About half (53.92%) of WaSt children had low MUAC, and all were underweight. Younger children aged <36 months had more WaSt, particularly males. Males with WaSt had higher median MUAC than females . A WAZ <−2.60 threshold detected WaSt with excellent sensitivity (99.02%) and high specificity (90.71%). MUAC threshold <13.20 cm had good sensitivity (81.58%) and moderate specificity (76.15%) to detect WaSt. WaSt prevalence of 5% is a public health concern, given its high mortality risk. All children with WaSt were underweight and half had low MUAC. WAZ and MUAC could be useful tools for detecting WaSt. Prevalence monitoring and prospective studies on WAZ and MUAC cut‐offs for WaSt detection are recommended. Future consideration to integrate WAZ into therapeutic feeding programmes is recommended to detect and treat WaSt children.We assessed prevalence of concurrently wasted and stunted (WaSt) and explored the overlaps between wasted, stunted, underweight and low mid‐upper arm circumference (MUAC) among children aged 6–59 months in Karamoja, Uganda. We also determined optimal weight‐for‐age (WAZ) and MUAC thresholds for detecting WaSt. We conducted secondary data analysis with 2015–2018 Food Security and Nutrition Assessment (FSNA) cross‐sectional survey datasets from Karamoja. Wasting, stunting and underweight were defined as <−2.0 z‐scores using WHO growth standards. Low MUAC was defined as <12.5 cm. We defined WaSt as concurrent wasting and stunting. Prevalence of WaSt was 4.96% . WaSt was more prevalent in lean than harvest season (5.21% vs. 4.53%;  WaSt prevalence of 5% raises public health concerns. In future, therapeutic feeding programmes should consider using WAZ to detect WaSt for treatment. Half (53.92%) of WaSt children had low MUAC, and all WaSt children were underweight.Younger children aged <36 months, particularly males, had more WaSt at 1.77 ratios. Nonetheless, majority of WaSt children who had low MUAC were females. Further analysis of the WaSt sex difference may prove useful.Thresholds of WAZ <−2.60 and MUAC <13.20 cm detected WaSt at optimal performance. Prospective studies on WAZ and MUAC cut‐offs for WaSt detection in different populations are recommended.1Childhood undernutrition remains an important public health problem. Globally, stunting (height‐for‐age z‐scores [HAZ] <−2.0) and wasting (weight‐for‐ height z‐scores [WHZ] <−2.0) affects about 151 and 51 million children under 5 years, respectively  and in November/December for the nonlean (postharvest) food security seasons. The FSNA is a population‐based cross‐sectional survey adapted from the Standardized Monitoring and Assessment of Relief and Transitions (SMART) survey protocol  <−2.0.Stunted: height for age z‐score (HAZ) <−2.0.Underweight: weight for age z‐score (WAZ) <−2.0.Severity of anthropometric deficits were defined as: no deficit ≥−2; moderate is <−2 and ≥−3 and severe <−3.WaSt case was defined as concurrent WHZ<−2.0 and HAZ <−2.0  and/or MUAC <12.5; severe acute malnutrition (SAM) as severe wasting (WHZ <−3) and/or MUAC <11.5 cm and moderate acute malnutrition (MAM) as moderate wasting (WHZ ≥−3 to −2) and/or MUAC ≥11.5 cm and ≤12.5 cm  statistic to assess the probability of a higher median of WHZ and HAZ in wasted‐only, stunted‐only compared with WaSt cases , the age distribution varied statistically  between the children in our study sample and those excluded for having incomplete anthropometric data. The flowchart  in the study sample than underweight 25.96%), wasting (12.03%) and low MUAC  of the children had at least one form of anthropometric deficit Figure . The pre3.4p = .0182; Table The prevalence of wasting (12.57%) was higher in the lean season than in the harvest season (11.11%). The prevalence of stunting ranged from 31.00% in 2016 to 36.14% in 2018. There were more children with WaSt in the lean season than in the harvest season  followed by low MUAC (20.72%) were the most prevalent forms of undernutrition in children aged 6–11 months Figure . Wasting3.6n = 1,634), there were more WaSt‐only cases  compared with those who had both WaSt and low MUAC  among the males (Table p < .001). The median MUAC among children with WaSt and low MUAC was slightly higher in males than in females  and stunting (37.00% vs. 30.25%). WaSt was also more frequent in males  than in females . The overall male to female WaSt prevalence ratio was 1.77 . WaSt prevalence peaked at 12–23 months in both sexes. Among children aged 12–23 months, WaSt was more prevalent in males than females  with probability of superiority of 51.43% . The CLES showed a slightly superiority WHZ (wasted‐only) > WHZ (WaSt), although the difference was not statistically significant. The median HAZ in stunted‐only children compared with WaSt children was significantly different . The probability of HAZ in a random stunted‐only case being above HAZ in a WaSt case was 58.07% . The CLES is >50% and the 95% CI does not include 50% that showed a clear ‘superiority’ of HAZ (stunted‐only) > HAZ (WaSt). WaSt children also had a significantly lower median WAZ than children with wasting only  or stunting only . Children with WaSt had a lower median MUAC compared with children with wasting only  and stunting only  and a high specificity (90.71%) with an area under the receiver operator characteristic curve (AUC) of 0.980  of the children with WaSt had low MUAC. An overlap between WaSt and low MUAC implies a child had simultaneously experienced the four common anthropometric deficits: wasting, stunting, underweight and low MUAC. Also, all WaSt children were underweight, as previously reported (Myatt et al., In the present analysis, the proportion of children with different forms of anthropometric deficits were associated with age. Both underweight and low MUAC were common among children aged 6–11 months. However, the proportion of children with low MUAC declined thereafter, underweight rate was higher at age 12–23 months but decreased steadily after 24 months. Wasting was common among children <24 months but less among >24 months old. Stunting was most prevalent beyond age 24 months. The higher underweight rate in the age group 36–48 months may indicate higher stunting rather than wasting rate (Waterlow, Younger children aged <36 months particularly males were more likely to have WaSt. The estimated WaSt male:female prevalence ratio is similar to findings reported in an earlier study (Myatt et al., Currently, the reasons for the observed sex difference in WaSt prevalence remain unclear and require further investigations. In a subpopulation analysis, we found that fewer males were WaSt and had low MUAC compared with those with WaSt‐only, whereas more females had WaSt and low MUAC than those with WaSt‐only. The sex disparities in childhood undernutrition could be due to the sex differences in body composition mainly muscle mass and body fat distribution measured by the different anthropometric indicators (Admassu et al., In the present analysis, children with WaSt were more severely stunted than stunted‐only children. The level of wasting did not differ between wasted‐only and WaSt cases. This is contrary to previous studies where WaSt cases were both severely wasted and stunted compared with wasted‐only or stunted‐only cases (Garenne et al., Thresholds of WAZ <−2.60 and MUAC <13.20 cm were useful for detecting WaSt cases with optimum performance. WAZ <−2.60 cut‐off detected WaSt cases at an excellent sensitivity and a high specificity. These WAZ and MUAC cut‐offs were similar to the thresholds found in an earlier study (Myatt et al., Few published data on anthropometric indicators for screening and detecting WaSt exist. A recent study reported that WAZ <−2.60 and MUAC <13.30 cm performed considerably better than chance to identify WaSt cases with the areas under the ROC curves of 0.9726 for WAZ and 0.8759 for MUAC (Myatt et al., 4.1The dataset used in this analysis was collected based on a standardized protocol during each round of the survey. However, our study is not devoid of limitations usually associated with retrospective analysis. Random error is a common challenge associated with a fixed sample size of a historic dataset. Nonetheless, random error associated with secondary dataset was likely minimized because of the large sample size of the dataset and the probability proportional to population size sampling procedure used in the FSNA surveys (Hulley et al., 5WaSt prevalence of 5% found in Karamoja is a public health concern given the increased risk of death associated with WaSt. All four anthropometric deficits, underweight, wasting, stunting and low MUAC, occurred simultaneously in half of the WaSt cases. Some of the WaSt cases did not have low MUAC. Our analysis suggests that WAZ and MUAC could be useful tools to detect WaSt cases with optimal performance. However, future prospective studies on WAZ and MUAC cut‐offs for WaSt detection in different populations would be required. Routine prevalence reports are useful for decision‐making. Future consideration to include MUAC into GMP and IMCI and WAZ into therapeutic feeding programmes to detect WaSt for treatment is recommended.The authors declare that they have no conflicts of interest.GAOOA, HW, MM, AB, JC, BKM, JNK and CAK conceptualized and designed the research study. GAOOA, HW, RA, JC, JNK, MM, AB and CAK analysed and interpreted the data. GAOOA, HW, JNK and CAK drafted the initial manuscript. GAOOA, HW, BKM, JNK, EM, IO, ALO, RA, JC, MM, AB and CAK reviewed and finalized manuscript.


