Periostin is a matricellular protein and is a useful marker in respiratory diseases. However, the roles of periostin in patients with obstructive sleep apnea (OSA) remain unclear. Several in vitro studies have suggested that mechanical stress, hypoxia, impaired metabolism, and kidney injury, which often accompany OSA, may upregulate the expression of periostin. Meanwhile, serum periostin level has been negatively associated with body mass index (BMI) in the general population. In this study, we hypothesized that a high level of serum periostin despite being overweight/obese may discriminate severe OSA or OSA with comorbidities from mild OSA with obesity alone. We aimed to clarify the roles of periostin in patients with OSA to assist in elucidating the heterogeneity of OSA with comorbidities.Among patients diagnosed as OSA, we examined the associations between serum periostin levels and clinical indices, including the severity of OSA, BMI, and comorbidities, using a multifaceted approach. The serum periostin levels and clinical indices were assessed after 3 months of continuous positive airway pressure (CPAP) treatment.n = 53), the level of serum-free fatty acids and the frequency of albuminuria were higher in patients with high serum periostin level of ≥87 ng/mL, which was the highest quintile among all participants, than in those with low serum periostin levels . In patients with severe OSA and high serum periostin levels, the levels of serum periostin and urinary albumin significantly decreased after 3 months of CPAP treatment.In 96 patients with OSA, serum periostin level was negatively correlated with BMI, albeit marginally, and tended to be higher in severe OSA than in others when adjusted for BMI. Cluster analysis identified four clusters, including two severe OSA clusters, one of which was characterized by high serum periostin levels and the presence of comorbidities, including albuminuria. In a comparative analysis of severe OSA cases (Elevated serum periostin in patients with OSA despite being overweight/obese may be an indicator of severe OSA with comorbidities, particularly albuminuria. Obstructive sleep apnea (OSA) is a major public health concern and has been to shown to increase in prevalence with obesity, which is its most important risk factor . OSA oftSerum periostin, which is an extracellular matrix protein, is a robust marker of T helper type 2 (Th2) inflammation, particularly in asthma –12. InteThese findings suggested that the serum periostin levels in patients with OSA may reflect a combination of its downregulation by obesity and upregulation by hypoxic stimulation or metabolic comorbidities. Therefore, we hypothesized that high serum periostin levels in patients with OSA, despite being overweight/obese, may discriminate severe OSA or OSA with comorbidities from mild OSA with obesity alone. To test this hypothesis, we aimed to clarify the associations between serum periostin levels and the sleep metrics of OSA and its comorbidities, including dyslipidemia or kidney injury, which have not been examined previously. This may assist in understanding the heterogeneity of OSA with comorbidities.The participants were recruited from a cohort of clinically stable patients aged between 20 and 80 years and who were diagnosed as OSA based on the findings of overnight polysomnography (PSG) at the Sleep Laboratory of Kyoto University Hospital between March 2014 and April 2015. Patients with dominant central sleep apnea, history of previous treatment for OSA, premenopausal status, hemodialysis or uncontrolled comorbidity, or ongoing systemic corticosteroid treatment were excluded, as described previously .This study was approved by the ethics committee of Kyoto University and was registered in the UMIN Clinical Trials Registry (Registry ID UMIN000012639). Written informed consent was obtained from all participants.90), high (≥3%) oxygen desaturation index (3% ODI), and high arousal index (AI) are described in the Supplementary Methods  and on the follow-up PSG 3 months after CPAP initiation. Patient history was taken at baseline. Physical examinations, blood tests, and urinalysis were performed at baseline and on follow-up PSG. Blood samples were collected between 06:30 and 07:00 after the completion of PSG. Sample analyses included blood glucose and serum levels of insulin, lipids, and periostin. Serum periostin levels were measured using an enzyme-linked immunosorbent assay at Shino-Test Corporation . Using a 12-h overnight urine sample, urinalysis and analysis of urine albumin and creatinine were performed . The deg90, 3% ODI, and AI; and the other clinical indices were evaluated. Changes in the measurements from baseline to after 3 months of CPAP therapy were examined.The associations between serum periostin levels and the severity of OSA; sleep metrics, including CT2 test, or Wilcoxon rank sum test, as appropriate. To determine correlations between continuous variables, the linear least squares method was used. For statistical analysis, the common logarithm of the serum periostin level was used. To assess the associations among serum periostin, severe OSA, and comorbidities, cluster analysis was performed. The detailed methods are described in the Supplementary Methods . Comparisons between two or more groups were performed using t-test, χOf 188 patients in total, 97 met the inclusion criteria but 1 withdrew from the original study and rejected the secondary use of specimens . Therefop < 0.05) but was negatively correlated with BMI, albeit marginally  and those with mild to moderate OSA (n = 43) (p = 0.28), but it tended to be higher in patients with severe OSA when adjusted by BMI (p = 0.07) . In addition, serum periostin level was not associated with the presence of major comorbidities, including COPD (p = 0.13), asthma (p = 0.32), and allergic rhinitis (p = 0.93)  and a higher prevalence of the comorbidities: DM (p < 0.01), albuminuria (p < 0.01), hypertension (p = 0.03), and asthma (p = 0.01).Subsequently, to comprehensively understand the associations among serum periostin levels, severe OSA, and comorbidities, cluster analysis was performed. This identified four clusters Table , includin = 41), the severe OSA with high periostin group (n = 12) exhibited significantly higher free fatty acid levels (p = 0.03) and prevalence of albuminuria (p = 0.04) (Table p = 0.07) and asthma (p = 0.06).The patients with severe OSA were divided into two groups, using a cutoff serum periostin level of 87 ng/mL, which was equivalent to the highest quintile of serum periostin among all participants. Compared with the severe OSA alone group n = 1, the sep < 0.01, Fig. Finally, among the entire study population, patients with severe OSA or high 3% ODI exhibited positive interactions with albuminuria for serum periostin levels (p = 0.04). Significant decrease in the serum periostin level after CPAP treatment was seen in the severe OSA with high periostin group (p = 0.04) but not in the severe OSA alone group (p = 0.38). The serum periostin level did not decrease in subjects with moderate OSA . Second, the presence of hypertension or dyslipidemia was defined according to the intake of antihypertensive or lipid-lowering medications. However, even if based on the diagnosis on self-reported disease (Table p = 0.03). Furthermore, the findings of the present study indicated that the presence of OSA in obese asthmatics should be suspected when the serum periostin level is elevated disproportionally to their BMI. Because OSA is a well-known comorbidity in obese patients with asthma [The present study had several limitations. First, besides its retrospective nature, this study did not discuss certain severe comorbidities of OSA, such as arrhythmia and heart failure. This was due to an insufficient number of subjects with these comorbidities. Similarly, this study included few subjects with COPD, which is an important confounder of the severity of OSA and hypoxia . Howeverse Table , we founse Table . Even afh asthma  and conth asthma , 41, serh asthma .In conclusion, patients with severe OSA were clustered into two groups, severe OSA with and without comorbidities. In addition, high serum periostin in patients with OSA, despite being overweight/obese, may be an indicator of severe OSA with comorbidities, particularly albuminuria.Additional file 1: Supplementary methods. Definitions of the severity of obstructive sleep apnea (OSA) and of high/low for the other sleep parameters. The definitions severity of OSA and high/low sleep parameters in the current study. Cluster analysis. The detailed description of cluster analysis. Definitions of comorbidities. The detailed definition of the presence of major comorbidities in the current study. Supplementary results. Characteristics of four clusters obtained according to cluster analysis. Cluster 1 was characterized mainly by younger age, male population, dyslipidemia, and high BMI but low prevalence of severe OSA. Among the four clusters, the patients in this cluster had the lowest serum periostin levels. Cluster 2 composed mainly of subjects with mild to moderate OSA and those with abnormal glycometabolism. Clusters 3 and 4 were characterized by the accumulation of severe OSA cases. Albuminuria was present in eight subjects in cluster 4 and in no subject in cluster 3. Of the eight subjects, six were positive for microalbuminuria (four with DM) and two exhibited macroalbuminuria (one with DM). Compared with cluster 3, cluster 4 had higher mean ± SD serum periostin level ; higher number of subjects with high serum periostin levels of ≥87 ng/mL, which was the highest quintile of serum periostin (p = 0.02); and higher prevalence of asthma. Figure S1. Serum periostin levels in patients with/without comorbidities. The presence of major comorbidities was not associated with serum periostin levels. The black bars and the numbers in the figure represent the mean serum periostin level. Figure S2. Distribution of the four clusters on the axes of apnea–hypopnea index and serum periostin levels. The details of each cluster are described in Table Figure S3. Serum periostin level in OSA. Overweight/obesity has negative impacts on the serum periostin level in OSA, as observed in the general population. However, high intermittent hypoxia contributes to high serum periostin level. Furthermore, severe OSA and comorbid albuminuria show positive interactions for serum periostin level. A high serum periostin in patients with OSA despite being overweight/obese may indicate a phenotype of OSA . Table S1. Clinical effects of CPAP in patients with moderate OSA. The serum periostin level did not decrease in subjects with moderate OSA after CPAP treatment (p = 0.75).


