This review aims to inform future guidelines by examining which anthropometric criteria best identify infants <6 m at high risk of mortality/morbidity.There is increasing global focus on small and nutritionally at-risk infants aged <6 months (<6 m). Current WHO guidelines recommend weight-for-length We searched Medline, EMBASE, CINAHL, Global Health, Cochrane Library and POPLINE for studies conducted in low- and middle-income countries and published between 1990 and October 2020. We included studies reporting anthropometric assessment of nutritional status in infants <6 m and assessed the association with subsequent morbidity or mortality.z-score (WAZ) were better at identifying infants at risk of mortality/morbidity. MUAC-for-age z-score did not perform better than using a single MUAC cut-off. Suggested MUAC cut-offs for this age group varied by context, ranging from 10.5 to 11.5 cm. The assessment for reliability showed that length was difficult to measure, making WLZ the least reliable indicator overall.A total of 19 studies were included in the final review, covering 20 countries, predominantly in sub-Saharan Africa. WLZ had poor reliability and poor prognostic ability to identify infants at risk of death. Mid-upper arm circumference (MUAC) and weight-for-age Evidence from our review suggests that a change in current practice is necessary. To better identify small and nutritionally at-risk infants <6 m WAZ and/or MUAC rather than WLZ should be used. Future research should explore possible benefits for programme coverage, impact and cost-effectiveness. Research should also examine if context-specific MUAC thresholds are needed. These infants are especially vulnerable to the short- and long-term health risks associated with anthropometric deficits. They face significantly higher risk of morbidity and mortality than older malnourished children, and in the long-term, potentially impaired physical and cognitive development, including greater susceptibility to cardiovascular and metabolic non-communicable diseases.37Approximately 8.5 million infants less than 6 months old (<6 m) globally are wasted. There is growing appreciation that apparent wasting in this age group needs to be taken more seriously, understood and better addressed. Whilst supporting exclusive breastfeeding is the main route to appropriate nutrition in this age group, only 37% of infants <6 m are exclusively breastfed in low- and middle-income countries. Even then, exclusive breastfeeding may not fully protect an infant against undernutrition since many other factors can contribute to inadequate weight gain. Cultural and socioeconomic influences, maternal nutritional deficits, maternal mental health problems, breastmilk composition (particularly oligosaccharides and bioactive proteins), acute and chronic infections, enteropathy, birth defects and prenatal nutrition can all negatively influence nutrition and growth in infants.12 The current COVID-19 pandemic poses additional problems in these areas. National and international food and health systems that were already fragile are now under significantly greater stress, with associated practical difficulties finding, assessing and supporting at-risk mothers and infants.Current wasting interventions focus on children 6 to 59 months with infants <6 m overlooked or neglected in many treatment programmes. guidelines on the ‘Management of Severe Acute Malnutrition in infants and children’, published in 2013, include a chapter focussing on the unique needs of infants <6 m. The guidelines recommend using low weight-for-length z-scores (WLZ) to identify small and nutritionally at-risk infants <6 m for admission to treatment programmes. However, this guidance was not based on direct evidence review but on conventions used among older children. In a 2015 systematic prioritisation of research questions to improve the management of malnourished infants <6 m flagged ‘defining wasting in this age group’ as the top priority question out of 60 listed.Current World Health Organization (WHO) A small but growing number of studies have explored the prognostic value of different anthropometric criteria, as well as their applicability and feasibility in front-line nutrition programmes.18 National and international guidelines are currently being updated to reflect those new perspectives and priorities. Evidence to inform these updates is urgently needed. Our systematic review therefore aims to synthesise latest evidence on which anthropometric criteria best identify infants at-risk of morbidity and mortality.Since the publication of the 2013 WHO guidelines and the 2015 prioritisation review, there has been growing appreciation that anthropometric case definitions of malnutrition should focus on those best able to predict clinically significant events – notably mortality and morbidity.Our systematic review protocol was pre-registered with PROSPERO (#CRD42019141047). The London School of Hygiene and Tropical Medicine Research Governance & Integrity Office also reviewed the methodology but since we only