Malaria is threatening more than half of Afghanistan population. Asymptomatic malaria is notable problem against malaria controlling strategies. In this study we evaluated the asymptomatic malaria status in Nangarhar Province, Afghanistan in 2017.Overall, 296 finger blood samples were taken on DNA Banking Cards and microscopic slides from asymptomatic individuals in Jalalabad city. We used a novel post real time PCR high resolution melting analysis beside microscopy and semi-nested multiplex PCR to evaluate status of asymptomatic malaria in this city.P. falciparum, P. vivax and mixed infection of P. falciparum and P. vivax, respectively.The prevalence of asymptomatic malaria in Jalalabad city was determined 1.7% (5/296), 7.43% (22/296) and 7.78% (26/296) by microscopy, Seminested multiplex PCR and qRT-PCR-HRM, respectively. Out of 26 positive cases were detected by qRT-PCR-HRM, 21, 1 and 4 cases were detected Our data indicating on existence of significant number of asymptomatic reservoirs that assists in prolonged endemicity of the disease. On the other hand, the molecular methods are better alternatives for microscopy especially for monitoring of asymptomatic cases of malaria. Plasmodium genus  are causative agents of human malaria. Despite many efforts towards eliminating malaria, there were 212 million new cases and 429000 deaths in 2015. Malaria had always been one of public health complications in Afghanistan. More than half of the population live in malaria-endemic regions. Plasmodium vivax is the dominant species (70%–95%) and P. falciparum located in the second place (5%–30%) (Five species of place 5%–0% (3).Malaria severity varies from symptomatic , to asymptomatic. Symptomatic malaria has been at the core of epidemiological studies' attention thanks to its mortality and morbidity. The manifestations of complicated malaria encompass cerebral malaria, convulsions, malarial anemia, haemoglobinuria, hypoglycemia, metabolic acidosis (associated with respiratory distress), acute pulmonary edema, acute renal failure, jaundice, circulatory collapse, hyperparasitaemia, high fever electrolyte disturbance, and/or spontaneous bleeding. On the other hand mild or uncomplicated malaria offers manifestations like fever, chills and sweats, headache, vomiting, watery diarrhea, anemia, jaundice, and splenomegaly , 5.Plasmodium parasite of any density who displays no fever or other acute symptoms and have not received recent antimalarial drugs is called asymptomatic. Therefore, asymptomatic carriers play a role as a reservoir that leading to persistence of malaria transmission within their localized populations  analysis is a cost-benefit implement for post-real-time analysis of PCR products. It does not require post PCR manipulating and detect multiplex species simultaneously . However, the molecular approaches like nested PCR and real-time PCR offer detection of aneously , 9.There are some works carried out in Afghanistan especially on drug resistance evaluation, risk prediction, vector controlling and epidemiological studies –12.Unfortunately there has been no attempt to evaluate the asymptomatic malaria status in Afghanistan so far. In this study we were planning to evaluate the asymptomatic malaria status in Nangarhar province by make a use of real-time PCR-HRM.A cross-sectional study was performed by available sampling method in 2017. In this study, referred individuals to the Nasiri laboratory were checked whether they have been suffered malaria since at least 6 months ago or possessing any symptoms of malaria, especially body temperature. These patients had issues other than malaria and febrile individuals were omitted from the study after checking by a physician. Out of them, only 296 individuals were selected as not malaria affected individuals. Then 296 finger blood samples were taken on DNA Banking Cards  and microscopic slides. Nangarhar province  is one of the hyper malaria endemic regions that located in the East of Afghanistan.Approval of ethics application with appropriate experimental protocols was taken from Medical Ethic Committee of Tarbiat Modares University, Tehran, Iran. All the protocols used in this study were in accordance with the approved guidelines (IR.TMU.REC.1395.401) and informed agreement were taken from all people participated in this study.Microscopic analysis was performed on prepared thick and thin blood films under ×100 lens with oil immersion. Totally 200 good fields were examined for detection of parasites and a double check procedure was performed for identification of mixed cases. For quality control 10% of negative cases and all of positive cases were double-checked bye a skilled microscopist. For final confirmation, the slides and their pictures were sent to the National Malaria Center, Tehran University of Medical Sciences, Tehran, Iran.DNA extraction was performed on DNA banking cards (DBCs). After punching 2 mm discs from the DBCs, 3–4 discs were applied for DNA extraction by the SinaPure DNA extraction kite . We added 3-4 discs to a sterile 1.5 or 2 polypropylene tube then add 400 μl Lysis buffer and vortex at max speed for 1 min. In the next step we added 300 μl Precipitation solution and vortex at max speed for 30 sec. The solution was transferred to a spin column with collection tube by pipetting and centrifuged at (12.100×g) for 1 min. The collection tube was discarded. Column spin were placed in new collection tube then 400 μl Wash buffer I was added and centrifuged at 12.100×g for 1 min. After discarding the flow-through. The spin-column Washed with 400 μl of Wash buffer II twice at centrifugation of 12.100×g for 1 min. The flow-through was discarded and the column carefully transferred to a new 1.5 ml tube. 30 μl 65 °C preheated elution buffer or TE buffer was placed in the center of the column, lid closed and incubate for 3–5 min at 65 °C. Thereafter, we centrifuged at 12.100×g for 1 min and eluted DNA were kept in −20.Plasmodium species. In the second round the PLF primer, 2 μl of diluted product of first round and four primers of plasmodium spp.  were added into 12.5 μl of “2X Taq Master Mix ReD” 2So4, 3 mM Mgcl2, 0.2% tween 20, 0.4 mM dntPs, 0.05 U/μl Taq DNA polymerase, inert red dye and stabilizer), to reach final concentration of 25 μl. The products of the second round  were seen in 2% agarose gels.Two rounds of semi-nested PCR (SNM-PCR) were performed . BrieflyPlasmodium species as described by Chua . Then the pTG19-T vector was employed to clone the amplicones directly through TA cloning technique . The colony PCR was performed by make use of universal primers; M13F: AGGGTTTTCCCAGTCACGA and M13R: GAGCGGATAACAATTTCACAC for confirmation of the cloning . For amplification, 0.1 μM of each primers (forward primer 5′-GRAACTSSSAACGGCTCATT-3′ and reverse primer 5′ -ACTCGATTGATACACACTA-3′), 4μl  of extracted DNA and 4 μl of 5X Hot Firepol® EvaGreen® HRM Mix  were used in qPCR 8-strip tubes .The thermal program included an initial denaturation at 95 °C for 15 min, followed by 40 cycles of amplification consisting of 95 °C for 15 sec (denaturation step), and 60 °C for 1 min . The amplicons were then subject to a melt program raised from 60 °C to 95 °C. During HRM, the amplicons were denatured before development of melting curves in the inflexion point, where changes in fluorescence concerning changes in temperature (dF/dT) were recorded with a ramp of 0.3 °C/sec. Ultimately, obtained melting curve profiles were analyzed by making a use of HRM software for Windows ® version 3.0.1. (Applied Biosystems).P. falciparum and P. vivax 18S rRNA sequences with the neighbor-joining algorithm using Molecular Evolutionary Genetics Analysis (MEGA) software, version 7.0 . Editing and aligning of sequences with reference sequences from GenBank, were performed by using two commercial software packages (Sequencher v. 4.4 and CLCMainWorkbench5.5). The phylogenetic tree was created for sion 7.0 .For analysis of the data in the present work, the chi-squared test performed by making use of software package SPSS, ver. 14.0. In addition, this software package was used for determining and comparing of the Sensitivity and Specificity among different diagnosis methods. The significance level was considered by 95% confidence interval.P=0.631) and age (P=0.694) in taking asymptomatic malaria.Among 296 participants, there were 175 (59.1%) male and 121 (41.9%) female with a mean age of 23 ((IQR: 5–71 and SD: 11.02). The statistical analysis showed that there were no significant association between sex , SNM-PCR  and real-time PCR-HRM , respectively  observed in specificity . Comparison of results for microscopy with three real-time PCR based methods that have been used in this study and two others is shown in The performance of molecular tests was assessed using microscopy as the gold standard. Real-time PCR-HRM has higher sensitivity  and specificity ; besides, the positive predictive values (PPV) and negative predictive values (NPV) were 100% and 92.7%, respectively . On the Plasmodium species from Jalalabad city were compared with other species in GenBank using the neighbor-joining algorithm  were seen in all of these techniques. Compared with these real-time PCR-based methods, microscopy showed unremarkable agreement, with kappa values. This is while real-time PCR-HRM showed more powerful performance in detection of true positives and true negatives , 26.There are many studies on asymptomatic malaria all over the world. Iran located in the west of Afghanistan is a country in the elimination phase. Studies in this country were done mostly in the southern regions and indicating on the absence of asymptomatic carriers. Although there are no data about asymptomatic malaria in Pakistan (south and east of Afghanistan), there are some works in India highlighted the existence of high prevalence of the reservoirs, especially in eastern populations. In Africa, there is a high prevalence of the asymptomatic malaria in African countries –33.P. falciparum (78.8 %) that is a warning for National Malaria and Leishmaniasis Control Program. Meanwhile, while our study was conducted, the most cases of malaria were P. vivax and rarely P. falciparum were observed in Jalalabad. This hidden malaria will assist in persist of severe form of malaria in the region, so it is worth doing an active case detection of asymptomatic infections  are big problems in front of establishing molecular methods in Afghanistan. Therefore, for promoting the malaria control program capacities the improvement of infrastructures is essential.P. falciparum is the common form of asymptomatic malaria in Nangarhar province. This should be taken into consideration by malaria managers, however, the existence of mixed infection and P. vivax should not be neglected. On the other hand, the molecular methods are better alternatives for microscopy especially for monitoring of asymptomatic cases of malaria.