Iron deficiency (ID) is a major public health burden in African children and accurate prevalence estimates are important for effective nutritional interventions. However, ID may be incorrectly estimated in Africa because most measures of iron status are altered by inflammation and infections such as malaria. Through the current study, we have assessed different approaches to the prediction of iron status and estimated the burden of ID in African children.We assayed iron and inflammatory biomarkers in 4853 children aged 0–8 years from Kenya, Uganda, Burkina Faso, South Africa, and The Gambia. We described iron status and its relationship with age, sex, inflammation, and malaria parasitemia. We defined ID using the WHO guideline (ferritin < 12 μg/L or < 30 μg/L in the presence of inflammation in children < 5 years old or < 15 μg/L in children ≥ 5 years old). We compared this with a recently proposed gold standard, which uses regression-correction for ferritin levels based on the relationship between ferritin levels, inflammatory markers, and malaria. We further investigated the utility of other iron biomarkers in predicting ID using the inflammation and malaria regression-corrected estimate as a gold standard.The prevalence of ID was highest at 1 year of age and in male infants. Inflammation and malaria parasitemia were associated with all iron biomarkers, although transferrin saturation was least affected. Overall prevalence of WHO-defined ID was 34% compared to 52% using the inflammation and malaria regression-corrected estimate. This unidentified burden of ID increased with age and was highest in countries with high prevalence of inflammation and malaria, where up to a quarter of iron-deficient children were misclassified as iron replete. Transferrin saturation < 11% most closely predicted the prevalence of ID according to the regression-correction gold standard.The prevalence of ID is underestimated in African children when defined using the WHO guidelines, especially in malaria-endemic populations, and the use of transferrin saturation may provide a more accurate approach. Further research is needed to identify the most accurate measures for determining the prevalence of ID in sub-Saharan Africa.The online version of this article (10.1186/s12916-020-1502-7) contains supplementary material, which is available to authorized users. Iron deficiency (ID) is the commonest nutrient deficiency affecting over 2 billion people worldwide and is a major public health burden in African children , 2. ID hReliable and accurate estimation of the prevalence of ID is essential in planning, monitoring, and targeting effective interventions. The gold standard method for estimating iron status is to stain bone marrow aspirate for iron, but this is invasive and impractical in population surveys . To defiA range of alternative markers of iron status have been proposed to determine iron status, but these also have limitations. Soluble transferrin receptor (sTfR) is only mildly increased during the inflammatory response , but itsIn the current study, we have measured a wide range of iron markers in 4853 African children and described their relationship with age, sex, underweight, inflammation, and malaria parasitemia. We then utilized the regression-correction approach proposed by BRINDA , 16 to pThis study included community-based cohorts from Kenya, Uganda, Burkina Faso, South Africa, and The Gambia.Participants were members of an ongoing rolling cohort evaluating malaria immunity in children as described elsewhere . Within ISRCTN32849447) to determine whether anthelminthic treatment during pregnancy and early childhood was associated with differential response to vaccination or incidence of infections such as pneumonia, diarrhea, or malaria [The Entebbe Mother and Baby Study is a prospective birth cohort study that was originally designed as a randomized, double-blind, placebo-controlled trial  [Infants born in Chris Hani Baragwanath Hospital living in Soweto, a non-malaria-endemic region in Johannesburg, South Africa, were recruited from vaccine trials coordinated by the Respiratory and Meningeal Pathogens Unit (ru.com/) . This stAll children aged 2 to 6 years and living in the West Kiang region of The Gambia were recruited during the malaria season to assess the effects of genetic variants on hemoglobin concentrations and iron status . Iron bi1-antichymotrypsin (ACT)) are shown in Additional file Plasmodium falciparum and other Plasmodium species were examined using Giemsa-stained thick and thin blood smears. All assays are based on single measurements per child.The assayed biomarkers of iron , zinc protoporphyrin (ZPP), and hemoglobin) and inflammation . Iron biomarkers  were ln-transformed to normalize their distributions. Differences in biomarker means between age groups were tested using two-tailed Student’s β1), and for the crude association between malaria and ferritin (β2), and multivariable linear regression was applied to estimate adjusted regression coefficients for associations between inflammatory marker level and ferritin (β3) and between malaria parasitemia and ferritin (β4). In the third step, the regression coefficients estimated in step 2 were used to calculate adjusted ferritin values using Eq.  using the same thresholds that were applied to the uncorrected ferritin levels in the WHO recommendations  and comA total of 4853 children, 1484 Kenyan, 1374 Ugandan, 348 Burkinabe, 894 South African, and 753 Gambian, aged between birth and 8 years were included in this study. Table Table Concentrations of ferritin, hepcidin, BIS, and TSAT decreased during the first year of life and increased thereafter, indicating that ID is most prevalent at about 1 year of age  as a gold standard to test the diagnostic utility of various markers of iron status Fig. . TSAT ouThrough this study, we have described iron status and estimated the prevalence of ID in more than 4800 children living across the African continent using a wide range of iron biomarkers. Among infants, ID increased from birth to approximately 1 year, and then decreased with increasing age. Males were generally more iron deficient than females up to 3 years of age. Underweight children had increased ID and erythropoietic drive. Inflammation and malaria parasitemia were associated with independent and substantial increases in ferritin concentrations and were also associated with other biomarkers of iron status. We found that WHO-defined ID underestimated the burden of ID in African children compared to regression correction, which predicts ferritin levels in the absence of inflammation and malaria. Of the other iron markers that we tested, TSAT had the best diagnostic properties when compared against the gold standard of regression correction.Age, sex, and being underweight were associated with iron status in young African children. We found that iron stores decreased rapidly during infancy and reached a nadir at around 1 year of age, supporting the argument that newborns have higher iron stores that are prenatally accumulated but decrease with demand for iron during rapid growth and low iron supply from breast milk and complementary foods . Iron stInflammation and malaria parasitemia substantially altered measures of iron status in agreement with previous studies , 38, 39.The underestimate of prevalence of ID by the WHO was largest in cohorts that had a high burden of malaria and other infections, for example, 27.0% and 21.4% of Burkinabe and Kenyan children, respectively, were misclassified as iron replete compared to 8.5% of South African children. The unidentified burden of ID increased with age in line with increasing prevalence of malaria parasitemia, and we observed higher ferritin levels among children with malaria parasitemia at every decile of CRP. Malaria parasitemia also increased ferritin levels independently of inflammation in multivariable analyses. In agreement, a study in Burkinabe children found that adjustment for asymptomatic malaria, in addition to inflammation, led to a 11.9% absolute increase in the prevalence of ID . Taken tWe then evaluated the diagnostic utility of uncorrected iron biomarkers in predicting ID regression-corrected for inflammation and malaria. A TSAT < 11% best predicted regression-corrected ID indicating its potential usefulness in estimating the prevalence of ID in our study populations. TSAT is calculated from measured serum iron and either transferrin or UIBC, all of which have standard assays that are easy and inexpensive to perform. TSAT< 11% performed well over a range of CRP concentrations, in children with both malaria and inflammation, and across populations. In support of our findings, Aguilar et al. showed that TSAT had a high sensitivity (81%) in predicting bone marrow ID in 180 anemic (hemoglobin < 11 g/dL) children in Mozambique although specificity was low (40%) and an optimal cut-off was not derived . In contThere were a number of important limitations of our study. The cross-sectional nature of our data limited us from analyzing longitudinal effects of inflammation, malaria, and nutritional status on iron status. Moreover, we did not measure α-1-acid glycoprotein (AGP), which has been shown to be a better marker for adjusting for inflammation in regression-correction analyses . NeverthIn this large-scale study including more than 4800 children in five countries across Africa, we explored a wide range of iron biomarkers to more accurately estimate prevalence of ID in countries with a high burden of childhood infections including malaria. There has been a long-standing concern regarding the challenge of using iron biomarkers to accurately estimate prevalence of ID in African populations [Additional file 1:Table S1. Laboratory assays for iron and inflammatory biomarkers by study site.Additional file 2:Table S2. Associations between age, sex, inflammation, malaria, and nutritional status.Additional file 3:Table S3. Regression-corrected and uncorrected prevalence of iron deficiency defined by additional iron biomarkers by study site.Additional file 4:Figure S1. Multivariable regression models of the predictors of iron biomarkers with additional adjustment for underweight. Error bars indicate 95% confidence intervals and values indicate effect size (95% CI). Inflammation was defined as C-reactive protein > 5mg/L or α1-antichymotrypsin > 0.6g/dL (in The Gambia). Malaria was defined as P. falciparum parasitemia. BIS, body iron stores; sTfR: soluble transferrin receptor; TSAT, transferrin saturation; ZPP, zinc protoporphyrin (measured in The Gambia only). Underweight data were missing in South Africa.Additional file 5:Figure S2. Comparison of prevalence of iron deficiency after additional regression-correction for age, sex and underweight. Graph letter ‘a’ indicates prevalence using WHO definition, ‘b’ correcting for malaria only, ‘c’ correcting for inflammation only, ‘d’ correcting for both malaria and inflammation, and ‘e’ correcting for malaria, inflammation, age, sex, and underweight. Error bars indicate 95% confidence intervals. No malaria in South Africa and anthropometry data were unavailable.Additional file 6:Figure S3. Relationship between estimated prevalence of iron deficiency and inflammation / malaria. The graph shows the prevalence of iron deficiency in children with and without inflammation and / or malaria. Error bars indicate 95% confidence intervals. Inflammation was defined as C-reactive protein > 5mg/L or α1-antichymotrypsin > 0.6g/dL (in The Gambia). Low ferritin was defined as ferritin <12 μg/L in children <5 years or <15 μg/L in children ≥5 years. WHO adjustment involved using a higher cut-off (30 μg/L) of ferritin for children with inflammation according to WHO-defined ID. Malaria was defined as P. falciparum parasitemia. Regression-correction uses ferritin levels corrected for the effects of inflammation and malaria in defining ID. TSAT, transferrin saturation.Additional file 7:Figure S4. Meta-analysis of optimal cut-offs of transferrin saturation in predicting iron deficiency regression-corrected for inflammation and malaria. Corrected iron deficiency was defined using the WHO definition with ferritin levels that were adjusted for malaria and inflammation using regression-correction. ES, effect size.