use publicly available sources decided that no formal ethical approval was required. We considered anthropometric measures as risk factors for morbidity and mortality, and also reviewed quality, reliability and validity of measurements. Studies that only compared how 1 anthropometric criterion predicted another anthropometric criterion were excluded since this design has no gold standard measure. We based our search strategy on a previous literature review exploring admission and discharge criteria for malnourished infants <6 months.Supplemental Annex 1. The last search was run on 15th October 2020.The following databases were searched: Medline, EMBASE, CINAHL, Global Health, Cochrane Library and POPLINE. The search was filtered to include only articles in English, research in humans and publications since 1990. Additional studies were found through reviewing the reference lists of the identified publications. Full list of search terms can be found in All initial results were screened by the first author based on their title and abstract; any studies that could not be excluded were then evaluated for inclusion based on the full text. Data were extracted from eligible studies using a standardised form to capture information regarding participants and setting; indicators measured; outcomes reported; and results. Due to the small number and heterogeneity of studies, the analyses are presented as a narrative synthesis. The presentation of the evidence follows a comparison of the different indicators based on the conceptual framework by Myatt et al, focussing on the prognostic properties of each indicator and their quality.Quality assessment was done based on the ‘Methods for the development of NICE  public health guidance’.z-score (WAZ) and 9 included weight-for-length z-score (WLZ). Three studies compared all 3 indicators. Other criteria investigated were MUAC-for-age z-score (MUAC-FA) (n = 4), length-for-age z-score (LAZ) (n = 7), BMI-for-age z-score (BMI-FA) (n = 1) and weight-velocity for age (WV-FA) (n = 1). Of the 7 studies analysing infants <6 m separately, 2 were set in a hospital,23 and 5 in the community.2428 Of the 5 studies analysing infants <6 m together with infants up to 24 months of age, 1 was set in a hospital and 4 in the community.3033After removing duplicates, 7375 titles and abstracts were identified; 150 full texts were reviewed; and 18 studies were identified for final inclusion in the analysis see . Almost Supplemental Annex Table 1). The highest risk of bias was found in the domains of Study Attrition and Study Confounding, due to a lack of detailed reporting about how the authors dealt with loss to follow-up and confounding variables.Overall, the studies showed a low to moderate risk of bias across different domains  but none collected details about the causes of death.Our review found that WLZ performed poorly when assessed for its prognostic ability and reliability. Six studies27 MUAC-FA z-score did not perform better than using a single MUAC cut-off. The most appropriate cut-off for this age group varied by context, ranging from 10.5 to 11.5 cm. In Kenya, Mwangome et al found a threshold of <11.0 cm to be statistically optimal for identifying inpatient mortality risk and < 11.5 cm for identifying those at risk of post-discharge mortality . In Burkina Faso <11.5 cm identified infants at risk of mortality reliably, but also captured a large proportion of infants overall (17%). A cut-off of <11.0 cm identified 6.8% of infants, which may be more reasonable depending on the available resources. However, in The Gambia, <11.0 cm captured 14% of the population. In a study in Nepal, MUAC <11.5 cm predicted mortality in 1 ethnic group  but not another .MUAC and WAZ identified infants at risk of mortality and morbidity better than WLZ.3640 Ezeofor et al reported WLZ had the lowest sensitivity under 3 months and overall detected only half of all cases of ‘undernutrition’ , while WAZ had the highest positive predictive value at all ages (0-6 months). Two studies specifically note the issue that WLZ cannot be calculated on infants less than 45 cm25; this only affected 0.2% of infants in a community-based study but affected 15% in a hospital-based study. Ayele et al reported measurement errors for weight, length and MUAC by members of a rural community in children 0 to 69 months in Ethiopia and found slightly higher errors for smaller children than for larger ones. The relative Technical Error of Measurement (%TEM) for MUAC (1.48 [95% CI 1.26-1.69]) was higher than for height (0.80 [0.68-0.92]) and weight  in children <2 years to be slightly less reliable than weight (%TEM 0.79). No studies formally assessed acceptability, cost or simplicity of measurements which are additional factors to consider, as per Myatt et al’s framework.In assessments of measurement reliability, length was generally more difficult to measure accurately, meaning WLZ is often the least reliable of the indicators.Our review found a small number of high-quality studies assessing the relationship between