The recent expansion of tools designed to accurately quantify malaria parasite-produced antigens has enabled us to evaluate the performance of rapid diagnostic tests (RDTs) as a function of the antigens they detect—typically histidine rich protein 2 (HRP2) or lactate dehydrogenase (LDH).Plasmodium falciparum malaria. Furthermore, antigen dynamics after successful anti-malarial drug treatment were evaluated in these samples using the Q-Plex Human Malaria Array (4-Plex) to quantify antigen concentrations.For this analysis, whole blood specimens from a longitudinal study in Bancoumana, Mali were used to evaluate the performance of the ultra-sensitive HRP2-based Alere™ Malaria Ag P.f RDT (uRDT). The samples were collected as part of a transmission-blocking vaccine trial in a high transmission region for Plasmodium LDH (pLDH), with the latter decaying more rapidly. Motivated by these differences in rates of decay, a novel algorithm that used HRP2:pLDH ratios to predict if an individual had active versus recently cleared P. falciparum infection was developed. The algorithm had 77.5% accuracy in correctly classifying antigen-positive individuals as those with and without active infection.The uRDT had a 50% probability of a positive result at 207 pg/mL HRP2 [95% credible interval (CrI) 160–268]. Individuals with symptomatic infection remained positive by uRDT for a median of 33 days [95% confidence interval (CI) 28–47] post anti-malarial drug treatment. Biphasic exponential decay models accurately captured the population level post-treatment dynamics of both HRP2 and These results characterize the performance of the ultra-sensitive RDT and demonstrate the potential for emerging antigen-quantifying technologies in the field of malaria diagnostics to be helpful tools in distinguishing between active versus recently cleared malaria infections. Plasmodium falciparum malaria, and RDTs targeting P. falciparum infections typically detect HRP2, while LDH is a constitutive enzyme expressed by all Plasmodium species. RDTs can specifically detect P. falciparum infections through antibodies targeting subspecies epitopes on the LDH antigen, as well as all Plasmodium species through conserved epitopes. Of note, HRP2-based RDTs can exhibit cross-reactivity with HRP3 due to antigenic similarity.Malaria is most commonly diagnosed in humans by either microscopy or rapid diagnostic tests (RDTs), which detect antigens produced by malaria parasites , 2. The Until recently, malaria RDT evaluation programmes have focused on benchmarking malaria diagnostic tests against parasite density measured either by microscopy or nucleic acid tests, although antigen concentrations of standard samples have recently begun to be included –5. RDTs ® and Pfs230-EPA/Alhydrogel® individually and in combination for safety and functional immunogenicity in malaria-exposed Malian adults . The vaccine was given on a 0, 1, 6 month schedule in the 1st year with a booster dose 12 months after dose 3. Given the longitudinal nature of the study cohort, the population level antigen dynamics for both HRP2 and LDH were investigated, as well as relative antigen abundance post-treatment, as determined with the Q-Plex enzyme-linked immunosorbent assay (ELISA).This report describes the performance of the ultra-sensitive Alere Malaria Ag P.f RDT (uRDT) on samples collected in a phase 1 clinical trial malaria vaccine study in Bancoumana, Mali. This trial assessed Pfs25M-EPA/AlhydrogelPlasmodium falciparum. This trial investigated the safety and immunogenicity of Pfs230D1M-EPA/Alhydrogel® and Pfs25M-EPA/Alhydrogel, both transmission-blocking vaccines against P. falciparum. Transmission-blocking vaccines were administered on study days 0, 28, 169, and 540. Blood smears were prepared before each vaccination, at least monthly post vaccination, or when clinically indicated. Starting 1 week after the third and fourth vaccinations, blood smears were prepared twice a week for 6 weeks at the same time that subjects underwent direct skin feeding assays with colony-raised Anopheles coluzzii to assess malaria parasite transmission. Whole blood samples to be analysed in this study were collected at per-protocol scheduled blood draws 1-6 weeks after the fourth vaccination  during peak malaria transmission season (July–December). Between 1 and 21 mL of whole blood was collected at each blood draw for each study participant, all who agreed to have blood samples stored for future research prior to enrollment. Individuals who at any point presented with symptomatic malaria, defined as any parasitaemia by blood smear or RDT positive result with symptoms consistent with malaria, were treated with anti-malarial drugs artemether–lumefantrine (Coartem or Laritem) for uncomplicated malaria and artemether for severe malaria. Whole blood samples for individuals treated with anti-malarials were collected at the same per-protocol frequency as the remainder of the study cohort. Microscopy-positive asymptomatic individuals were not treated, per Malian National Policy on Malaria Control Guidelines.Individuals in this analysis were part of a double-blind, randomized, phase 1 clinical trial conducted by the Laboratory of Malaria Immunology and Vaccinology (LMIV)/National Institute of Allergy and Infectious Diseases (NIAID)/National Institutes of Health (NIH). Bancoumana is a rural village 60 km from Bamako, Mali, with high prevalence of Frozen whole blood samples (n = 622) were sent to PATH’s laboratory  for further evaluation. Two ultra-sensitive HRP2-based Alere Malaria Ag P.f RDTs (uRDT), product number 05FK140, lots 05LDB005A and 05LDB004A, were used to test in duplicate each specimen, all of which had been stored at − 80 °C. The test required 5 µL of whole blood and was run following the standard workflow outlined in Das et al. . A finalPlasmodium LDH (pLDH) concentrations were quantified using the Q-Plex Human Malaria Array (4-Plex), which quantifies pLDH by detecting pan epitope  at time of blood draw visits. Out of 160 participants, 110  were positive by ultra-sensitive rapid diagnostic test (uRDT) and 99  were positive by microscopy at least once during the sampling window. Furthermore, 101  of these participants presented with symptomatic infection at least once. Of the 166 microscopy-positive whole blood specimens, 155 (93%) were The performance of the uRDT was evaluated against microscopy as well as HRP2 and all-malaria pLDH confirmed by Q-Plex Table . There wComparable uRDT positivity and HRP2 concentration data exists for two recently published studies conducted in asymptomatic individuals, one in Uganda  . DetailsP. falciparum.Compared to a small subset of samples in Mali for which conventional RDTs (co-RDTs) were run in the field (n = 35), the uRDT was on average more than tenfold more sensitive in its LOD than the co-RDT: 50% probability of detection was achieved at 207 pg/mL HRP2 for the uRDT compared to 3140 pg/mL HRP2 for co-RDT. However, both tests had higher detection thresholds than expected (80–100 pg/mL for uRDT and 800 pg/mL for co-RDT) . Out of 2 = 0.61 and R2 = 0.29, respectively) among all microscopy-positive samples  and for pLDH was 101.8 pg/mL (geometric SD 17.7). The correlation for the transformed log10 value of each antigen (pLDH and HRP2) with log10 parasitaemia by microscopy was poor, but was higher for pLDH than HRP2  estimated that median time to uRDT negativity was 33 days post-treatment (95% CI 28–47)  were still positive by uRDT, with the highest proportion 43.3%) uRDT-positive and microscopy-negative  and only 8 days for pLDH  to avoid uncertainty in model predictions once median antigen values declined below the limit of quantification. Using criterion that minimized both AIC and BIC, a biphasic exponential model best estimated population level antigen decay  with persistent antigenaemia (HRP2 and/or pLDH positive). The relationship between the ratio, HRP2 concentration, and recent treatment history is visualised in  were then selected such that sensitivity and specificity were maximized. If HRP2 is > 100 pg/mL, the optimal threshold (HRP2:pLDH ratio) for classifying whether samples come from individuals with cleared infection is 8.99, above which cleared infection due to recent anti-malarial treatment is predicted [area under the curve (AUC): 0.92] , HRP2:pLDH ratios were calculated. Samples from six study participants treated twice during this time frame were included as separate samples with differing days of treatment. The median HRP2:pLDH ratio was 0.348 [interquartile range (IQR): 0.07–2.11] pretreatment and 3.41 (IQR: 0.28–51.0) post-treatment Fig. a. HRP2:p in Fig. b. Recent92] Fig. c. Here t92] Fig. d. OveralFinally, there were 11/614 samples (1.8%) from eight unique individuals positive by direct skin feeding (DSF) experiments. On average, the HRP2:pLDH ratio was lower (P = 0.02) for individuals with a positive DSF result in reference to microscopy and DSF negative samples  evaluate performance of the uRDT compared to other diagnostic methods, (2) investigate how relative antigen concentrations can classify infections, and (3) better understand the post-treatment dynamics of pLDH and HRP2.Comparing the HRP2 threshold at which there was a 50% probability of detection by uRDT with the same values from two other studies in Uganda and Myanmar , 8 resulpfhrp2/3 deletions [pfhrp2/3 deletions [In this Malian population, pLDH had a stronger correlation with parasitaemia than HRP2, with the constraint that parasitaemia was quantified by microscopy and not quantitative polymerase chain reaction (qPCR). This is consistent with findings that residual HRP2 lingers after parasite clearance, whereas pLDH has a shorter half-life and is more indicative of active infection , 23. Thieletions , 26. In eletions , 25.Several first-order kinetics models have previously been used to fit HRP2 dynamics , 26. HerP. falciparum infections in the future, a standardized protocol for dealing with pLDH values of 0 pg/mL (or < LOQ) in HRP2:pLDH calculations will need to be defined.One of the concerns accompanying introduction of the uRDT is that due to HRP2 persistence, ultra-sensitive HRP2-based diagnostics may lead to overtreatment due to individuals with recently cleared infections testing positive and being treated with anti-malarial drugs when there may be another infection or illness causing fever . The abiP. falciparum setting and to adults over 18, whereas children under 5 years carry the majority of the global malaria burden [This analysis was limited to individuals in a high-transmission a burden . qPCR daa burden . FurtherP. falciparum cases. Further studies analysing a range of longitudinal datasets that include anti-malarial drug history are required to improve upon and validate this approach.A deeper understanding of HRP2 and pLDH antigen dynamics in malaria endemic populations will greatly inform the understanding of the performance of RDTs and the utility of RDTs in malaria interventions beyond case management. The data presented here, only considering the relative proportions of two malaria antigens (HRP2 and LDH), suggests that antigen dynamics can be used to differentiate active infections from recently treated infections in Additional file 1: Figure S1. Scatterplot showing correlation between parasite count by microscopy and HRP2 concentration quantified by Q-Plex ELISA (R2 = 0.29) for all samples P. falciparum positive by microscopy (> 0 parasites/µL). Abbreviations: ELISA, enzyme-linked immunosorbent assay; HRP2, histidine rich protein 2; pLDH, Plasmodium lactate dehydrogenase. Figure S2. Scatterplot showing correlation between parasite count by microscopy and pLDH concentration quantified by Q-Plex ELISA (R2 = 0.61) for all samples P. falciparum positive by microscopy (> 0 parasites/µL). Table S1. Antigen ratios by direct skin feed (DSF) infectivity status. Geometric means for the two antigens of interest, HRP2 and pLDH, along with the geometric mean HRP2:pLDH ratio for study participants that were DSF-positive (n = 11) or DSF-negative (n = 603), grouped by microscopy result. Individuals positive by DSF had a significantly lower HRP2:pLDH ratio (p = 0.02) on average in reference to DSF and microscopy negative individuals. Abbreviations: geo, geometric; HRP2, histidine rich protein 2; pLDH, Plasmodium lactate dehydrogenase; SD, standard deviation.