Although WHO recommends cotrimoxazole (CTX) discontinuation among HIV patients who have undergone immune recovery and are living in areas of low prevalence of malaria, some countries including Uganda recommend CTX discontinuation despite having a high malaria burden. We estimated the prevalence and factors associated with malaria parasitaemia among adults living with HIV attending hospital outpatient clinic before and after discontinuation of CTX prophylaxis.Between March and April 2019, 599 participants aged 18 years and above, and attending Kitgum hospital HIV clinic in Uganda were enrolled in a cross study. A standardized questionnaire was administered and physical examination conducted. A finger-prick blood sample was collected for identification of malaria parasites by microscopy. The prevalence of parasitaemia was estimated and compared among participants on and those who had discontinued CTX prophylaxis, and factors associated with malaria parasitaemia assessed.3) was also associated with increased risk of having parasites .Of the enrolled participants, 27 (4.5%) had malaria parasites and 452 (75.5%) had stopped CTX prophylaxis. Prevalence of malaria parasitaemia was significantly higher in participants who had stopped CTX prophylaxis (5.5% versus 1.4% p = 0.03) and increased with increasing duration since the discontinuation of prophylaxis. Compared to participants taking CTX, those who discontinued prophylaxis for 3–5 months and >5 months were more likely to have malaria parasites  = 1.64, 95% CI 0.37–7.29, p = 0.51, and aPR = 6.06, 95% CI 1.34–27.3, P = 0.02). Low CD4 count (< 250cells/mmPeople from malaria endemic settings living with HIV have a higher prevalence of malaria parasitaemia following discontinuation of CTX compared to those still on prophylaxis. The risk increased with increasing duration since discontinuation of the prophylaxis. HIV patients should not discontinue CTX prophylaxis in areas of Uganda where the burden of malaria remains high. Other proven malaria control interventions may also be encouraged in HIV patients following discontinuation of CTX prophylaxis. Malaria and HIV are among the major health problems affecting developing countries , 2. TogeIn 2001, the WHO recommended cotrimoxazole (CTX) as part of the minimum package of care for PLHIV in Africa . This reOne trial in Uganda assessed the safety of discontinuing CTX in stable patients on ART and found that patients were at increased risk of malaria , howeverA cross-sectional study was conducted between March and April 2019. The study was carried out at the HIV outpatient clinic of Kitgum Hospital, the largest public health facility in Kitgum district that offers health care services free of charge to its clients with support from the government of Uganda. The HIV clinic at the hospital receives patients from across the Acholi sub-region  and some from South Sudan. During the course of the study, the HIV clinic was open four days a week (Monday to Thursday) from 8am to 5pm, close to 50 patients were seen per clinic day, and had about 3,000 people living with HIV registered.According to the 2018/2019 Uganda malaria indicator survey, the prevalence of microscopic parasitaemia in children under five years in Kitgum district is estimated at 12% . Other iWe consecutively enrolled 599 people living with HIV who attended the Kitgum hospital HIV clinic. Participants were included in the study if they: 1) visited the clinic during the study period, 2) were aged 18 years and above, 3) were either on or off CTX for at least 3 months, 4) provided written informed consent. Participants were excluded if they declined to have a finger prick blood sample to be collected. A pre-tested questionnaire was administered by study staff to all enrolled participants including data on participant socio-demographics  and clinical characteristics . Data on clinical characteristics was abstracted from the patient’s files at the time of the interview. The questionnaire was pretested among the first 30 participants of the study. The results of the participants among whom the pre-test was done were not included in the analysis. A finger prick blood smear was collected onto a frosted slide which was labelled with a participant’s identification number. All smears were air dried, and stored in slide boxes, and transferred to the molecular research laboratory of the Infectious Diseases Research Collaboration for staining and reading.Thick blood smears were stained with 2% Giemsa for 30 minutes and evaluated for the presence malaria parasites. Parasite densities were calculated by counting the number of malaria parasites per 200 leukocytes , assuming a leukocyte count of 8,000/μl. A thick blood smear was considered negative if examination of 100 high power fields revealed no malaria parasites. For quality control, all slides were read by a second microscopist and a third microscopist settled any discrepant readings .The study was designed to test the hypothesis that participants who had discontinued CTX prophylaxis for at least three months would have a higher prevalence of malaria parasitaemia than those on prophylaxis. Using the sample size of two proportions and setting the prevalence of parasitaemia in the CTX prophylaxis group at 4% and that in the group that had discontinued prophylaxis at 13.2% with the estimated proportions of participants being 53% and 47% on CTX and off CTX respectively . We needAll data were collected using standardized questionnaires and double-entered using Microsoft Access . Analyses were performed using Stata, version 14 . Descriptive characteristics included proportions for categorical variables, and mean (SD) or median (IQR) values for continuous variables. The prevalence of malaria parasitaemia was defined as the number of individuals with a positive malaria smear divided by the total number of participants tested, and was stratified by whether they were on prophylaxis, had discontinued prophylaxis between 3–5 months or had discontinued prophylaxis for more than 5 months. The prevalence of parasitaemia among those on CTX prophylaxis was compared to the prevalence among those who had discontinued CTX prophylaxis using Pearson’s chi-square test. To test for difference in parasite density between groups , Wilcoxon rank-sum (Mann-Whitney) test was used. Univariable and multivariate associations between malaria infection and potential associated factors were assessed using robust (modified) Poisson regression. Measures of association were reported as prevalence ratios with their 95% confidence intervals (CI). Given the relatively large sample size and in order to eliminate all potential confounders, a saturated model was used in the multivariate analysis and p-value of 0.05 was considered significant. Sensitivity analysis comparing the model that only included significant outcomes to the saturated model showed no difference in the estimates generated.Ethical approval was obtained from the Makerere University School of Medicine Research and Ethics Committee (#REC REF 2019–061). Administrative permission to conduct the study was obtained the Kitgum hospital board and written informed consent was obtained from all the research participants.Out of 600 people living with HIV screened, 599 (99.8%) were enrolled in the study. Only one patient was screened but not enrolled because he declined to provide consent as he did not want to have a finger-pick blood sample collected . All the3. On the other hand, 411/452 (90.9%) participants who had stopped CTX had CD4 results and of those, 86.1% (354/411) had a CD4 count <250cells/mm3. In addition, 92.2% (552/599) of the participants in this study had viral load results. Of the 105 participants on prophylaxis and with viral load test results, 55.2% (58/105) had a non-detectable viral load result. Similarly, 5.4% (24/447) of the participants who had a detectable viral load had stopped CTX prophylaxis.Of the enrolled participants, 491 (82.1%) reported having used an insecticide treated net (ITN) the night before the survey, and 547 (91.3%) reported having had their house sprayed with an insecticide within the last one year. Most participants (97.8%) were still on the first line of ART treatment. Of the 599 participants enrolled, 452 (75.5%) had discontinued CTX prophylaxis. Mean duration since CTX discontinuation was 4.5 months (1.2). Participants who had discontinued CTX prophylaxis were older and were more likely to have undetectable viral load than those on prophylaxis. The overall number of participants that had CD4 results in this study was 85.3% (511/599). Of the 147 participants still taking CTX prophylaxis, 68.0% (100) had CD4 results. Of the 100 participants on CTX prophylaxis and having CD4 results, 83.0% (83/100) had a CD4 count ≥250cells/mmμL of blood while the highest was 8,500 parasites /μL of blood. The mean parasite density was 1,790 parasites /μL of blood. The median (IQR) parasite density was 1,200  parasites /μL.The overall prevalence of malaria parasitaemia was 4.5% (27/599). On stratifying by CTX prophylaxis, the prevalence of parasitaemia increased with increasing duration since discontinuation of prophylaxis. Participants who were still on CTX prophylaxis had the lowest prevalence of parasitaemia (1.4%), followed by those who had stopped for 3–5 months (2.9%). Participants who had discontinued prophylaxis for more than 5 months had the highest prevalence of parasitaemia of 14.3% . The low3 were more likely to have parasites than participants with CD4 count of 250 cells/mm3 or higher .Factors that were significantly associated with malaria parasitaemia among the study participants included status of CTX prophylaxis and CD4 count . The risIn the unadjusted analyses, no IRS use in the previous 1 year  and not using an ITN the previous night  were associated with an increased risk of parasitaemia, but these associations were not statistically significant in the adjusted analysis.3) had higher parasite densities  when compared to participants with CD4 counts of 250 cells/mm3 , p value = 0.012  was associated with increased risk of having parasites in the study population.This study explored the prevalence and factors associated with malaria parasitaemia among people living with HIV and attending Kitgum Hospital HIV clinic six months following the recent policy recommendation to discontinue CTX prophylaxis in Uganda in September 2018 . FindingAlthough the malaria prevalence estimated from this study population is comparatively lower than the 12% prevalence in the district  and prevPlasmodium interrelate with the host's immune system, which may result in HIV replication and thus increased morbidity and mortality [CTX, although grouped under antibiotics has been shown to be effective in the treatment and prevention of malaria infections irrespective of whether they have HIV or not with evidence from studies done in Côte d'Ivoire, Uganda and Nigeria , 11, 30.ortality . To mainortality . Unfortuortality . The fin3) was associated with a fourfold increase in risk of having malaria parasites independent of whether CTX was discontinued. Similarly, participants with low CD4 counts had significantly increasing parasite densities in comparison to participants with higher CD4 counts. From this study however, it is unclear whether the low CD4 is the risk factor or the effect of having the malaria parasites. Both HIV and malaria destroy important cells required for proper immunological functioning of the body [3 among other considerations [We also note that a low CD4 count (<250 cell/mmthe body  and intethe body –35. Thisthe body  which iserations . Immune erations , 38.Uganda has rolled out several effective malaria control interventions including IRS, use of ITNs and malaria case management with an ACT. Our study findings show that participants who reported using a bed net the night before the survey and those who had IRS within the last one year were more likely to be protected from malaria infections than those not using protective measures, although the findings were not significant at multivariate analysis. Bed nets protect against malaria by providing a barrier against mosquito bites and when treated with insecticides kill the mosquitos on contact. When effectively and consistently used, bed-nets have been shown to reduce the risk of malaria infection in the populations living in malaria endemic countries , 40. PreOur study was not without limitations. The period for data collection was limited to three months and at one HIV clinic. Given the short duration of the study, we did not capture how the estimates change in the different malaria seasons and with changing patient health seeking behaviours. We however believe that even when the strength of association may differ, the conclusion would have been the same had the study period been extended. Furthermore, this study was designed in a cross-sectional fashion hence making it impossible to establish a causal relationship because information about the exposure and the outcome was collected at the same point in time. In addition, the study used microscopy for the detection of the parasites and may have missed sub microscopic infection that would have been picked up by the more sensitive molecular methods . DespiteIn conclusion, our study findings indicate that people with HIV and living in malaria endemic settings who discontinue CTX prophylaxis have a higher risk of having malaria parasitaemia than those on prophylaxis, and the risk increases with increasing duration since discontinuation of the treatment. We recommend that HIV infected patients living in areas of high malaria burden should not stop CTX prophylaxis even following immune recovery. There is also need to provide and encourage the use of other proven malaria control interventions in HIV patients when CTX prophylaxis is discontinued in order to reduce their risk of morbidity and mortality due to malaria.


