue or commune of residence and hsRDT positivity in Artibonite  the acceptability and cost effectiveness of EAGs surveys in low malaria transmission settings.In Artibonite, the only focus that was identified had been missed by the previous national surveys, which is unsurprising because they are not designed to detect confined foci in countries with low transmission. However, this particular focus was also missed by the passive surveillance system, which is unexpected since some cases were symptomatic and sought treatment at the nearest health facility. This illustrates the advantage of obtaining household-level spatial information as an add-on to venue-based sampling; it enabled sub-catchment identification of malaria foci. This is particularly relevant in the context of rural Haiti, where there are no residential addresses and where localities are vaguely defined—a reason why locating cases identified by passive surveillance is difficult without further investigation. The distance participants traveled to the venue suggests that assuming the venue reflects the surrounding area is not always true, and defining the venue catchment areas is important to accurately assign the spatial risk of transmission. Although the travel time question was less precise than physically tracing the household, the conclusions were consistent in suggesting the catchment was not restricted to the immediate area and provides a programmatic alternative if geolocating participants is not feasible. Importantly, the inclusion of spatial information identified sub-catchment malaria foci that would benefit from targeted interventions, confirming that the primary risk factor for being infected in this area is related to the location where participants lived. This finding is consistent with the notion of ongoing transmission in both study regions.In this study, overall parasite infection prevalence (measured by hsRDT) was 0.8% in Artibonite, compared to 7.0% in Grand’Anse. The performance of the new hsRDT was only slightly better than that of cRDT  [While this study was not designed to assess malaria risk factors, it is noteworthy that there were very few variables associated in the models with greater odds of having a positive hsRDT among those included in the EAG surveys. Residing in a high burden commune was significantly associated with a higher odds ratio, but only for children recruited in schools in Grand’Anse. Arguably, this association could have been blurred in Artibonite, where the overall prevalence was lower and half of the cases were identified in a hotspot. These results suggest that as transmission decreases, malaria indicators at the smallest administrative unit commonly used for programs in Haiti  could become less and less precise due to higher spatial heterogeneity. The presence of a current or recent fever was the only variable that was significantly associated with a higher risk of a malaria parasite infection in all populations studied. This association is not well-known by the Haitian population, as recently highlighted in a qualitative study . Fever iites/μl) . New diaContrary to many low-transmission settings, history of travel was not associated with increased odds of malaria infection in this study, which is consistent with the fact that travel concerned mainly trips to low-transmission areas . While hWith the exception of churches, the surveys were easy to implement in EAGs. Preparatory work required a census of all health facilities and schools in the study area, with their GPS coordinates. This information was already available and was obtained thanks to the relevant governmental authorities. Therefore, contrary to household surveys, it was not necessary to perform a population census, nor to recruit participants by walking house-to-house. On the other hand, further actions were required to obtain spatial information of the participants’ residences. While several methods were tested in this study to collect this information, the most practical and easy way is likely to track the participants to their homes. If the EAG approach were to be used for programmatic purposes , it would imply tracking only participants with a positive RDT. In this study, community health workers or community members were easily trained and involved for this purpose. Finally, while several information procedures had to be followed in schools to recruit children without their parent’s direct consent, this requirement would not apply in a non-research context . Surveys can also be repeated in the same panel of EAGs for active surveillance or for impact evaluation of a specific intervention. The lapse that would be required between the different rounds of surveys will vary depending on several factors, notably the local malaria epidemiology and seasonality, the rationale of the study, and the characteristics of the intervention.This study had some important limitations. By design, the samples obtained in these venues are not representative of the underlying population. Therefore, results cannot be directly extrapolated to the general population. However, refusal rates in these studies were < 1% and studies in other settings have highlighted that EAG studies, while biased, are able to provide a reasonable approximation of community-level malaria prevalence/burden and provide sufficient information to inform program activities . IdeallyWe conducted surveys to estimate malaria prevalence in three different EAG populations in two regions of Haiti with different underlying malaria transmission intensity. Spatial and demographic variations in hsRDT prevalence were observed within and between each setting, offering increased spatial granularity of malaria transmission compared to routine confirmed malaria cases reported at the facility level and national surveys. In low-transmission settings like Haiti, EAG surveys provide a convenient alternative for targeted surveillance to identify non-care seeking and/or asymptomatic malaria infections below the health facility population catchment level. The increased granularity of information can then be used for data-driven decision-making and more tailored responses for reducing transmission and reaching the ultimate goal of malaria elimination. By identifying a malaria focus at an elevation of 650 m, this study notably found that malaria transmission was possible in areas usually not targeted by programmatic or modeling efforts.Additional file 1. Summary Euclidean straight-line distance (meters) between household location and sampling venue for the subset of individuals with spatial coordinates available, according to hsRDT positivity by venue types  and study location .Additional file 2. Mode of travel and self-reported time it took to arrive at the venue on the day of the study.Additional file 3. Results comparison between cRDT and hsRDT, by region and type of venue.