Plasmodium species infections among pregnant women attending antenatal care has not been yet studied at the study area. Therefore, the aim of this study was to assess the prevalence of asymptomatic Plasmodium species infections among pregnant women attending antenatal care at Fendeka town health facilities.Malaria in pregnancy remains a major public health problem especially in sub-Saharan Africa. In malaria endemic areas, majority of pregnant women may remain asymptomatic but still associated with complications on the mother and her foetus. They also serve as reservoirs and act as transmitters of infection. Despite these effects, the prevalence of asymptomatic Plasmodium species infections. Univariate logistic regression was employed to assess factors associated with asymptomatic Plasmodium species infection. Variables with P-value < 0.25 in the univariate logistic regression were selected for multivariate logistic regression analysis model. Odds ratios with 95% confidence intervals were calculated and P- values < 0.05 were considered as statistically significant.Health facility based cross -sectional study was conducted from February to March 2019. A total of 331 participants were enrolled by using convenient sampling technique. Socio-demographic and associated factors were collected by a face to face interview. All the 331 samples were tested using rapid diagnostic tests (RDTs) and microscopy. However, only 83 dried blood spot (DBS) samples out of 331 participants, were collected by using systematic random sampling technique for molecular analysis. Data was analysed using SPSS version 20. Descriptive statistics were used to determine the prevalence of asymptomatic Plasmodium species infections were detected using: RDTs, microscopy and real-time PCR altogether. The asymptomatic Plasmodium species infection prevalence was 17 (5.1%), 30 (9.1%) and 15(18.1%) using RDTs, microscopy and real-time PCR, respectively. Asymptomatic Plasmodium species infections were more likely to occur in primigravida , secundigravida , rural inhabitants  and in participants who did not use indoor residual spray (IRS) for the last one year .Overall, 37 (11.2%) asymptomatic Plasmodium species infection was 11.2%. Pregnant women who reside in the rural area, primigravidae, secugravidae and those who did not utilize indoor residual spray for the last one year were at high risk of infection. Therefore, routine laboratory diagnosis of asymptomatic Plasmodium species infection among pregnant women should be adopted as a part of the antenatal care.The prevalence of asymptomatic  Plasmodium species [P. falciparum and P. vivax pose the greatest threat in the world. The disease is transmitted to people mainly via the bites of infected female Anopheles mosquitoes [falciparum and P. vivax, respectively [P. falciparum to P. vivax in highland areas was reported by Gemeda et al [Malaria is a serious public health problem in tropical and subtropical regions of the world and caused by five  species , 2. Of tsquitoes . Sixty pectively . On the da et al .Globally, 125 million pregnant women are at risk of getting malaria every year. Of these, most cases and deaths are in Sub-Saharan Africa .Plasmodium species in pregnant women remain asymptomatic [Plasmodium species infection (API) is detection of Plasmodium species in the blood without any clinical symptoms of malaria. However, there is no standard definition for asymptomatic Plasmodium species infection [P. falciparum and only a few reports are available for other Plasmodium species in stable malaria transmission areas [In stable malaria transmission areas, most infections with ptomatic . Asymptonfection , 8. In aon areas .Plasmodium species infection during pregnancy causes placental infection and anaemia [Asymptomatic  anaemia ,11. Plac anaemia ; and ass anaemia ,13. Plac anaemia ,15. This anaemia ,17.Ethiopia has done lots of efforts to improve health status of pregnant women. According to the unpublished data from the Jawi district health office, the antenatal care coverage of the study area was greater than 85%. One of the efforts that has been done at the study area is to prevent pregnant women from infectious diseases like malaria.Plasmodium species and transmit the disease to the population [Ethiopia has launched malaria elimination program by 2030 and doing lots of efforts. To achieve this goal, currently the country used three major malaria prevention methods: early diagnosis and prompt treatment, vector control measures like use of indoor residual spraying (IRS) and insecticide treated bed nets (ITNs) methods and adoption of surveillance . Moreovepulation . These cPlasmodium species infection is common in stable malaria transmission area, this hypothesis holds true at Jawi district. In addition, there is no data that showed the magnitude and associated factors for asymptomatic Plasmodium species infection among pregnant women at the study area. Therefore, the aim of this study was to assess the prevalence of asymptomatic Plasmodium species infection and associated factors among pregnant women attending antenatal care at Fendeka town health facilities, Jawi district north west Ethiopia.Since asymptomatic 0 c-37.6 0 c and the altitude ranges from 648-1300m above sea level. The number of malaria reported cases in 2018/19 were 24,434 . Moreover, Tana Beles integrated sugar development project is found at the study area and there is year-round transmission of malaria. According to 2007 national central statistical census report, the total population of the district was 79,090; of whom 41,407 were men and 37,683 women [Health facility based cross-sectional study was conducted from February to March 2019 at Fendeka town Jawi district northwest Ethiopia . Jawi diPlasmodium species infections and the independent variables were: age, education level, occupation, marital status, family size, residence , Gravidity , gestational age , malaria prevention strategies .The dependent variable was asymptomatic Plasmodium species in the absence of any clinical symptoms of malaria .The detection of A woman who is pregnant for the third time and above.Plasmodium species infection [Sample size was calculated using single population proportion formula; using 9.4% prevalence of asymptomatic nfection ; and marUsually, pregnant women attend their follow up at the antenatal care clinic (ANC) to check whether the presence or absence of problems and/or anomalies related to pregnancy. As they reached at the ANC, the midwife gave their identity card and examined the status of their pregnancy. After checking their status and if they were normal, the pregnant women were recruited regardless of their appointment period. Then, the midwife requested their willingness to participate in the study. On volunteered pregnant women, socio-demographic characteristics like sex, residence, age, educational level, occupation, marital status, family size, and associated factors like gravidity, gestational age, ownership and utilization of ITNs and use of IRS, previous use of antimalarials during pregnancy and clinical questions like fever, shivering and sweating were collected via a face to face interview by the trained midwives.Blood sample was collected from participants pricking at their ring finger for RDTs, thick and thin blood films and DBS using Whatman filter paper. For RDTs, 5μl of blood was added using micropipette to the sample well and 2 drops (60μl) of buffer solution to the buffer well according to the manufacturer’s instruction. For thick and thin blood films preparation, 6 μl and 2 μl blood were used using an automatic pipette and smeared on a slide. In addition, each of the thick and thin films was dried overnight, and the thin films were fixed by dipping in absolute methanol for 10–20 seconds. Then, slides were stained with 3% Giemsa for 30–45 minutes . For DBSParasite detection by CareStart™ Malaria Pf / Pv (HRP2/pLDH) Ag Combo RDTs was done based on manufacturer instructions. Five microliters of capillary blood were added using micropipette to the sample well and 2 drops (60μl) of buffer solution to the buffer well and then after waiting for 20 minutes, the results were reported based on the manufacturers instruction Plasmodium parasites by experienced laboratory professionals working at Jaw health centre and hospital. Thick blood films were considered positive when sexual and asexual forms were detected and considered as negative after observing 200 high power fields without detecting any parasite.Thin and thick stained smears were examined under light microscope (Olympus CX-21) with 100x magnification for detection and identification of Finally, for molecular analysis, 3-mm punches of the DBS were punched out and placed in to 1.5-ml micro-centrifuge tubes. The punches inside the tube were treated with ATL buffer, proteinase K, AL buffer and 