Malaria transmission has recently fallen in many parts of Africa, but systematic descriptions of infection and disease across all age groups are rare. Here, an epidemiological investigation of parasite prevalence, the incidence of fevers associated with infection, severe hospitalized disease and mortality among children older than 6 months and adults on the Kenyan coast is presented.Plasmodium falciparum was used in the community and the OPD, and microscopy in the hospital. Crude and age-specific incidence rates were computed using Poisson regression.A prospective fever surveillance was undertaken at 6 out-patients (OPD) health-facilities between March 2018 and February 2019. Four community-based, cross sectional surveys of fever history and infection prevalence were completed among randomly selected homestead members from the same communities. Paediatric and adult malaria at Kilifi county hospital was obtained for the 12 months period. Rapid Diagnostic Tests (CareStart™ RDT) to detect HRP2-specific to Parasite prevalence gradually increased from childhood, reaching 12% by 9 years of age then declining through adolescence into adulthood. The incidence rate of RDT positivity in the OPD followed a similar trend to that of infection prevalence in the community. The incidence of hospitalized malaria from the same community was concentrated among children aged 6 months to 4 years . Only 3.7% (12/316) of deaths were directly attributable to malaria. Malaria mortality was highest among children aged 6 months–4 years at 0.57 per 1000 person-years . Severe malaria and death from malaria was negligible above 15 years of age.Under conditions of low transmission intensity, immunity to disease and the fatal consequences of infection appear to continue to be acquired in childhood and faster than anti-parasitic immunity. There was no evidence of an emerging significant burden of severe malaria or malaria mortality among adults. This is contrary to current modelled approaches to disease burden estimation in Africa and has important implications for the targeting of infection prevention strategies based on chemoprevention or vector control. Parasite exposure, age and immunity are intimately related . In malaPlasmodium falciparum continues to infect millions of people in sub-Saharan Africa (SSA) every year . ArcMap version 10·5  was used to develop the map in Fig. versus dry seasons .Between March 2018 and February 2019, 6479 participants between the ages of 6 months–98 years were enrolled over four cross-sectional surveys. The overall RDT positivity, herein regarded as parasite prevalence (PR), across all four survey rounds was 9.9%  and was 13.7%, 10.7%, 7.2% and 9.0% during each cross-sectional survey, respectively ; after adjusting for site, month of enrollment, long lasting insecticide use (LLINs), and gender. Malaria parasite prevalence was lower in the older age groups compared to children aged 6 months–4 years ; with the lowest risk being reported among participants aged 50 years and above . The age-pattern of PR showed a strong age-dependency, gradually increasing from childhood and reaching level of 12% by 9 years of age before declining from late adolescence into adulthood as shown in Fig. Of the enrolled participants, 348  had either measured or reported fever. Fever was associated with a higher risk of malaria infection  compared to participants without fever; after adjusting for site, age, month of enrollment, LLINs use and gender.During the 12-month surveillance period, 28,134 febrile patients ≥ 6 months of age sought treatment in one of the six out-patient health facilities shown in Fig. The overall RDT positivity rate (TPR) was 43.2% . The median age of febrile patients who tested positive for malaria  was significantly lower compared to the non-malaria fevers  (p < 0.001). Febrile children aged 5–14 years had a higher risk of testing RDT positive  compared to febrile children aged 6 months–4 years; after adjusting for site, month of enrollment, LLINs use, and gender. Other age groups had a lower risk of RDT positivity  compared to febrile children aged 6 months–4 years. TPR did not differ during the wet versus dry season .During the surveillance period there were 71,562 person-years of observation in residents of the study area aged ≥ 6 months. The annual period prevalence and incidence rate of RDT positivity followed a similar trend to that of infection prevalence in the community; initially increasing from childhood, plateaued in school-aged children (7–13 years) and peaked around 10 years before declining in late adolescence and adulthood  were female and 13% (n = 80) had been admitted in the paediatric high dependency unit (HDU). The overall median age of all-cause admission was 12 years . Among the 76 malaria admissions, the majority (57%) presented with one or more criteria for severe malaria; 33 (43%) cases had uncomplicated malaria. The ratio of children with cerebral malaria to severe malaria anaemia was 5:2. There were no cases of severe malaria among patients aged 9 years and above.The age-pattern of incidence of malaria admissions is shown in Fig. A total of 316 deaths were recorded among the population of the study area between March 2018 and February 2019. Thirty-six deaths (11%) were among children aged 6 months–4 years, 22 (7%) among 5–14 years, 61 (20%) among 15–49 years and 197 (62%) deaths among those aged 50 years and above.versus those missing VA data (p = 0.09). Using the MICE algorithm, there were 2 cases  aged 6 months–4 years ascribed malaria based on the distribution of deaths among known causes.Of the 316 deaths, 55 (17%) died during hospital admission including 3 malaria deaths and 8 2.5%) died < 30 days post-discharge with one diagnosed with malaria during admission. Four (1.3%) individuals had been admitted over the preceding 24 months, where three had been diagnosed with terminal cancer and one with epilepsy  , it was Despite a low transmission intensity, the age-pattern of infection was consistent with patterns previously described across a wide range of endemicities , 47. InfFrom the age of 30 years, prevalence steadily decreased to very low levels through to old age, suggesting a continued acquisition of immunity to infection throughout adulthood Fig. a, similaOver 12 months, 12,143 fevers associated with malaria infection presented to the six health facilities shown in Fig. The age-pattern of the period prevalence and incidence of fevers associated with malaria rose in childhood to peak at 10 years and declined thereafter to a low level throughout adulthood Fig. b. The hiThe age-specific incidence of severe malaria showed a very different age-profile to either community-based or passively detected fever prevalence of malaria infection. The incidence of hospitalized malaria from the same community was concentrated among children aged 6 months to 4 years. There were three cases of non-severe, hospitalized malaria described in adults aged, 18, 31, 88 years, none of these presented with severe malaria Fig. c. DespitThe present study area has also been subject to a transition in the intensity of malaria transmission over the last 20 years , 48. ThiDuring the study period there were 316 deaths recorded within the community aged 6 months and above. The overall malaria-specific mortality rate was estimated to be 0.17 per 1000 population above 6 months p.a. . With this approach, there were no malaria deaths among children 6–11 months of age, 6 (50%) of the malaria deaths occurred among children aged 1–4 years, 6 (50%) among children aged 5–14 years, and there with no deaths attributed to malaria among the non-pregnant population aged above 15 years  project relies on VA data from DSS sites in sub-Saharan Africa, which have shown different malaria-specific age patterns –59. BetwIn the absence of clinical and parasitological data, the definition of mortality events directly attributed to malaria are fraught with difficulties , leadingDeaths that had contact with diagnostic facilities provide unique information on causes of death. Relying solely upon a computerized algorithm to attribute malaria as a cause of death from VA responses misses opportunities to review underlying causes available in free-text commentaries, notably in the identification of important unambiguous causes, including HIV and SCD. The approach taken here maximizes the amount of information available, and by comparing with hospital presentations with severe malaria, allows a more plausible approach to defining the contribution of malaria mortality by age.P. falciparum prevalence setting. Nevertheless, RDTs are now widely used in clinical and survey settings, but it is important to note that comparisons with other data on microscopy  between May 2018 and January 2019 and total monthly rainfall over the 12-month surveillance period (black dashed line).Additional file 2. Varying confirmatory evidence to attribute deaths.