anthropometric indicators of nutrition status and subsequent risk of mortality in infants <6 m. Most found MUAC and/or WAZ to be the best predictors of short-term mortality, as well as having lower measurement errors than length-based measurements. There was little benefit of using a MUAC-FA measure over a fixed threshold.z-score calculations of WAZ and WLZ add opportunity for additional errors, and magnifies existing errors in weight or length, especially for WLZ which combines the 2 measures. An additional problem is that WLZ cannot be computed for infants shorter than 45 cm – hence some missing values for this indicator. Whilst the WHO reference shows that by 4 weeks of age, 99% of infants are longer than 45 cm, those that are not are among the most vulnerable ones.42 Based on these findings, it is difficult to justify continuing to recommend WLZ as the standard indicator for nutritional vulnerability in infants <6 m.The current standard indicator for identifying at-risk infants <6 m is WLZ. We found 8 studies that investigated the predictive ability of WLZ for mortality and in all the studies WLZ was either not associated with subsequent mortality, performed equally well as the other indicators or showed the worst performance. Additionally, several studies investigated the reliability of measuring length in infants and all found it to be a consistently less reliable measure than weight. This could be due to several factors including: the physiological tendency of young infants to flex their knees and hips, making proper flat positioning on a measuring board challenging; healthcare workers’ relative unfamiliarity handling young infants; the fact that infants are active and difficult to hold still. 40 WAZ has also been previously dismissed as a useful indicator as it is ineffective at differentiating between deficits in height versus deficits in weight, however recent studies in older children (6-59 months) have highlighted the higher risk of death in children with multiple nutritional deficits and WAZ is better at identifying these children than WLZ.43 Use of WAZ rather than WLZ would also improve practicalities of assessing risk at a community level, although MUAC is the most useful measure in this regard.For assessing nutritional risk in older children (6-59 months), the common alternatives to WLZ are MUAC and occasionally WAZ. This review found that WAZ was a predictor of mortality in 6/6 studies. Weight is routinely assessed in infants in growth monitoring programmes, so WAZ also has the advantage of familiarity and that scales are widely available and health workers are trained to use them. Age used to be perceived as problematic to ascertain accurately but increasing numbers of infants worldwide have date of birth recorded and even where not available it is easier to calculate than in older children since shorter recall periods are needed.44 MUAC is the cheapest indicator to measure and requires only minimal training.45 It is also well accepted by mothers as infants do not need to be undressed and can remain in the arms of the caretaker. It can also be accurately conducted by mothers themselves at home.48 These benefits strongly suggest that MUAC should be included in the nutritional assessment of infants <6 m in order to improve efficiency, coverage and effectiveness of identification; it is already recommended by the recently updated MAMI Care Pathway . Future research is needed to represent a wider range of contexts and identify context-specific MUAC thresholds. Ideally, this research should compare indicators against clinical outcomes rather than against each other.For MUAC, studies of its predictive value, precision, accuracy and independence of age were found. All studies of infants <6 m showed an association between MUAC and mortality. The 3 studies that analysed whether MUAC was independent of age found its performance was either not influenced by age or performed equally well when compared to MUAC-FA. The cut-off threshold used has a large impact on specificity and sensitivity for identifying at-risk infants and optimal cut-off values seemed to vary by context. Statistically optimal cut-off values ranged from 11.5 to 10.5 cm, each with consequences to be considered for types of intervention and trade-offs between sensitivity and burden on treatment programmes.This is the first systematic review examining anthropometric criteria specifically for infants <6 m, and is not without limitations. Significant heterogeneity in results, including which anthropometric indicator was analysed, at what age, how frequently and with what case definition, limits the comparability of findings. Four of the studies were also relatively old, having taken place more than 20 years ago. This may affect their generalisability to current populations. Another limit to the generalisability, is that some of the highest burden countries for wasting are not represented, such as those in the African Sahel region, and only 1 study for South America and 1 study from South-East Asia were found.Future research