96% ethanol, mixing thoroughly by vortexing, brief centrifuge and incubation with consecutive additions. The mixtures were transferred into QIAamp Mini spin column and with subsequent addition of wash buffer AW1 and AW2, DNA was extracted. The DNA was eluted in 100μl of elution buffer, aliquoted and stored at -20°C until running PCR assay . The PCRPhoto -induced electron transfer PCR (PET-PCR) Primer sequence.5’-GGC CTA ACA TGG CTA TGA CG-3’.Original Genus 18sFor, 5’-agg cgc ata gcg cct ggC TGC CTT CCT TAG ATG TGG TAG CT-3’.Original Genus FAM 18sRev, 5’-ACC CCT CGC CTG GTG TTT TT-3’.Falciparum For, 5’-agg cgg ata ccg cct ggT CGG GCC CCA AAA ATA GGA A-3’.Falciparum Rev, HEX-5’-GTA GCC TAA GAA GGC CGT GT-3’.P. vivax For, 5’- agg cgc ata gcg cct ggC CTG GGG GAT GAA TAT CTC TAC AGC ACT GT-3’.P. vivax Rev, HEX-Plasmodium Genus and P. falciparum or P. vivax was performed in a 20μl reaction containing 2X TaqMan Environmental Master Mix buffer, forward and reverse primers for the Genus and P. falciparum multiplex assays and P. vivax singleplex assay, and 5μl of DNA samples. Samples were run in duplicate on manually loaded 96-well PCR plates. The reactions performed under the following cycling conditions: initial hot-start at 95oC for 15 minutes, followed by 45 cycles of denaturation at 95 0C for 20 seconds and annealing at 60 0C for 40 seconds for Genus and P. falciparum multiplex assays.Amplification of P. vivax single plex assay; initial hot-start at 95oC for 15 minutes, followed by 45 cycles of denaturation at 95 0C for 20 seconds and annealing at 60 0C for 40 seconds and then at 72 0C for 30 seconds. The correct fluorescence channel was selected for each fluorescently labelled primer set and the cycle threshold (CT) values recorded at the end of annealing step. Any sample with a CT value of 40.5 or below was considered positive [P. falciparum multiplex assays were performed for all samples. P. vivax single-plex assay was performed for Genus positive and P. falciparum negative samples.For positive . The GenPlasmodium species identification. Moreover, the same microscope mark (CX21) was used to minimize the possible instrumental error. The questionnaire were prepared in clear and understandable way. The clarity, understandability, and flow of each question were assessed properly. Data were collected under intensive supervision by the principal investigator (PI). Finally, the discordant results were rechecked by the PI along with other experienced laboratory professionals who did not participate in the initial identification and preparation process.Before the data collection, training was given to the data collectors by the principal investigator how to collect the sample and fill the questionnaire. Socio-demographic characteristics and associated factors were collected by midwives.; while experienced laboratory professionals were involved for RDTs, thin and thick blood film preparation, DBS sample collection and Ethical approval was obtained from the ethical review board of college of medicine and health sciences, Bahir Dar University. Then, permission letters were also obtained from Amhara public health institute, Zonal health department, district health office and from both health facility managers. After explaining the purpose of the study clearly, written informed consent was obtained from each pregnant woman. The data and sample collected from each study participant were used only for this study and confidentiality was always maintained. Finally, positive results were communicated to the attending midwives and received anti-malaria treatment.Plasmodium species infection among pregnant women. Univariate logistic regression analyses were performed to assess the association between dependent and independent variables. To filter out the confounding effects, variables with P-value < 0.25 in the univariate logistic regression model were further analysed in multivariate logistic regression analysis. Odds ratios at 95% confidence intervals were calculated and variables with P-value < 0.05 were considered as statistically significant.Data was analysed using SPSS version 20 statistical package. Descriptive statistics was used to assess the prevalence of asymptomatic Table 1).A total of 331 pregnant women were participated and of these 208 (62.8%) were rural dwellers. Most of the participants 111(33.5%) age were between 21–25 years with the median age of 25 years and standard deviation of (SD ± 5.35). Majority 203 (61.3%) of study participants could not read and write and farmers accounted for 174 (52.6%). Most 313 (94.6%) of pregnant women were married and 164 (49.5%) of them had 3–6 family sizes. Among the participants, 154 (46.5%) were multigravida and 151 (45.6%) were at their third trimester. Two hundred fifteen (65%) of the participants possessed ITNs; among these 154 (71.4%) had 1 ITNs at their home; and 197 (91.6%) of them had used by hanging it on their beds. Forty-seven (23.9%) of the participants used ITNs only in rainy season. Among the participants, 253 (76.4%) of them used IRS for the last one year (Plasmodium species infections was 17 (5.1%), 30 (9.1%) and 15 (18.1%) using RDTs, microscopy and real-time PCR, respectively. Of the 15 samples detected by PCR, 8 (53.4%), 5 (33.3%) and 2 (13.3%) were P. falciparum, P. vivax and mixed infections, respectively (Table 2). This Table depicted that for the detection of asymptomatic Plasmodium species infection, PCR was better as compared to RDTs and microscopy.The prevalence of asymptomatic Table 3).Residence, age, gravidity and IRS usage for the last one year had P-value < 0.25 by univariate logistic regression analysis. Moreover, residence, gravidity and IRS usage for the last one year depicted statistically significant association at P- value < 0.05 .In multivariate logistic regression analysis; residence, gravidity and IRS usage for the last one year had a statistically significant association with asymptomatic Plasmodium species infection could be an obstacle in malaria elimination program due to the low health seeking behaviours of infected individuals and absence of standardized diagnostic tests. As a result, they become reservoirs for sustainable malaria transmission [Asymptomatic smission . EspeciaRapid diagnostic tests detected and identified 5.1% of the APIs. This result is in line with study done in Nigeria 4.8% . But, itPlasmodium species infections among pregnant women. This result is consistent with studies done in south Ethiopia 9.1% [Microscopy detected and identified 9.1% of asymptomatic pia 9.1% , Congo 7pia 9.1%  and Nigepia 9.1% . Howeverpia 9.1% , and othpia 9.1% , 31, Burpia 9.1%  and Congpia 9.1% . In contpia 9.1% . These dpia 9.1% , 29, 31,Plasmodium species infection. It is consistent with the study done in Congo 19% [Real-time PCR detected and identified 18.1% of the asymptomatic ongo 19% . On the ongo 19% . These vPlasmodium species were identified as P. falciparum, P. vivax and mixed infections, respectively. This result is in line with MoH report that states 60% and 40% of the Plasmodium species in Ethiopia are P. falciparum and P. vivax, respectively [Sixty-two percent, 33% and 5% of the ectively .Plasmodium species infection compared to urban dwellers. This could be explained by the fact that Plasmodium species infection is usually higher in the rural environment where Anopheles mosquito breeding and malaria transmission is more intense [Factors associated with APIs depicted that residence, gravidity and IRS use for the last one year were statistically significant with APIs. Rural dwellers were 4.51 times more likely exposed to  intense , 33.Plasmodium species infection than multigravida. This finding agrees with the studies done in south Ethiopia [Plasmodium species infection was significantly higher in primigravida than multigravida [Women in their first pregnancy were 4.51 times and second pregnancies were 3.87 times more likely developed asymptomatic Ethiopia , NigeriaEthiopia , 31 and Ethiopia . In addiigravida ,35.Pregnant women whose house did not spray IRS for the last one year were 3.13 times more likely develop APIs as compared to those pregnant women whose house sprayed IRS. This result is inconsistent with study finding in south Ethiopia that IRS usage was inversely associated with prevalence of APIs . This diThe limitation of this study was unable to diagnose all participants by real-time PCR due to limited resources and the data were collected during the dry season which do not address the wet season. Moreover, we did not take sample from placenta.Plasmodium species infections among pregnant women. Pregnant women who reside in the rural area, primigravida, secugravida and those did not use IRS for the last one year were at higher risk of API infection.This study showed significant number of asymptomatic S1 File(SAV)Click here for additional data file.