Approximately 6% of children hospitalized with severe falciparum malaria in Africa are also bacteremic. It is therefore recommended that all children with severe malaria should receive broad-spectrum antibiotics in addition to parenteral artesunate. Empirical antibiotics are not recommended currently for adults with severe malaria.Blood cultures were performed on sequential prospectively studied adult patients with strictly defined severe falciparum malaria admitted to a single referral center in Vietnam between 1991 and 2003.Staphylococcus aureus in 2, Streptococcus pyogenes in 1, Salmonella Typhi in 3, Non-typhoid Salmonella in 1, Klebsiella pneumoniae in 1, and Haemophilus influenzae type b in 1. Bacteremic patients presented usually with a combination of jaundice, acute renal failure, and high malaria parasitemia. Four bacteremic patients died compared with 108 (12.9%) of 836 nonbacteremic severe malaria patients . In patients with >20% parasitemia the prevalence of concomitant bacteremia was 5.2%  compared with 0.65%  in patients with <20% parasitemia, a risk ratio of 8.1 (2.2–29.5).In 845 Vietnamese adults with severe falciparum malaria admission blood cultures were positive in 9 ; In contrast to children, the prevalence of concomitant bacteremia in adults with severe malaria is low. Administration of empirical antibiotics, in addition to artesunate, is warranted in the small subgroup of patients with very high parasitemias, emphasizing the importance of quantitative blood smear microscopy assessment, but it is not indicated in most adults with severe falciparum malaria. Blood cultures were positive in 9 of 845 sequentially studied Vietnamese adults with severe falciparum malaria. In contrast to children, concomitant bacteremia in adults with severe malaria is uncommon and does not warrant use of empirical antibiotics in all patients. Malaria is associated with bacterial infection, but the relationship is complex . In endeThis investigation took place in the severe malaria ward of the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam, during 2 sequential studies of adult patients admitted with strictly defined severe falciparum malaria. The first, conducted between 1991 and 1996, was a double-blind comparison of intramuscular quinine and intramuscular artemether , and thePlasmodium falciparum on a peripheral-blood smear, were older than 14 years, were not in the first trimester of pregnancy, were not intravenous drug users, had received less than 3 g of quinine or 2 doses of artemisinin or a derivative in the previous 48 hours, were not allergic to the study drugs, and had 1 or more of the following: Glasgow Coma Scale score less than 11 , anemia  with a parasite density greater than 100 000/μL, jaundice  with a parasite density greater than 100 000/μL, acute kidney injury , hypoglycemia , more than 10% parasitemia, and systolic blood pressure less than 80 mm Hg with cool extremities (indicating shock). These criteria are similar to the World Health Organization–endorsed definition [Patients were included in the studies if they (or an accompanying relative) gave informed consent, they had asexual forms of On enrollment, patients were examined and weighed and baseline blood samples were taken for full blood count, clotting studies, biochemistry, arterial pH and blood gases, blood cultures, and thin- and thick-film malaria parasite counts. A full history was taken from the patient or attendant relatives and a full clinical examination performed including a detailed neurologic assessment. A urinary catheter was inserted. Patients were managed by a dedicated team according to standard recommendations . AntimaloC for 7 days. Blind subculture was performed at 24 and 48 hours and at 7 days or whenever physical growth was observed in the bottles. In September 1997, a BACTEC  culture system was introduced. Aliquots of blood (5–8 mL) were inoculated into BACTEC plus aerobic bottles and then incubated for 5 days in a BACTEC 9050 automated analyzer. Bottles that gave a positive signal were subcultured.Between 5 and 15 mL of blood was taken for blood cultures . Between 1991 and 1997 a manual blood culture system was used . Each 5-Haemophilus influenzae or Neisseria meningitidis was suspected, and onto Sabouraud’s agar if a yeast or mold was suspected . Plates were incubated at 37oC in air (blood agar) or 5% CO2 (chocolate agar) for 48 hours or 30oC in air (Sabouraud’s agar) for 5 days. Organisms were identified by standard methods including API identification kits  when necessary. Specific antisera were used to identify Salmonella serogroups, including Vi for Salmonella enterica serovar Typhi (S. Typhi). Staphylococcus epidermidis, or other skin commensals were considered contaminants.Subcultures were plated onto fresh sheep blood agar and heated blood (chocolate) agar if Proportions were compared using Fisher’s exact test using Epiinfo .S. Typhi [Salmonella [, Staphylococcus aureus [Streptococcus [, H. influenzae type b [Klebsiella pneumoniae [Burkholderia cepacia, but this was regarded as a contaminant, and the patient recovered uneventfully without receiving antibacterial treatment. The usual clinical presentation in these bacteremic patients was with the hepatorenal syndrome of fever, jaundice, and acute kidney injury accompanied by high parasitemia. One patient was unconscious .Blood culture results were available for 845 adult patients admitted with severe falciparum malaria. Of these, 9 were positive for pathogens, a prevalence of 1.07% 95% CI, .37–1.76%). The organisms cultured were 7–1.76%. s aureus , Group AFour of the 9 patients with severe malaria and concomitant bacteremia died, as discussed in the following:Staphylococcus aureus.A 47-year-old male farmer admitted with 20.1% parasitemia, jaundice, and shock died 2.5 hours after admission. Blood cultures subsequently grew S. Typhi.A 24-year-old male farmer with a 7-day history of fever had generalized convulsions followed by coma on the day of admission. His parasitemia was 0.9%. He had clinical signs of pneumonia, for which he was given ceftriaxone, but he died 29 hours later without regaining consciousness. Blood cultures subsequently grew H. influenzae type b.A 28-year-old male soldier with a 5-day history of fever was admitted with 24.9% parasitemia, jaundice, and acute renal failure. He died 16 hours later. Blood cultures subsequently grew K. pneumoniae, and amikacin was added. After a protracted course he developed nosocomial pneumonia and he died 24 days later.A 48-year-old male builder with a 6-day history of fever presented with 24.6% parasitemia, hyperlactatemia, acute oliguric kidney injury, jaundice, pulmonary edema, and upper gastrointestinal bleeding. Hemofiltration was started immediately. His parasitemia rose to 54% within 8 hours of admission. He received artemether and, because sepsis was suspected clinically, he was given ceftriaxone. On day 2, blood cultures grew P = .022).The overall mortality of patients with severe malaria but no concomitant bacteremia was significantly lower, 12.9% (108/836). The risk ratio for death in patients with concomitant bacteremia was 3.44 . Thus, the prevalence of concomitant bacteremia in patients with more than 20% parasitemia was 5.2%  compared with 0.65%  in patients with less than 20% parasitemia, a risk ratio of 8.1 . Mortality in nonbacteremic patients with more than 20% parasitemia was 18% (13 of 72). Leukocytosis, which may also occur in very severe malaria infections, and other hematological or biochemical indices were not useful as indicators of concomitant bacteremia. Only 1 of the 8 bacteremic patients with a differential white blood cell count performed on admission had a neutrophilia.The 9 patients admitted with severe malaria and concomitant bacteremia were slightly older  than the other 836 patients . They were also more likely to be hyperparasitemic; the median (range) parasite count was 501 144/μL (39 564 to 1 765 936/μL) compared with 81 766/μL (12 811 to 316 512/μL) in the patients with nonbacteremic severe malaria. Four of the 9 bacteremic patients had more than 20% parasitemia compared with 72 of 836 nonbacteremic patients  in 1991 and 10% (33/334) in 2000.Between 1991 and 2000, during which 90% of the patients in this series were recruited, hospital , comprisSalmonella infections  have been associated specifically with falciparum malaria infections in African children [S. Typhi and it is noteworthy that S. Typhi was also the most common cause of community-acquired bacteremia identified in Ho Chi Minh City during this period [In this large prospective study of Vietnamese adults admitted to the hospital with strictly defined severe falciparum malaria the rate of concomitant bacteremia was low. This contrasts with large studies in African children with severe malaria in whom concomitant bacteremia is sufficiently common (more than 5 times more common than in adults in this series) , and thechildren , 19. In s period , 21. AltPfHRP2 or P. falciparum DNA [These results contrast markedly with a recent study from Myanmar in which 15% (13 of 87) of adults hospitalized with a primary diagnosis of malaria were bacteremic . Malariaarum DNA . As paraarum DNA . Semi-quarum DNA . They ararum DNA  and explarum DNA . HoweverAlthough this study in Vietnamese adults was long, large, and detailed, it has several limitations. Empirical use of antibiotics before admission to the hospital is common in Asia (and was reported in 35% of the Myanmar series) and could have obscured some bacterial infections. However, prior antibiotic use was unusual in the low-income rural population at risk of malaria in Vietnam between 1991 and 2003, so is unlikely to be a significant confounder. Blood culture is intrinsically insensitive, so bacteremia was probably underestimated. The target blood volume cultured (10 mL), which was the same in both the Myanmar and Vietnam series, is not maximally sensitive , 31. It High parasitemia was clearly a risk factor for concomitant bacteremia. Of the 76 patients with more than 20% parasitemia , 4 (5.2%) were also bacteremic. This group has a high mortality, so giving broad-spectrum antibiotics empirically to patients with very high parasitemias is justified. Thin blood smear assessment should be performed on all patients admitted with severe falciparum malaria. Overall, however, in contrast to children in areas of higher malaria transmission, the incidence of concomitant bacteremia in adults with severe malaria is low and does not warrant the use of empirical antibiotics in all patients.