Z-score , weight-for-age Z-score , height-for-age Z-score , or mid-upper arm circumference . There was no statistically significant evidence of a difference in the relationship in communities receiving azithromycin or placebo. During the low transmission season, there was no evidence that malaria parasitemia and impaired nutritional status co-occur in children.The relationship between malaria and malnutrition is complicated, and existence of one may predispose or exacerbate the other. We evaluated the relationship between malaria parasitemia and nutritional status in children living in communities participating in a cluster-randomized trial of biannual azithromycin compared with placebo for prevention of childhood mortality. Data were collected during the low malaria transmission and low food insecurity season. Parasitemia was not associated with weight-for-height  Conversely, some studies have shown a protective effect of malnutrition against malaria, which may be mediated by iron or other nutritional deficiencies.6 Given the complexity of any potential relationship, it is possible that the relationship may differ in different geographic or seasonal settings or in the presence of interventions that affect one or both conditions.In Niger, the high malaria transmission season overlaps with periods of high food insecurity and malnutrition.8 Although studies of mass azithromycin distribution for trachoma control have yielded mixed results,12 a study of biannual mass azithromycin distribution for child mortality demonstrated a significant decrease in malaria parasitemia in communities receiving azithromycin compared with those receiving placebo.13 Any relationship between malaria and malnutrition could potentially be affected by distribution of azithromycin to children given the overall effect in a reduction in malaria parasitemia.Azithromycin has modest activity against malaria by targeting the plasmodial apicoplast.Here, we investigated the relationship between malaria parasitemia and malnutrition in the same communities in which azithromycin was shown to decrease malaria parasitemia in Niger compared with biannual placebo to assess the relationship between parasitemia and nutritional status in the low transmission season and any effect of the azithromycin intervention on that relationship.clinicaltrials.gov NCT02048007).14 No mass azithromycin distributions for trachoma occurred within 5 years before the study, and the area was not receiving seasonal malaria chemoprevention at the time of the study. Thirty communities were randomly selected from the same pool as the main MORDOR study for more intensive monitoring and similarly randomized to biannual distributions of azithromycin or placebo. After the biannual census and before treatment, children were monitored with anthropometric assessments and blood smears for malaria parasitemia. Ethical approval was obtained from the Committee on Human Research at the University of California, San Francisco, and the Institutional Review Board of the Nigerien Ministry of Health.Data arose from the Macrolides Oraux pour Réduire les Décès avec un Oeil sur la Résistance (MORDOR) study, a cluster-randomized trial in which communities in Boboye and Loga, Niger, were randomized in a 1:1 fashion to either biannual azithromycin distribution or placebo to children aged 1–59 months  and thin and thick smears for malaria parasitemia. Malaria parasitemia was considered the presence of malaria parasites on thick smear. Laboratory personnel were masked to treatment arm. Anthropometric measurements, including weight, height, and mid-upper arm circumference (MUAC) were measured in all children. Children were weighed standing if able or in the arms of a caregiver , with heavy garments and jewelry removed before weight measurements. Recumbent length was measured in children who were not able to stand and standing height in children who could stand . Weight and height were measured in triplicate, and the median was used for analysis. Weight-for-height Z-scores (WHZ), HAZ, and weight-for-age Z-scores (WAZ) were calculated based on 2006 WHO standards.15 Wasting, underweight, and stunting were defined as WHZ, WAZ, and HAZ scores of < −2 SD, respectively.A random sample of 40 children aged 1–59 months per community were randomly selected from the census for monitoring. Children were monitored in March 2015, June 2016, and April 2017. The 12-month monitoring visit occurred after two rounds of study treatment and the 24-month visit occurred after four rounds of study treatment. Each child had a finger stick to estimate hemoglobin with HemoCue Hb 20116Children were offered treatment if they were between 1 and 59 months of age and weighed at least 3,800 g. A single directly observed oral dose of study drug (azithromycin 20 mg/kg or matching placebo) was administered to all eligible children approximately every 6 months. Children were offered treatment after all monitoring activities were completed. Dosing was calculated based on height-stick approximation for children who could stand or by weight for children who were unable to stand.We used mixed effects