Plasmodium and dengue virus (DENV) infectious species could have serious and fatal outcomes if left undiagnosed and without timely treatment. The present study aimed to determine the pooled prevalence estimate of severe malaria among patients with co-infection, the risk of severe diseases due to co-infection, and to describe the complications of severe malaria and severe dengue among patients with co-infection.Co-infection with both CI) of severe malaria among patients with Plasmodium and DENV co-infection was estimated with a random-effects model to take into account the between-study heterogeneity of the included studies. The risks of severe malaria and severe diseases due to co-infection were estimated with the pooled odds ratio (OR) and 95% CI with a random-effects model.Relevant studies published between databases between 12 September 1970 and 22 May 2020 were identified and retrieved through a search of the ISI Web of Science, Scopus, and MEDLINE. The pooled prevalence and 95% confidence interval . Patients with co-infection had a higher risk of severe diseases than those with DENV mono-infection . Patients with co-infection had a higher risk of severe dengue than those with DENV mono-infection . The most severe complications found in severe dengue were bleeding (39.6%), jaundice (19.8%), and shock/hypotension (17.9%), while the most severe complications found in severe malaria were severe bleeding/bleeding (47.9%), jaundice (32.2%), and impaired consciousness (7.43%).Of the 5653 articles screened, 13 studies were included in the systematic review and meta-analysis. The results demonstrated that the pooled prevalence estimate of severe malaria among patients with co-infection was 32% (95% Plasmodium and DENV co-infection. Physicians in endemic areas where these two diseases overlap should recognize that patients with this co-infection can develop either severe malaria or severe dengue with bleeding complications, but a greater risk of developing severe dengue than severe malaria was noted in patients with this co-infection.The present study found that there was a high prevalence of severe malaria among patients with CRD42020196792.The protocol of this study was registered at PROSPERO:  Plasmodium and dengue virus (DENV) infection in humans are caused by the bites of infected female mosquitoes, including Aedes aegypti and Aedes albopictus for DENV transmission and Anopheles mosquitoes for Plasmodium transmission [Plasmodium can cause malaria in humans, including P. falciparum, P. vivax, P. malaria, P. ovale, and P. knowlesi [smission , 2. Fiveknowlesi , whereasknowlesi .Plasmodium infection in humans also manifests a wide range of signs and symptoms, ranging from asymptomatic to severe malaria [Malaria and dengue are widespread throughout the tropics and subtropics with an estimated 390 million cases of dengue per year , and an  malaria . Severe  malaria .Plasmodium and DENV is common where the specific ecosystem, Environmental, and climatic factors support both infections in an individual [P. falciparum or P. vivax and DENV co-infection in different countries [P. knowlesi malaria and DENV co-infection in Sabah, Malaysia, was also reported [In endemic areas where malaria and dengue overlap, co-infection with dividual . Previouountries –15. Recereported . It is oreported . Althougreported .Plasmodium and DENV co-infection is crucial for clinicians in the design and management of treatment strategies for severe disease and its complications. Our previous systematic review and meta-analysis demonstrated that malaria parasitaemia, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in DENV and Plasmodium co-infection were lower than those in Plasmodium mono-infection, while the platelet count and haemoglobin levels were significantly higher in patients with co-infections [Plasmodium infections can lead to life-threatening complications, they need to be diagnosed early and accurately to permit timely management. Currently, a limited number of meta-analytic studies report on the impact of co-infection on disease severity. An understanding of the prevalence of severe complications among patients with co-infections is necessary for clinicians in designing and managing treatment strategies. Therefore, the primary objective of the present study was to determine the pooled prevalence estimate of severe malaria among patients with co-infection. The secondary objective was to determine the risk of severe malaria, severe dengue, and severe diseases  caused by Plasmodium and DENV co-infection. The third objective was to describe the complications of severe malaria and severe dengue among patients with co-infection. The tertiary objective was to describe the complications of severe malaria and severe dengue among patients with co-infection.An understanding of fections . Since bPlasmodium OR malaria) AND dengue AND (severe OR complicated OR complication)” were used to retrieve the related studies. The details of the search and search terms are provided in Relevant studies published between 12 September 1970 and 22 May 2020 were identified and retrieved through a search of the ISI Web of Science, Scopus, and MEDLINE databases. The search terms “ were extracted. The extracted data were transferred from the Excel datasheet to Stata version 14.0  and Review Manager version 5.3  for statistical analysis.One author (MK) extracted data from the included studies into Excel 2010 , which were cross-checked by the second author (KUK). Information on authors, years of publication, study areas, years of the survey, study designs, age range, sex, participants, and the number of individuals with severe isolated dengue, severe isolated malaria, severe malaria due to Two authors  assessed the methodological quality of the included studies independently using the Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomized studies in meta-analyses ; the NOSCI) of severe malaria among patients with Plasmodium and DENV co-infection were estimated with the “metaprop” command in Stata, with a random-effects model to take into account between-study heterogeneity among the included studies [OR) and 95% CI by a random-effects model, as the studies had considerable amounts of heterogeneity. The results of the meta-analysis were plotted and demonstrated in forest plots, which were generated by Review Manager version 5.3 . The heterogeneity of the included studies was calculated with Cochran’s Q test and I2 statistics. The heterogeneity of the included studies was noted if the I2 was greater than 50%. The complications of severe malaria and severe dengue among patients with co-infection were demonstrated as frequencies and percentages.For the primary outcome of the present study, the pooled prevalence and 95% confidence interval  among the included studies; therefore, a random-effects model was used to produce the pooled prevalence of severe malaria among patients with co-infection. Overall, the pooled prevalence of severe complications/severe malaria of the included studies was 32% (95% CI: 0.18–0.47)  [CI: 1–18%) [From the meta-analysis of the pooled prevalence, there was statistical heterogeneity I = 92.3% 43–0.90) , while t: 1–18%) .Fig. 2TPlasmodium mono-infection (296/845) had an equal risk of developing a severe disease    had an equal risk of developing severe malaria   and those with on 302/84 had an ePlasmodium mono-infection (701/1930) had a greater risk of developing severe dengue   and those with According to the WHO 2015 criteria for severe malaria, most of the severe complications of patients with severe malaria were bleeding , jaundice , shock/hypotension , severe anaemia , impaired consciousness , metabolic acidosis or acute respiratory distress , acute renal failure , prostration , multiple convulsions , and pulmonary oedema . No case of hypoglycaemia was found.According to the WHO 2009 criteria for severe dengue, most of the severe complications of patients with severe dengue were severe bleeding/bleeding , jaundice , impaired consciousness , shock , respiratory distress , renal failure , and multiple convulsions .The publication bias among the included studies was assessed through the visualization of the symmetry of the funnel plot. The results demonstrated that the funnel 340 plot seemed asymmetrical, indicating some publication 341 bias due to the small study effect among the included 342 studies Fig. .Plasmodium and DENV infection are endemic in most tropical and subtropical countries, which are also popular tourist destinations, and these infections are increasingly encountered in travellers who return from areas where malaria is endemic, such as Southeast Asia, Latin America or the Caribbean [Plasmodium and DENV co-infection (32%). This information indicated that co-infection with Plasmodium and DENV impacted the severity of disease in patients with these co-infections. The meta-analysis of severe malaria between patients with co-infection and those with Plasmodium mono-infection demonstrated an equal risk of severe malaria between the two groups. This finding might be because most of the included studies were conducted in an adult population, while only one study was conducted in a paediatric population [P. falciparum and DENV co-infection with P. falciparum as the main cause of severe malaria [Plasmodium mono-infection was comparable, the impact of co-infection on the severity of malaria was reported individually in studies by Epelboin et al., 2012 [Plasmodium mono-infected group.aribbean –36. Thosaribbean , 38, andaribbean . The prepulation . As mostpulation , this re malaria . Althougl., 2012  and Magal., 2012 , as a siThe present study demonstrated a significantly higher risk of severe dengue in patients with co-infection than in those with DENV mono-infection. These results indicated that the severe manifestations of co-infection resembled those of dengue. The previous study has supported the results of the present study that the clinical features of dengue are predominant over those of malaria in patients with co-infection . A previPlasmodium mono-infection [Plasmodium mono-infection, indicating a good outcome [Plasmodium and DENV co-infection were reported by Ahmad et al., 2019 [The definitions of severe complications of severe malaria and severe dengue were guided by the 2014 WHO criteria  and the nfection , 40. Int outcome . The prel., 2019 , Barua al., 2019 , and Kaml., 2019 . The deal., 2019 .Plasmodium and DENV co-infection or mono-infection were reported in four studies, while the other included studies reported the numbers of patients with severe complications of each disease separately. As one patient can develop more than one complication during hospitalization, the results of the pooled prevalence estimates of severe malaria tend to be higher than usual and, therefore, need to be carefully interpreted by readers. Second, hypotension was not included in the comparison of severe diseases between co-infection and mono-infection. Although hypotension is not a 2009 WHO criterion for severe dengue, it is an early sign of shock and is used to determine whether patients need to be treated accordingly. Third, a limited number of studies reported the severe manifestations of these two diseases in the research databases, which caused the low number of included studies in the present meta-analysis.The present meta-analysis had several limitations. First, the numbers of patients with severe diseases caused by Plasmodium and DENV co-infection should be confirmed before the final diagnosis. Further well-designed prospective studies are needed to understand the effect of co-infection on the severity of the disease.The manifestations of these two diseases are clinically indistinguishable. In addition, the diagnosis of one disease in a febrile patient in an endemic area does not preclude infection with the other parasite. Therefore, the possibility of Plasmodium and DENV co-infection. Physicians in endemic areas where these two diseases overlap should recognize that patients with this co-infection can develop either severe malaria or severe dengue with bleeding complications, but a greater risk of developing severe dengue than severe malaria was noted in patients with this co-infection.The present study found that there was a high prevalence of severe malaria among patients with Additional file 1: Table S1. Search terms.