Microscopy performed on stained films of peripheral blood for detection, identification and quantification of malaria parasites is an essential reference standard for clinical trials of drugs, vaccines and diagnostic tests for malaria. The value of data from such research is greatly enhanced if this reference standard is consistent across time and geography. Adherence to common standards and practices is a prerequisite to achieve this. The rationale for proposed research standards and procedures for the preparation, staining and microscopic examination of blood films for malaria parasites is presented here with the aim of improving the consistency and reliability of malaria microscopy performed in such studies. These standards constitute the core of a quality management system for clinical research studies employing microscopy as a reference standard. They can be used as the basis for the design of training and proficiency testing programmes as well as for procedures and quality assurance of malaria microscopy in clinical research. Plasmodium species and stages, and estimate parasite density [Microscopy continues to play an important role in malaria diagnosis and in research studies. While the advent of rapid diagnostic tests have reduced its importance as a primary diagnostic test in routine practice in some countries, microscopy remains an essential tool to support clinical research, severe malaria case management and monitoring of anti-malarial treatment efficacy . In the  density . Microsc density , 4. A pa density , though  density . In the Standardization of methods enables direct comparisons of results from studies conducted at varying points in time and location while improving the quality and reliability of results generated in individual studies. This is particularly relevant to preparing and staining slides and for the detection, identification and quantification of malaria parasites in thick and thin blood films given their importance in determining the endpoints and outcomes of malaria trials. Various factors beyond the quality of the blood film and the reading process can also influence microscopy results. These include stochastic variation such as the distribution of parasites throughout a film, the workload and expertise of readers, how slides are declared negative or parasite density is estimated, and how discrepant results are handled, all of which can influence outcomes, particularly relating to parasite quantitation and detection of low-density infections.Guidelines and procedures for quality assurance in malaria microscopy have already been published and are an excellent basis for standardizing and improving clinical malaria microscopy –10. HoweThe minimum quality assurance standards for research malaria microscopy proposed in this article would enable better comparison of results between studies conducted by various institutions. The standards have been reviewed by a committee of experts under the auspices of the Special Programme for Research and Training in Tropical Diseases (TDR) at the World Health Organization (WHO) and are the basis of the Research Malaria Microscopy Standards (ReMMS) manual . An assoThe generation of accurate, precise and reproducible microscopy data can only be accomplished from correctly prepared, stained and examined blood films, which in turn depend on the inputs of skilled, well-trained and motivated laboratory personnel. Attaining and maintaining high standards in the preparation and staining of blood films should be routine given the availability of correct tools, standard operating procedures, and materials (see later sections of this article) but the examination of slides requires a different approach. A research microscopist is expected to be able to detect, identify and count human malaria parasites in Giemsa-stained peripheral blood films while reliably differentiating parasites from other features normally found in the film and from artefacts or debris that may be indistinguishable from parasites to the inexperienced eye. Extensive training and experience are required to attain the necessary level of proficiency, but it is hard to standardize their content and duration such that this level of proficiency can be assured. Work practices, workload and the working environment can further affect microscopists’ performance. Minimum competency standards to be attained in formal testing as a pre-requisite to performing microscopy for research studies can however be defined with reference to the particular requirements of research microscopy.Plasmodium falciparum malaria parasites are of prime importance to ensure patients with malaria receive the most appropriate anti-malarial treatment. The ‘competence level’ of microscopists is defined based on competence in detecting parasitaemia, species identification and parasite quantification, with no specific criteria defined for diagnostic sensitivity or specificity. As also indicated in the WHO Malaria Microscopy Quality Assurance Manual, specialized microscopy in clinical trials would require a more thorough assessment with an expanded slide set to obtain more robust estimates of sensitivity, specificity and other diagnostic performance criteria for each microscopist being tested.Standardized assessments for competency in malaria microscopy are currently based on a slide set described in the WHO Malaria Microscopy Quality Assurance Manual . This slThe proposed slide set for assessing the competency of research microscopists comprises 130 slides  and feedback directly influence the performance of microscopists (see later sections) as does the volume of work. Microscopists’ performance and workloads need to be carefully monitored and managed to ensure that operator fatigue does not adversely affect the quality of microscopy. A research microscopist may be expected to read approximately 40 slides per day depending on the proportion of positive slides and on the parasite density on those slides. More precise estimates of ideal workloads have been calculated elsewhere, but would need to be adapted to take into account the expected numbers of positive slides at patient recruitment and on follow-up according to the research protocol as well as the increased number of fields to be read before declaring a slide negative .BOX 1: Recommended slide set for Competence Assessment of Research MicroscopistsDetailed lists of equipment and materials required for the preparation, staining and examination of slides have been published in various malaria microscopy guides and manuals , 9. ThesBOX 2: Recommended equipment and materials for research malaria microscopy • Slide template see Fig. : The tem• Micropipette to measure 5–10 µL volumes• Giemsa stain, buffer pH 7.2—commercial preparations preferable or methods from WHO manuals or standard operating procedures.• pH meter calibrated for measurements at pH 7.2• Synthetic immersion oil, refractive index 1.5• Binocular microscopes with 10 × FN 20 oculars, 100 × oil-immersion plan-achromat objective, inbuilt electric light source.• Grid reticle (recommended if objectives are not of plan-achromat type).BOX 3: Minimum requirements for preparing blood films and labelling slides• Labelling minimum requirement: Study + subject ID, date and time of blood collection.• Sampling: Blood from fingerprick or venepuncture is acceptable but should be consistent within a study and stated in the protocol and study report.• Thick films to be spread using a known volume of blood spread over fixed area, e.g., use of micropipette to measure 6 µL of blood and a template to prepare thick films of 12 mm diameter.• Thin films to be spread using 2–5 µL of blood to obtain a thin film with a feathered edge.• Slides must be completely dried before staining—e.g., minimum of 1 h at 37–40 °C (may need to be longer for anticoagulated blood) or overnight at ambient temperature with low humidity.• Thin films to be fixed with absolute methanol.Preparing standard thick blood films can be easily accomplished by spreading a known volume of blood over a defined area on a slide as has previously been recommended for reference slides for slide banks see Fig. , Box 3 . The useParasite loss is usually not seen from thin films since they are fixed with methanol before staining but measures need to be taken to enhance parasite detection and density estimations in the thick film, given reports of 30–90% parasite loss during the staining process , 13. VarFinally, it is critical to ensure that slides can be unequivocally identified through correct, clear and consistent labelling to be able to link the microscopy results to study subjects and other related data without which the conclusions from the research may not be reliable. At a minimum, slides must be labelled with a study and anonymized subject ID as well as the date and time of blood sample collection. Further aids to identifying slides and detecting and resolving errors in labelling include follow-up time-points and unique codes.P. falciparum and Schüffner’s dots in P. vivax more clearly visible and allows for staining large numbers of slides simultaneously in batches at low cost per slide. The ‘slow’ staining method with 3–5% working Giemsa solution and 45–60 min of staining is recommended for slides that may need to be stored for long periods or require repeat reading [All commonly used methods for staining blood films for malaria microscopy are based on the ‘Romanowsky effect’, wherein an acidic and a basic stain are combined to produce differential colouring of the parasite nucleus and cytoplasm . The sta reading , 8. ThisMalaria microscopy is a procedure for the detection, identification and quantification of parasites per unit volume of blood. This implies that a standard volume of blood must be examined before a slide can be declared negative or to be able to estimate the parasite density in a given blood sample. Some early researchers used the time spent to examine blood films as a way of standardizing the volume of blood examined . While tFor research applications, it is common to read up to 200 HPFs or count up to 2000–2500 WBCs on the thick film before declaring a slide parasite-negative. This limit of detection is theoretically as low as 3–4 parasites per µL  of the parasites along with a parasite count against HPFs or WBCs. Depending on the requirements of the study, microscopists may choose to count parasites separately by species and stages, each against at least 40 HPFs or 500 WBCs. If parasites are seen after the first 40 HPFs or 500 WBCs, counting may be stopped and the results reported per the exact number of HPFs viewed or WBCs counted. It may be difficult to distinguish species of individual parasites on the thick film, especially at low densities, and the parasite counts in mixed infections can be combined for reporting but each detected species should be clearly indicated. Depending on the requirements of the study, gametocytes may need to be reported and counted, whereas schizonts are counted along with other asexual parasite forms .• Parasites must be counted in a minimum of 40 HPFs or against 500 WBCs in the thick film or against 2000 RBCs in the thin film.• Results must be reported as raw figures, i.e. species of parasites, the number of asexual or sexual forms of the parasite over the number of WBCs or fields counted.• The most recently measured actual WBC or RBC counts for the study subject must be used for parasite density estimation.Quality control (QC), which in the context of microscopy usually means re-checking of slides already examined by a microscopist, is a critical component of any quality management system since it provides feedback on the quality of outcomes obtained from routine processes implemented under everyday conditions. For malaria microscopy, QC must also include assessments of the quality of blood films and staining as poor quality slides can directly affect the ability of the microscopist to detect, identify and count parasites. Deviations from pre-defined tolerances can help to identify needs for adjustments or refinements to established procedures and corrections to individual outcomes, while also indicating if more rigorous QC protocols are required. In malaria microscopy, such tolerance limits have been defined for detection, identification and estimation of parasite density and are described below. These limits have also been defined in Box Definitions of acceptable tolerance in the detection and identification of parasites are straightforward—if results from two independent reads on the same slide are not in agreement with respect to the presence of parasites or their species then a tiebreaker read is needed. Defining the tolerance for variations in parasite density requires more sophisticated methods. The implementation of such QC criteria or protocols can be facilitated and made systematic by using computing aids which standardize the thresholds for cross-checking by a third microscopist and acceptance criteria for managing outlying results across studies, producing more predictable variances when comparing paired reads for concordance –31. The BOX 5: Guidelines for internal and external quality control (IQC and EQC) of malaria microscopy• All microscopists performing QC reads must be blinded to the slide details as well as the initial results.