should address these limitations and examine the different anthropometric indicators in a wider range of current settings. Though it is plausible that simpler indicators, notably MUAC might facilitate improved programme coverage and impact and contribute to greater cost-effectiveness of treatments, this needs to be formally explored. Can MUAC be used as a stand-alone indicator or is combination with WAZ better to identify higher risk infants? Towards this, more data is needed to determine MUAC cut-off recommendations. One cut-off for the whole 0 to 6 m age range is unlikely but perhaps there could be: 1 cut-off for early infancy; another for 6 weeks (age of first vaccination) to 6 months. Future research should also examine how proposed new indicators might improve links with other programmes: WAZ with growth monitoring programmes; MUAC embedded into vaccination and community programmes for example. Finally, it is important to document outcomes for infants who might not be identified using the new criteria, for example, the small numbers with low WLZ but normal MUAC and normal WAZ.While further research remains necessary, we found good quality evidence to underpin changes to current policy and practice relating to small and nutritionally at-risk infants <6 m. To better identify infants at high risk of mortality and morbidity, WAZ and/or MUAC should be used in preference to current WLZ criteria. MUAC-for age does not add value but more data is needed to determine what MUAC cutoffs should be used. Future research is needed to explore the performance of the various indicators in a wider range of contexts and to document their impact on programme coverage, impact and cost-effectiveness.Click here for additional data file.Supplemental material, sj-docx-1-pdi-10.1177_11795565211049904 for Anthropometric Criteria for Identifying Infants Under 6 Months of Age at Risk of Morbidity and Mortality: A Systematic Review by Christoph Hoehn, Natasha Lelijveld, Martha Mwangome, James A Berkley, Marie McGrath and Marko Kerac in Clinical Medicine Insights: Pediatrics


Iron fortification and micronutrient initiatives, specifically, vitamin A, and zinc supplementation are the most cost-effective developmental strategies against malnutrition and health emergencies in pre-school children. Iron-deficiency among pre-school children have been documented, however, studies evaluating the impact of immunoglobulin G (IgG) isotype responses among iron-fortified pre-school children in malaria endemic communities has not been assessed. We evaluated the impact of iron fortification on the IgG responses to GLURP R0, GLURP R2 and MSP3 FVO malaria-specific antigens among pre-school children in malaria endemic areas.-controlled, double-blinded, cluster-randomized trial study was conducted in Wenchi Municipal and Tain District of Bono Region. The trial was registered at ClinicalTrials.gov-registered trial (Identifier: NCT01001871). Ethical approval was obtained and informed consent were sought from each participant parents/guardian. For the current objective, 871 children aged 6–35 months were screened, from which 435 children received semi-liquid home-made meals mixed with 12.5 mg of iron daily (intervention group), and 436 received micronutrient powder without iron (placebo group) for 5 months. Standardized clinical and epidemiological questionnaires were administered and blood samples taken to measure IgG responses to GLURP R0, GLURP R2 and MSP3 FVO recombinant antigens using the Afro Immunoassay (AIA) protocol.This community-based, placebop > 0.05). After the intervention, there was no significant difference in the IgG response against GLUP R0, GLUP R2 and MSP3 FVO between the iron-containing micronutrient and placebo groups (p > 0.05). The iron-containing micronutrient powder group who were iron-sufficient or iron replete had significantly higher IgG response to GLURP R0 and GLURP R2 compared to iron-deficient and iron-deficiency anaemia in the same group (p < 0.05). The IgG responses to all the three malaria specific antigens were low among children without malaria episode but high among those with two and four episodes due to exposure differences.Baseline anthropometry, malaria diagnosis, anaemia and iron status, demographic features and dietary intake were identical among the groups (Iron fortification did not influence antibody response against endogenous malaria specific antigens among pre-school children in malaria endemic areas, however, IgG response to malaria specific antigens were high among children with sufficient iron status. Malaria is a major cause of morbidity and mortality among pre-school children and a main contributor to anaemia –3. In 20in vitro parasitaemia against malaria have gone through phase 1 vaccine trials and their efficacies are being tested in both phase 2 and 3 vaccine trials among pre-school children in Sub-Saharan Africa [Parasitized erythrocytes have numerous antigenic surface proteins that illicit antibody response –7. Asexun Africa , 8–11.Escherichia coli and the recombinant MSP-3 