Malaria diagnosis by quantitative PCR confirmed the presence of Plasmodium falciparum for 20.9% (79/378) samples while RDTs did not detect HRP2 antigen in 83.5% (66/79) of these samples. Quantitative PCRs targeting the pfhrp2/pfhrp3 genes confirmed the absence of both genes for 86.5% of P. falciparum strains. The very large number (86.5%) of falciparum parasites lacking the pfhrp2/pfhrp3 genes observed in this study, now justifies the use of non-HRP2 alternative RDTs in Djibouti. In this area and in most countries where HRP2-based RDTs constitute the main arsenal for falciparum malaria diagnosis, it is important to implement a systematic surveillance and to inform biologists and clinicians about the risk of malaria misdiagnosis. Further investigations are needed to better understand the mechanism of selection and diffusion of the pfhrp2/pfhrp3-deleted parasites.Following the diagnosis of a  A pfhrp2/pfhrp3 deletion was confirmed for this strain (data not shown).A case of imported pfhrp2/pfhrp3-deleted parasites in Djibouti, a study was carried out between January and May 2019. The flow chart for this study is described in Plasmodium diagnosis was assessed with HRP2-based RDT . Twenty-two percent (83/378) of blood samples were tested pan-pLDH positive. After extraction of DNA from the 83 pan-pLDH(+) blood samples using a DNeasy Blood & Tissue Kit® (Qiagen©), qPCR, as previously used [P. falciparum alone for 90.4% (75/83) samples, both P. falciparum and P. vivax for 4.8% (4/83) samples and P. vivax only for 4.8% (4/83) samples. Among the 79 P. falciparum PCR(+) samples, the RDT did not detect the P. falciparum HRP2 antigen for 83.5% (66/79) of samples  [pfhrp2/pfhrp3 ones with a limit of detection for β-tubulin qPCR of 15 parasites per µl compared to 5 and 1.67 parasites per µl for pfhrp2 and pfhrp3 qPCRs, respectively. The β-tubulin qPCR made it possible to rule out 5 β-tubulin PCR(-) samples that would have been false negative in pfhrp2/pfhrp3 qPCR due to a low quantity or quality of specific DNA. Two modified qPCRs were carried out in order to amplify the exon 2 of pfhrp2 and pfhrp3 genes [pfhrp2 and pfhrp3 was performed in a final reaction volume of 10 µL containing the DNA template, with 0.15 µM of each primer (pfhrp2-F1 and pfhrp2-R1 for pfhrp2 gene and pfhrp3-F1 and pfhrp3-R1 for pfhrp3 gene), 5 mM or 4 mM MgCl2, respectively and LightCycler® FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH). The qPCR conditions were identical for both genes with a denaturation at 95°C for 15 min, followed by 45 cycles of 95°C for 15 s, 55°C for 20 s and 72°C for 60 s. The pfhrp2/pfhrp3 qPCRs confirmed the absence of both genes for 86.5% (64/74) of the P. falciparum strains  and histidine-rich protein 2 (pfHRP2) only produced by P. falciparum [falciparum infection, 90% of the RDTs used in the diagnosis of malaria were only based on pfHRP2 detection [P. falciparum protein, the PfHRP3 antigen due to a high degree of similarity in the amino acid sequence [For more than a decade, rapid diagnostic tests (RDTs) have become essential to malaria diagnosis. A total of 276 million RDTs were sold globally in 2017, and in sub-Saharan Africa, RDTs accounted for an estimated 75% of all the diagnosis tests carried out for suspected cases . RDTs caetection . PfHRP2-pfhrp2 and/or pfhrp3. These pfhrp2/pfhrp3-deleted falciparum strains raise a diagnosis issue as a large number of falciparum-infected patients will go undetected by the pfHRP2-based RDTs. The public health consequence can be an increased risk of malaria morbidity and mortality due to delayed treatments, and an increase in the transmission of malaria.However, since 2010, false negative results with pfHRP2-based RDTs have been reported in the Amazon region and in some parts of Africa and Asia , usuallypfhrp2/pfhrp3 genes among the falciparum strains circulating in Djibouti. The lack of previous data makes it difficult to date this trend. However, the current warning of health professionals from Djibouti, who are concerned by a high level of negative RDTs despite clinical suspicions of malaria, suggests that this trend is recent.Our study identified a very high prevalence (86.5%) of parasites lacking pfhrp2/pfhrp3 genes as these strains are already present in these areas at a high frequency (4.8% and 62% in Ethiopia (2017) and Eritrea (2016), respectively) [Djibouti is a major transit point for migrants, predominantly Afar and Somalis and lesser numbers Ethiopians and Eritreans. These human migratory and commercial flows could be at the origin of the diffusion of parasites lacking ctively) ,11. Thispfhrp2/pfhrp3-deleted parasites are selected. The consensual assumption is the misdiagnosis and the selection of pfhrp2/pfhrp3-deleted strains by the exclusive use of HRP2-based tests over time, as previously observed in Eritrea [pfhrp2/pfhrp3-deleted parasites due to misdiagnosis would have given them a selective advantage compared to pfhrp2/pfhrp3 non-deleted strains. In Djibouti, malaria diagnosis is exclusively based on the use of RDT selective for the detection of P. falciparum and P. vivax but not for the Plasmodium species with pan-pLDH [pfhrp2/pfhrp3-deleted clone could have been indirectly selected due to the presence, on this same clone, of another mutation responsible for the resistance to an antimalarial used in this area. This scenario is unlikely in Djibouti since none of the 7 pfhrp2/pfhrp3-deleted strains tested in this study carried a mutation on the PfKelch13 protein (data not shown) [The second main issue is to understand how these  Eritrea  and pred Eritrea . So, thepan-pLDH . Furtherpan-pLDH . Anothert shown) .pfhrp2/pfhrp3-deleted parasites observed in Djibouti now justifies the use of non-HRP2 alternative RDTs like LDH- or aldolase-based RDTs. Indeed, the WHO recommends a policy switch to more effective WHO-approved non-HRP2 malaria tests when the prevalence of pfhrp2-deleted parasites meets or exceeds a 5% prevalence. Despite the costs and resources involved [A systematic country-wide surveillance might ensure that the prevalence found in this study reflects the national one. However, the high prevalence of involved , these ifalciparum imported malaria [falciparum infection. The high frequency of mutant parasites in Djibouti raises the question on the risk of malaria misdiagnosis for locals but also for travellers or soldiers as Djibouti’s strategic situation has made it a desirable location for foreign military bases.In most countries, HRP2-based RDTs constitute the main diagnosis arsenal for  malaria ,17. Cons


P = 0.042) and child body iron stores . Chorioamnionitis was associated with reduced risk of child parasitaemia . Periconceptional iron supplementation of young women did not alter body iron stores of their children. Higher child body iron stores and placental malaria increased risk of childhood parasitaemia.This study in Burkina Faso investigated whether offspring of young mothers who had received weekly periconceptional iron supplementation in a randomised controlled trial were at increased risk of malaria. A child safety survey was undertaken in the peak month of malaria transmission towards the end of the trial to assess child iron biomarkers, nutritional status, anaemia and malaria outcomes. Antenatal iron biomarkers, preterm birth, fetal growth restriction and placental pathology for malaria and chorioamnionitis were assessed. Data were available for 180 babies surviving to the time of the survey when their median age was 9 months. Prevalence of maternal iron deficiency in the last trimester based on low body iron stores was 16%. Prevalence of active placental malaria infection was 24.8%, past infection 59% and chorioamnionitis 55.6%. Babies of iron supplemented women had lower median gestational age. Four out of five children ≥ 6 months were iron deficient, and 98% were anaemic. At 4 months malaria prevalence was 45%. Child iron biomarkers, anaemia and malaria outcomes did not differ by trial arm. Factors associated with childhood parasitaemia were third trimester C‐reactive protein level , active placental malaria (OR 5.8; 1.0–32.5,  Iron deficiency prevalence in young mothers in Burkina Faso was low.Periconceptional iron supplements did not alter placental or child malaria risk.Malaria parasitaemia risk was higher in children with better iron status.Placental malaria increased risk of child malaria.Intensified efforts are needed to reduce risk of placental malaria which should, in turn, reduce risk of malaria and anaemia in young children.1Plasmodium falciparum infection risk early in pregnancy . The primary trial outcome was malaria prevalence at the first antenatal visit, but women were followed till delivery and gestational age, preterm birth, birthweight, placental malaria and neonatal deaths have been reported , or an identical capsule containing folic acid alone (2.8 mg) (n = 979) for up to 18 months, or till pregnancy occurred , were treated with oral quinine. A second scheduled IPTp‐SP dose was provided through routine antenatal care. Gestational age was estimated by ultrasound examination at ANC1 with a FF Sonic UF‐4100 (Fukuda Denshi) scanner, with measurement of crown rump length in the first trimester and by biparietal diameter, femur length and abdominal circumference afterward.Nulliparous participants had been individually randomised to receive, directly observed, either a weekly capsule containing ferrous gluconate (60 mg) and folic acid (2.8 mg) . Delayed umbilical cord clamping was routinely practised. Following hospital delivery, placental biopsies (2.5 × 1 cm) were excised from fetal and maternal sites at mid‐distance between umbilical cord insertion and the placental border and placed in 10% neutral buffered formalin. Rural health centres were also trained and equipped to take placenta samples. Placental biopsies were not available from home deliveries.2.4Haemophilus influenzae type B and polio at 2, 3 and 4 months; measles at 9 months; yellow fever at 12 months). Health cards were examined and reports of recent illness, fever and drug intake within the last 2 weeks were checked. Anthropometric measurements were performed in duplicate by two independent observers and mean values computed. Children were weighed to the nearest 10 g with an electronic scale . Length was measured to the nearest 5 mm using a measuring mat  and mid‐upper arm circumference to the nearest mm using a circumference tape . Clinical examination was completed with temperature. A venepuncture blood sample was collected. The volume of blood sampled differed by weight, 1 ml in EDTA and 4 ml in a dry tube for infants ≥ 5600 g and 0.5 and 3.0 ml, respectively, for lower weights. This was used for malaria microscopy and RDT for malaria, Hb, serum iron biomarkers , C‐reactive protein (CRP) and a reserve filter paper blood spot. Children diagnosed with malaria, or any other concurrent health problem, received free treatment according to National Guidelines, with appropriate follow‐up.In the wet season in October 2013, mothers who had delivered and remained in the study area were invited to