• QC checks performed prior to the initial examination of the slide and during re-checking must include assessments of slide quality per standard criteria. The slide(s) must be rejected if any issues that could affect parasite detection and/or counting are identified .• IQC: minimum 20% of all study slides, randomly selected, re-read for IQC; in case the study microscopists have been certified at Competency Levels 3 or 4, or are uncertified, up to 100% double-reads may be required depending on QC results.• Results from paired reads from the same slide are considered discrepant and require a third tie breaker read if:• Only one microscopist detects parasites;• The species of the parasites reported by the microscopists is (are) different;• The difference in parasite density for asexual parasites is greater than a pre-defined limit .• Source of Giemsa stain stock solution.• Microscope specifications and/or brand, model.• Number of fields examined before declaring slide negative.• Parasite density estimation method (‘WBC’ or ‘HPF’); WBC, RBC counts used in calculations .• QC protocols .kappa for species, proportion of slides with discordant parasite density as detected by external rechecking).• QC results  presented here, complemented by the ReMMS Manual and the Obare Calculator research microscopy standardization tool, are an attempt to make a set of standards and procedures freely available to the malaria research community, aimed at increasing the consistency and reliability of malaria microscopy performed for research studies (see Box 6) , 30, 32.Many research laboratories have already implemented quality management systems for microscopy or for the laboratory as a whole. However, a framework, such as the one described in the WHO Malaria Microscopy Quality Assurance manual for National Malaria Control Programmes, is lacking for microscopy in research. As a result, high-quality training and standard proficiency testing is hard to find outside certain reference centres . While tMicroscopy on Giemsa-stained blood films to detect, identify and quantitate malaria parasites is a technique which is already more than a century old and may be replaced in the future by methods based on polymerase chain reaction (PCR) or machine learning for micrographic image analysis for research and clinical applications , 35. PCRThe standards described here Fig.  and the


Malaria surveillance is a key pillar in the control of malaria in Africa. The value of using routinely collected data from health facilities to define malaria risk at community levels remains poorly defined.Four cross-sectional parasite prevalence surveys were undertaken among residents at 36 enumeration zones in Kilifi county on the Kenyan coast and temporally and spatially matched to fever surveillance at 6 health facilities serving the same communities over 12 months. The age-structured functional form of the relationship between test positivity rate (TPR) and community-based parasite prevalence (PR) was explored through the development of regression models fitted by alternating the linear, exponential and polynomial terms for PR. The predictive ranges of TPR were explored for PR endemicity risk groups of control programmatic value using cut-offs of low (PR <5%) and high (PR ≥ 30%) transmission intensity.Among 28,134 febrile patients encountered for malaria diagnostic testing in the health facilities, 12,143  were positive. The overall community PR was 9.9%  among 6,479 participants tested for malaria. The polynomial model was the best fitting model for the data that described the algebraic relationship between TPR and PR. In this setting, a TPR of ≥ 49% in all age groups corresponded to an age-standardized PR of ≥ 30%, while a TPR of < 40% corresponded to an age-standardized PR of < 5%.A non-linear relationship was observed between the relative change in TPR and changes in the PR, which is likely to have important implications for malaria surveillance programs, especially at the extremes of transmission. However, larger, more spatially diverse data series using routinely collected TPR data matched to community-based infection prevalence data are required to explore the more practical implications of using TPR as a replacement for community PR. Monitoring the intensity of malaria transmission in time and space is an important parameter to define the required combinations of intervention to accelerate control and elimination, measure impact and over time repurpose intervention and control ambitions –3.Plasmodium falciparum prevalence (PfPR2–10), 43,187 empirical survey estimates were used to provide 16,200,000 5 × 5 km predictions of malaria prevalence, disease and mortality in stable endemic areas of Africa between 2000 and 2017 [The prevalence of malaria infection among community residents, or school attendees, has been used for over a century as a marker of the quantity of malaria (endemicity) in a location , 5, and The sparsity of community-based prevalence surveys remains an important source of error in the predictions of malaria endemicity and disease burden in time and space across SSA . NationaA more continuous and spatially ubiquitous source of information is the prevalence of malaria infection among patients examined in health facilities. Traditionally, this was referred to as slide positivity rate and was recommended as a transmission surveillance metric when community-based prevalence fell below 2%; a point where community sampling became financially impractical , 20, 21.While the detection of all malaria cases represents a core intervention for elimination , the useAnopheles gambiae s.l and An. funestus s.s. [This study was conducted in the southern part of Kilifi Health and Demographic Surveillance System (KHDSS) located along the Kenyan coast . The stutus s.s. , 47 and ™) to detect HRP2 specific to P. falciparum. If the mRDT results were positive the patient received appropriate treatment as per the Government of Kenya guidelines for malaria-case management [The study in the health facilities was established as a partnership with the County Ministry of Health and was developed to reflect routine practices as far as was possible. The national standard treatment guidelines for malaria in Kenya specify that all patients presenting with fever should be investigated parasitologically  and the nagement . HRP2 banagement . The pat™), irrespective of their fever status. All participants with fever and/or a positive rapid test were advised to seek treatment at the nearest health facility.Four community-based prevalence surveys were undertaken during the facility surveillance period; May–June 2018, August 2018, October 2018 and December 2018– January 2019. During each survey round, random homesteads were selected and in the subsequent sampling frames, previously selected homesteads were excluded. A sample size of at least 4,341 participants was obtained based on the local prevalence estimated to be ≤ 30% , a preciData was entered electronically using laptops in the facilities and tablets in the community-based surveys by the study team on a PHP web-based interface and data saved onto MySQL database and synchronized onto a secure server.Analysis included only data for participants identified as residents of the catchment areas shown in 2–10 years using data from the entire community age-standardised to the 2–10 age group in each EZ as described elsewhere [The health facility TPR was defined as the number of positive diagnostic tests as a proportion of the total tests performed among febrile patients. The community parasite prevalence (PR) was defined as the proportion of participants tested found with a positive mRDT. To compute the TPR and PR for each EZ, the number of positive participants and total tests performed were aggregated at the EZ level. For each EZ, the facility TPR data were matched to two-month period around each community-based cross-sectional survey. To explore different temporal matches, the time interval was altered by 1) matching the health facility data to the exact same four-time periods when the community-based survey was conducted; 2) matching to the subsequent month i.e. lagging the health facility data by one month after the community cross-sectional survey; or 3) by using all the health facility data in each EZ versus all four community-based prevalence surveys in each EZ. TPR and PR values were compared using actual age-groupings and against the traditional PRlsewhere . The ass2, and Akaike Information Criteria (AIC). Regression diagnostics were performed including Cook’s D to assess for high leverage, which was indicative of influential EZ, and/or large residuals (outliers). To test the variability in the prediction performance of the models, 80% of the data were divided into train and 20% into test datasets, and the model was rerun on 100 randomly selected samples. The models developed by the training dataset were validated against the test datasets by comparing the accuracy measures i.e. the correlation between actual and predicted estimates and the error rates (mean absolute percentage error (MAPE), mean square error (MSE) and mean absolute error (MAE)). As a sensitivity analysis, the effects of using varying intervals of TPR in quantifying the relationship between TPR and PR using the best fitting model was explored.To explore the functional form of the relationship between TPR and PR in a more formal form, i.e. to define a function (F), that transforms an estimate of PR over any range , into a TPR over any range , i.e. F: PR→TPR, various models were considered. The relationship was assessed using a linear relationship and other flexible function forms using the polynomial and exponential transformations. A selection of the best fitting model was made using goodness of fit, measured as root mean square error (RMSE), adjusted Rhttp://www.diva-gis.org/gdata).Finally, the predictive ranges of TPR were explored for PR endemicity risk groups of programmatic value, i.e. cut-offs of low (PR <5%) and high PR ≥ 30%), used in national malaria stratification in Kenya –52 and T%, used iThe health facility surveillance did not impose any changes in the national treatment guidelines and data used in the analysis were gathered as part of routine care. Consent was waived by the ethics committee; therefore, individual patient consent was not sought. All the records were pseudo-anonymized at the point of data capture in the healthcare facilities, but linked to our demographic surveillance by an ID number. During the community surveys written informed consent was sought from participants ≥18 years of age or the parents/guardians for children aged 10 years and below. With parental guidance, children aged over 10 and less than 18 years were asked to sign an assent form. These documents were available in Kigiriama, Kiswahili, and English. This study was approved by the Kenya Medical Research Institute Scientific Ethics Review Unit (KEMRI/SERU/CGMR-C/106/3592) and the Oxford tropical research ethics committee (OxTREC Reference: 511–18).Between March 2018 and February 2019, 46,567 febrile patients ≥ 6 months of age sought treatment in one of the six out-patient health facilities shown in 2–10 years was 12.7% and ranged between 0.01% and 53.2% in the 36 EZs.During the four community-based prevalence surveys, 7,255 participants ≥ 6 months of age were approached for enrolment; 425 (5.9%) declined consent and 351 were excluded because they either had been enrolled in the health facility survey within the last 14 days (198), were pregnant (79) or had missing mRDT results (74). A total of 6,479 participants aged 6 months to 98 years were surveyed in the community with an average of 180 per EZ. The median age of those sampled was 12 years , 61% were female  and 348 2–10 years  and 5–9 years   attendees per EZ. Among this time-matched series, TPR varied significantly (p <0.001) across the 36 enumeration areas ranging between 17% and 73% and significantly differed across age groups . The cor10 years . The cor10 years . Stronge6, 0.74) . These c6, 0.74) .2–10 years, currently used to map malaria transmission in Africa.The subsequent analysis focuses on three age comparisons: 1) children aged < 5 years, as DHIS2 data is aggregated over the age groups below and above five years and household surveys measure malaria infection mostly in children aged 6 months to five years; 2) all ages, as this is also available from existing national DHIS2 platforms; and 3) TPR comparisons to age-standardized PR2), in all comparisons, except TPR0.5–4 years vs. PR0.5–4 years, where the linear model was the best fitting model  developed symptoms that required treatment in an out-patient health facility   . The linof ≥ 43% . Howeverall ages when PRall ages was below 10%, but smaller changes in the predicted TPRall ages when PRall ages was above 30%  and high transmission areas (rho = 0.01) [rho = 0.97) between clinic slide positivity data and community survey data from four villages compared at three different periods of observation [The relationships between TPR and PR shown here are consistent with other studies undertaken using similar designs. In three separate transmission settings in western Kenya, the correlation between malaria positivity rate among