linear and logistic regression models to estimate the relationship between malaria parasitemia and nutritional status. Because of the cross-sectional nature of the data, the goal of this analysis was to estimate associations between parasitemia and nutritional status rather than estimate causal effects. Children who had valid anthropometric and parasitemia measurements were included in analyses. Because children aged 1–59 months were randomly sampled at each time point, we did not have longitudinal measurements for most children. Furthermore, because samples were collected in 1-year intervals, we felt that any relationship between malaria parasitemia and nutritional status measurements would be unlikely to be correlated over such a long time period. For these reasons, all analyses were cross-sectional analyses conducted at each sample collection time point . A separate model was built for each time point. Models were built predicting nutritional status indicators  as continuous variables and dichotomized as wasting (WHZ < −2 SD), stunting (HAZ < −2 SD), and underweight (WAZ < −2 SD). Models included fixed effects for malaria parasitemia status, the child’s age and gender, community treatment assignment, an interaction term between treatment assignment and malaria parasitemia, and a random effect for study community. All analyses were conducted in R version 3.5.3 .P = 0.0006) and lower WHZ and HAZ scores, although these were not statistically significant ; however, the CI was wide.In models adjusted for age, gender, and study arm, there was no association between malaria parasitemia and WHZ, HAZ, WAZ, or MUAC at months 0, 12, or 24 in either placebo or azithromycin communities . At 24 mWe were unable to find evidence of a relationship between malaria parasitemia and nutritional status in this population-based sample of children in Niger, and no evidence of a difference in the relationship in communities receiving or not receiving azithromycin after 12 or 24 months of biannual distributions. In the placebo communities, children with malaria parasitemia were more often stunted; however, the CIs were wide, and this finding could have been due to the chance given the number of comparisons in this analysis. Overall, the prevalence of malaria parasitemia was relatively low, likely because of the timing of data collection during the low transmission season. These data do not provide evidence that children with parasitemia are more likely to have worse nutritional indicators during the non-malaria and non-lean season in a region with highly seasonal malaria transmission.17 However, an effect on anthropometric outcomes has not been consistently shown in studies of mass azithromycin distribution for trachoma control.19 Azithromycin has some activity against malaria and has been shown in some studies of mass distribution for trachoma to affect parasitemia and splenomegaly.20 In the same study in which communities were measured at the same time points, azithromycin was effective at reducing the burden of malaria parasitemia. Azithromycin distribution could hypothetically affect the relationship between malaria parasitemia and nutritional status if it affected either condition individually. However, in this study, we saw no evidence of a relationship between parasitemia and nutritional status in either azithromycin or placebo communities.In some populations, antibiotics have been shown to cause weight gain in children.This study must be considered in the context of several limitations. First, this study included cross-sectional assessments of malaria parasitemia and nutritional status. Although a subset of children who were randomly selected for evaluation at multiple time points were followed longitudinally, the small sample size of this cohort precluded longitudinal analysis. Furthermore, samples were taken every 12 months. The presence of malaria parasitemia may not be expected to be associated with nutritional status 12 months later, and thus we did not attempt to conduct longitudinal analyses. We are, therefore, unable to comment on whether past infection or nutritional status influences current disease status. More granular measurements and larger sample sizes would be required for meaningful longitudinal analysis of this relationship. Given the highly seasonal nature of malaria and nutritional status in Niger, the results of this study are likely not generalizable to the malaria season or to settings without seasonal transmission.In conclusion, we were unable to find evidence supporting a relationship between malaria parasitemia and nutritional status in children living in communities participating in a large trial of mass azithromycin distribution for prevention of child mortality during the low malaria transmission season. Both malaria and undernutrition are major contributors to childhood mortality and morbidity in Niger. This study did not provide evidence that children with malaria parasitemia have worse nutritional status during the low transmission season compared with those without parasitemia, and interventions to address one during this season may not affect the other.