have their offspring assessed at the Clinical Research Unit of Nanoro (CRUN) Health Clinic. The follow‐up child assessment was performed by experienced paediatric nursing research staff and included: medical and vaccination history  and CRP by ELISA . Intra‐assay coefficients of variation (CVs) were all <10%. Ranges for normal controls were ferritin, 69.1–114.7 μg L−1; sTfR, 4.2–5.9 mg L−1; CRP, 5–8 mg L−1. Definitions of iron status were based on (1) adjusted ferritin using the internal regression correction approach  to log10 ferritin (μg L−1), which assesses both stored and functional iron and is possibly less affected by inflammation. Values > 5.6 in children derive from the cut‐offs sTfR > 8.3 μg ml−1 and ferritin < 30 μg L−1. This best predicted iron deficient bone marrow stores using the same assay as in the present study, in an area of high malaria transmission  using the regression‐adjusted ferritin estimate were calculated using the equation derived by Cook, Flowers, and Skikne (−1) = −[log10 (1000 × sTfR/ferritin) − 2.8229]/0.1207 [39]. Low BIS was defined as <0 mg kg−1. Plasma hepcidin was measured by competitive ELISA at an International Reference Laboratory , a third independent reading was made, with the mean of the two closest observations accepted as the true value.Blood samples were transported within 3 h from the clinic to the central project laboratory at CRUN. Sera aliquots were stored at −80°C. Hb was measured  and ZPP by fluorometry  on fresh whole blood. Anaemia in children ≥ 6 months was defined as Hb < 11 g dld Skikne : body ir2.6After the delivery of the placenta, it was placed in a receptacle fetal side upwards. Using scissors, a biopsy of 2.5 cm × 1 cm was excised at mid‐distance between the insertion of the umbilical cord and the placenta border and placed into a prefilled specimen container with 10% neutral buffered formalin . A 1 cm cross‐section from the umbilical cord was cut with scissors at about 5 cm from the insertion and placed into the same receptacle. After turning the placenta in order to expose the maternal side upwards, a second biopsy of about the same size was excised at half distance between the centre and the border of the placenta and placed into a second container with formalin. A 10 cm × 10 cm piece of the membrane was cut with scissors and placed into the same container.Formalin fixed specimens were stored for up to 3 months at room temperature in an air‐conditioned room (20°C) at the CRUN laboratory. After transport to the department of pathology at the National University Hospital Yalgado Ouedraogo in Ouagadougou, tissue samples were processed by experienced technicians according to standard histopathological procedures.Samples were embedded in paraffin wax following standard methods. Membranes were rolled to cylinders in order to obtain sufficient material to be cut and embedded. For each set of samples, paraffin sections 3 to 5 μm thick were placed on two slides, the fetal side of the placenta together with the cord on one slide, the maternal side of the placenta together with the membrane on another slide. Slides were prepared in duplicate to allow for different staining. De‐paraffinised sections were stained with haematoxylin and eosin and Giemsa stain. Histological examination of all samples was done by a specialised senior pathologist with light microscopy and under polarised light. Histological classification of placental malaria was based on the different significance of haemozoin and parasitised RBC in the intervillous space of the maternal side of the placenta as described by Ismail et al. : acute i2.7N (%) and compared between treatment arms using ordinary or logistic regression models adjusting for child sex and age at assessment. Age was fitted with a cubic spline function with 5 degrees of freedom, with sensitivity analyses confirming that this was a sufficient representation of the non‐linear age relationships. Results are presented as odds ratios for categorical outcomes or differences between arms for continuous variables with 95% CI. Where appropriate, outcomes were log transformed and the presented effect size can be interpreted as a ratio between arms.The sample size was determined from formal power calculations for the malaria trial endpoints  were added where these did not obscure the presentation.The associations between infant/maternal factors and infant malaria were assessed using logistic regression models for malaria outcomes against the relevant factors, adjusting for age and sex as above. The associations between iron biomarkers and malaria were visualised as scatterplots of the relationship between the biomarker and age for the children with and without malaria, with cubic spline regression lines added for each group. All analyses were performed in the R statistical environment version 3.6.2.8Clinicaltrials.gov on 27 September 2010: https://clinicaltrials.gov/show/NCT01210040.This child outcome analysis was conducted as a safety assessment of malaria risk in the first born offspring of young mothers enrolled in a randomised periconceptional trial of weekly iron and folic acid supplementation (PALUFER). The study received ethical approvals in Burkina Faso; National Ethics Committee (CERS Ref 015‐2020/CE‐CM) and the Comité d'Ethique pour la Recherche en Santé du Centre Muraz (015‐2010/CE‐CM); the United Kingdom Research Ethics Committee, Liverpool School of Tropical Medicine (LSTM/REC protocol 10‐55); the Institutional Review Board of the Institute of Tropical Medicine (IRB/AB/AC/016) and the Antwerp University Hospital Ethics Committee, Belgium (EC/UZA). The trial was registered with 33.1During the trial 478 pregnancies occurred, with 348 known deliveries occurring before the survey period Figure . Followi3.2Z score ≤ 2SD), 2.2% were referred for severe malnutrition (Z score ≤ 3SD) and three children had received recent haematinics. Malaria and iron biomarker outcomes are outlined in Table P < 0.001). Mean Hb concentration, iron biomarkers levels or anaemia prevalence in children did not differ between trial arms, nor were there significant differences for any malaria‐related outcomes. Median CRP concentration increased from 2.7 mg L−1 in children ≤ 6 months to 6.3 mg L−1 for older children (P < 0.001), but values did not differ by trial arm.Maternal characteristics at delivery and offspring characteristics by trial arm are shown in Table 3.3Parasite prevalence increased from low values at 1 month to approximately 45% by 4 months, with positive RDT prevalence increasing to 70% at this age. Clinical malaria prevalence increased to 20% by 7 months. Prevalence of all malaria infection parameters progressively increased with age  and BIS (P < 0.001). Similar iron biomarker differences were seen in relation to parasitaemia and/or recent malaria treatment  was 1.13  and for parasitaemia and/or recent malaria treatment 1.18 . Higher zinc protoporphyrin concentration was associated with malaria infection (P = 0.003). Gestational age at delivery, SGA, and child undernutrition were not associated with childhood malaria risk.The age‐specific pattern of child iron biomarkers is shown in Figure P = 0.042) was associated with active placental infection but was higher for parasitaemia and/or recent malaria treatment . Past placental malaria infection also predicted child parasitaemia and/or recent malaria treatment . Malaria parasitaemia at ANC1 marginally increased the risk of childhood parasitaemia . In children, parasitaemia risk was higher with evidence of maternal inflammation based on raised CRP  in the third trimester. Chorioamnionitis at delivery was associated with reduced risk of child parasitaemia (P = 0.038), although for child parasitaemia or recent malaria treatment this reduction was not significant.Child malaria infection prevalence grouped by categories of active and past placental infection are shown in Figure 4Periconceptional iron supplementation made no difference to placental malaria parasite prevalence, or in children, to malaria‐related outcomes or iron biomarker values. When combining trial arms, increased malaria infection prevalence was seen in children with better iron status. There was increased childhood malaria infection risk following active and past placental malaria, indicating that earlier gestational malaria was also a risk factor for malaria in young children, although the active infection had cleared by the time of delivery.−1), prevalence at ANC1 in our study was 12.8%  associated with malaria in pregnancy but via indeterminate mechanisms (Park et al., An early peak in malaria prevalence occurred at around 4 months of age followed by uniformly rising prevalence with increasing age. An early peak in prevalence was similarly associated with placental infection in a cohort of Cameroonian children (Le Hesran et al., Malaria risk in young children is also influenced by other maternal factors, although this study found no association with gestational age, use of IPTp, SGA or child undernutrition. Evidence for an association between IPT in pregnancy and malaria in infants is limited (Kakuru, Staedke, Dorsey, Rogerson, & Chandramohan, Lack of adjustment for malaria transmission intensity is an important limitation of the present study, as for nearly all published studies, which may be confounded by shared maternal and child exposures to infected mosquitoes (Kakuru, Staedke, Dorsey, Rogerson, & Chandramohan, No previous studies have longitudinally assessed both maternal and child iron status as determining factors for childhood infection risk in areas of high malaria transmission. Lack of efficacy of the primary intervention in mothers precluded better understanding of the effects of supplementation on the iron status of their offspring. Strengths of the present study include assessment of gestational age by ultrasound, lack of confounding due to maternal parity or age, inclusion of key determinants of maternal and child malaria and completion of this cross‐sectional survey during the peak month of malaria transmission (Natama et al., 5High prevalence of placental and child malaria was associated with better iron status in young mothers and children. Intensified efforts are needed to reduce risk of placental malaria which should, in turn, reduce risk of malaria and anaemia in young children.The authors declare that they have no conflicts of interest.BB and SG designed the research. BB was the Principal Investigator for the main randomised controlled trial and recipient of the grant. SG, SD, BB and HT conducted the field research. SD and OL conducted and supervised the laboratory research. SAR analysed the data. BB and SR wrote the paper. All authors reviewed and approved the final manuscript.Appendix S1. Further Trial DetailsClick here for additional data file.Figure A1. Participant flow diagramTable A1. Antenatal characteristics at ANC1 of mothers of children lost to follow‐up and of children included in follow‐up surveyFigure A2. Age specific child iron biomarkers in relation to malaria parasitaemia or malaria treatmentClick here for additional data file.