suspected patients from the health facilities and asymptomatic malaria positivity among school children was 0.78, 0.61, and -0.039 in the low, moderate, and high transmission settings, respectively . As with = 0.01) . A much ervation .Routinely collected TPR has several advantages as a surrogate measure of malaria transmission. There are obvious opportunity costs of using routine, rather than expensive survey data; data are spatially cosmopolitan rather than opportunistically sampled; available at continuous temporal resolutions; and provide granular data at district levels for district level decision making. In the present study, national guidelines  on test-Although an out-of-sample validation was performed, the confidence in the generalizability of the results is reinforced if it is validated in an external population. Furthermore, caution should be used in interpreting the function of best-fit regression. Noise in the estimation of either variable will lead to not just uncertainty in the estimate, but also to a slope that is biased towards zero due to the effect of regression dilution bias. Noise can be introduced by completely random errors (minimized by sample size) and other errors introduced by heterogeneity in transmission. It is possible that larger datasets would lead to the estimation of a steeper function linking PR to TPR, which would then lead to a more favourable impression of the utility of TPR in determining endemicity.Health facility-based surveillance with indicators like TPR remains an attractive measure which might be a crude reflection of transmission dynamics while at the same time, they are more operationally attractive compared with community-based surveys in terms of time and cost. However, a better understanding is required of the biological, clinical, social and epidemiological relationships between malaria infection and fever across all ages, and all health systems. Importantly, these studies must be undertaken across a wide range of endemicities to develop a more pragmatic usable criteria for NMCPs to use effectively in a stratified response to malaria control.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Loop-mediated isothermal amplification (LAMP) is a sensitive molecular tool suitable for use as a near point-of-care test for the diagnosis of malaria. Recent meta-analyses have detailed high sensitivity and specificity of malaria LAMP when compared to microscopy, rapid diagnostic tests, and polymerase chain reaction in both endemic and non-endemic settings. Despite this, the use of malaria LAMP has primarily been limited to research settings to date. In this review, we aim to assess to what extent commercially available malaria LAMP kits have been applied in different settings, and to identify possible obstacles that may have hindered their use from being adopted further. In order to address this, we conducted a literature search in PubMed.gov using the search terms (((LAMP) OR ) AND  OR (Plasmodium))). Focusing primarily on studies employing one of the commercially available kits, we then selected three key areas of LAMP application for further review: the performance and application of LAMP in malaria endemic settings including low transmission areas; LAMP for malaria screening during pregnancy; and malaria LAMP in returning travelers in non-endemic settings. Bacillus stearothermophilus (Bst) DNA polymerase with strand displacement activity ..®  or passed through a column that purifies the DNA via gravity  . Both prP. falciparum (depending on which extraction method is used) and 0.1 p/µL for P. vivax [Result readout with an Illumigene Malaria Illumipro-10™ incubator/reader measures turbidity induced by the formation of magnesium pyrophosphate. Ten samples can be analyzed per run in this platform . The detP. vivax ,31. A diP. vivax ,34.The Illumigene kit is preferably conducted on venous EDTA whole blood samples, making it possible to perform the LAMP assay with some delay. This blood sampling method can however be challenging in resource limiting settings . FurtherThe Loopamp Malaria Detection Kits have been endorsed by the Foundation for Innovative New Diagnostics (FIND) who has developed standardized procedures for their use ,35. The DNA can be extracted with the commercially available Loopamp PURE DNA Extraction Kit, consisting of a series of interlocking plastic components providing a closed system for preparation of a blood aliquot requiring only a heat block or incubator, micropipettes, DNase/Rnase free sterile pipette tips, and latex gloves . The LooThe LA-500 real-time turbidimeter  or the HumaLoop M  platform for sample preparation, amplification, and easy visual result reading under a built in UV light can hold 16 tests per run. If a simple heating block system is used then batches of up to 46 samples can be run together with a positive and negative control (the maximum number of tubes that fit on a regular heat-block), making it a useful tool for prevalence surveys in endemic areas ,42. FurtTM MALARIA Detection kits and two studies the Illumigene® (Alethia) malaria LAMP. Overall, LAMP has shown ≥95% pooled sensitivity and specificity for detecting both P. falciparum and P. vivax infections when used in endemic settings [The performance of LAMP has been evaluated in malaria endemic field settings in over 30 publications, of which 15 studies employed the Loopampsettings . The consettings ,47,48,49As malaria transmission declines in areas of successful malaria control, the relative proportion of asymptomatic and low-density malaria infections increases . These iThe improved analytical sensitivity of LAMP, resulting in the detection of significantly more infections compared to RDT or microscopy ,48,52,53Eight studies—all of which were conducted in sub-Saharan Africa—have applied commercial LAMP kits as a screening tool in malaria prevalence surveys. For instance, LAMP has been used in several studies in Uganda to investigate the effect of HIV infection on malaria incidence ; the effLAMP has also been applied for assessing risk factors and spatial clustering of asymptomatic malaria though active and reactive surveillance in low transmission settings ,61,62. IPlasmodium infection. Malaria prevalence was 16% and 34% by microscopy and LAMP, respectively and submicroscopic Plasmodium infection was found in 22% of the children [A single study performed in Congo has applied the Illumigene Malaria kit. Venous blood (4 mL) with EDTA was taken from 1088 children aged between 6 and 59 months for assessing the prevalence of anemia and its relationship with asymptomatic submicroscopic children .It is unlikely that LAMP will replace conventional diagnostic tools such as RDTs and microscopy in the point-of-care diagnosis of clinically symptomatic malaria in endemic settings ,9,33,64.P. vivax infections—requiring additional liver-stage treatment—are prevalent [P. falciparum and P. vivax, it is at an additional cost of 5.20 euros per additional test and it still does not provide a very detailed and/or accurate description of the Plasmodium species composition [P. knowlesi—which has the ability to cause severe disease even at low parasite densities—is present. Molecular testing for quality assurance of microscopy-confirmed cases in Indonesia recently found that microscopy was unable to identify or miss-classified up to 56% of confirmed malaria cases, half of which were later determined to be P. knowlesi infections [Malaria species identification is important in areas where co-endemicity of non-falciparum species occurs, e.g., in areas where revalent ,37. Althposition . This hafections . Althougfections .Taq polymerase inhibitors such as hemoglobin [The lower cost of the Loopamp Malaria Detection Kits, together with the endorsement from FIND, is likely the main reason as to why primarily these kits have been used in malaria endemic settings . The simmoglobin , and is moglobin ,42,68. Hmoglobin .Plasmodium species identification and parasite density quantification.An additional advantage with the Loopamp kits is the possibility of using dried blood spots on filter paper, as supposed to venous blood samples or whole blood from finger pricks ,28. ThisImproved throughput for screening of larger numbers of samples simultaneously would benefit routine prevalence surveys in areas aiming at malaria elimination ,42. HighFinally, the use of the naked eye or a UV-lamp for the read-out of LAMP results is comparably less objective than the use of specialized equipment such as the Illumipro-10™ incubator or the LA-500 real-time turbidimeter ,36,41,65P. falciparum parasites typically accumulate in the placenta, whereas the parasite density in the peripheral circulation is low. Low-density infections are also common for P. vivax, with dormant hypnozoite stages that may cause multiple relapses since the radical cure primaquine is contradicted during pregnancy [Malaria in pregnancy affects the health of the fetus, resulting in substantial adverse neonatal morbidity and mortality. The WHO therefore recommends intermittent preventive treatment in pregnancy (IPTp) in areas of moderate to high malaria transmission in sub-Saharan Africa . In loweregnancy ,72.LAMP has shown to greatly improve the detection of these low-density infections in maternal peripheral blood during pregnancy ,75,76,77Given their improved sensitivity, commercially available LAMP kits have recently been introduced as an additional parameter in clinical trials for assessing the effectiveness of malaria preventative measures during pregnancy ,78,79. TIt is currently understood that placental malaria is a major cause of adverse birth outcomes ,80,82; hFollowing this, some studies have assessed the relationships between longitudinal measures of submicroscopic parasitemia during pregnancy and placental malaria. Although data are limited, current findings have shown that women with only submicroscopic infection have an increased risk of placental malaria compared to women without any parasitemia . The risThe current recommendations for malaria testing in returning febrile travelers issued by theUnited States Center of Disease Control, is to provide a preliminary result by RDT followed by on average three consecutive thick and thin films spaced 6 to 8 h apart, to ensure that no parasites are present . RepeateP. falciparum malaria have high enough parasite densities to be detected by microscopy and RDT when attending health care in non-endemic areas [P. falciparum infections presented with parasite density below 100 p/µL, i.e., in the range of the detection limit of microscopy and RDT [P. ovale and P. malariae—typically present with low parasite densities and are therefore often missed [The Illumigene Malaria LAMP test has primarily been evaluated for use in non-endemic areas, both prospectively on blood collected at the primary visit ,84,85,86ic areas ,84,85,88ic areas ,89 and o and RDT . Furthern missed .The practicality of the Illumigene kit and accompanying equipment has proven to be useful in non-endemic settings . It has A cost analysis assessment of a novel algorithm for screening febrile patients with LAMP in a non-endemic setting, estimated that a single LAMP test with a high negative predictive value will provide a per-patient cost saving of USD$13 . DespiteThe need for highly sensitive, field adaptable diagnostic tools that can readily be used in low resource settings is apparent . AlthougP. falciparum infections in pregnant women might have on adverse birth outcomes requires further evaluation [Although LAMP in its current form and cost is unlikely to become a reality for point-of-care diagnosis of clinically symptomatic malaria in low resource settings ,9,33,44,aluation ,80,81, taluation ,77. Furtaluation ,34,83. Haluation .Malaria LAMP may also provide a powerful molecular alternative to enable the detection of low-density infections especially in areas where prevalence is declining . While tPlasmodium species differentiation, especially for P. knowlesi.Improving Improving the throughput of commercial kits, whilst providing objective read-out of results, and reducing the need for plastic consumables.Assessing the impact on transmission of applying LAMP in malaria elimination strategies.Assessing the impact on adverse birth outcomes of early detection of low-density malaria infections by LAMP during pregnancy.Detailed assessments of cost and cost-effectiveness of malaria LAMP in different transmission settings, especially for use in malaria elimination and antenatal care programs.Following is a suggested list of areas that require further exploration and/or development for commercially available LAMP kits:Despite LAMP being a promising near point-of-care tool for use in low resource settings, it is unlikely that commercially available LAMP kits will replace conventional diagnostic tools in the diagnosis of clinical malaria in endemic areas. They may, however, provide a sensitive molecular alternative for use in malaria elimination programs, as well as in malaria screening during pregnancy in low transmission settings, and in returning travelers in non-endemic settings. Nevertheless, further improvements of the already available commercial kits and detailed assessments of cost-effectiveness could improve their uptake on a larger scale.