of the FVO strain expressed in E. coli [Recombinant malaria antigens GLURP R0 protein containing the conserved non-repeat N-terminal region, (amino acids 25–514), GLURP R2 (amino acids 705–1178) of the carboxyl-terminal repeat region all expressed in  E. coli –14. GLUR E. coli –17.Recent iron supplementation strategies to prevent malaria and anaemia have only been partially effective and antibody responses to malaria specified antigens have been inconclusive, yielding conflicting results from malaria endemic and non-endemic areas , 19. The3 hinge region has been known to influence antigens specific to IgG3 variants that mitigate the response to malaria and indirectly prevent anaemia [Polymorphisms in the IgG anaemia . ImmunolIn addition, factors modulating immune responses against malaria vaccine candidates that may help provide possible explanations to the sub-optimal efficacies observed are not well understood. The current study assessed the impact of iron-containing micronutrient powder on malaria-specific isotype antigens IgG; GLURP R0, GLURP R2 and MSP3 FVO among pre-school children.-controlled, double-blinded, cluster-randomized trial study was conducted in Wenchi Municipal and Tain District of Bono Region. Ethical approval was obtained and informed consent were sought from each participant parents/guardian. For the current objective, 871 children, aged 6–35 months were screened, from which 435 children received semi-liquid home-made meals mixed with 12.5 mg of iron daily (intervention group), and 436 received micronutrient powder without iron (placebo group) for 5 months. Standardized clinical and epidemiological questionnaires were administered and blood samples taken to measure IgG responses to GLURP R0, GLURP R2 and MSP3 FVO recombinant antigens using the Afro Immunoassay (AIA) protocol. Children with extreme anaemia (haemoglobin 70.0 g / L), severe malnutrition (weight-for-length z-score -3.0), on iron supplements during the past 6 months or had any chronic disease were excluded  [Ethical approval was obtained from the Kintampo Health Research Centre Institutional Ethics Committee (KHRCIEC), (KHRC/IEC/FEA/2009-2), Ghana Health Service Ethics Committee. In addition, the Food and Drug Authority of Ghana and Hospital for Sick Children Research Ethics Board (REB), Toronto, Canada (REB File No.: 1000013476) also approved the study. The trial was registered at ClinicalTrials.gov-registered trial (1001871) . The stu1001871) , 22. QuaAbout 500 μl of finger or heel prick blood was collected from each participants into an ethylene diamine tetra-acetic acid (EDTA) tube at baseline (BL) and endline (EL) for haematological, malaria microscopy, acute protein process, and iron biomarker analyses as previously described , 22. Bri0C. Plates were then washed four times in washing buffer (PBS with 0.1% Tween-20 and 0.5 M NaCl) with 30 seconds incubation between each wash using the Biotek ELx 405 automated ELISA plate washer . The washed plates were padded dry on a tissue paper and blocked with 200 μL blocking buffer  and incubated at room temperature in a humidified chamber for 1 hour. Plates were then washed four times in washing buffer and serum samples diluted at 1:200 in serum dilution buffer  added at 100 μL / well in duplicates.The IgG responses to GLURP R0, GLURP R2 and MSP3 FVO recombinant antigens was determined using indirect enzyme-linked-immunosorbent assays (ELISA) with a little modification made in the concentrations of the coating antigens and secondary / detection antibodies as previously described , 23. BriThe plates with the samples were then incubated on a clinical rotator set at 200 rpm at room temperature for 2 hours in a humidified chamber after which they were washed four times in washing buffer and the appropriate secondary (detection) antibody for the specific antibody to be determined added at 100 μL / well. The plates with the conjugates were then incubated on a clinical rotator set at 200 rpm for 1 hour at room temperature in a humidified chamber after which they were washed four times in wash buffer and padded dry. The bound secondary antibody—(Peroxidase-labelled affinity purified antibody to human IgG (H+L)) was quantified by colouring with ready to use 100 μL / well of KPL ABTS Peroxidase Substrate System soln. A + soln. B in the ratio 1:1 and incubated in the dark for 30 minutes. Optical density (OD) was read at 405 nm with a reference at 620 nm in the microplate absorbance reader .p-values ≤ 0.05 were considered statistically significant. Kruskal-Wallis equality-of-populations rank test was used to determine whether total IgG isotype responses to the malaria antigens were associated to the risk of clinical malaria and anaemia in the cohort study.The clinical and epidemiological data were entered into a Visual Fox Pro version 9.0 (Microsoft) data management programme and imported into STATA version 14.0  and SigmaPlot version 11.0 for analysis. Study participants were grouped by their wealth indices into high or low socio-economic status. The distribution and comparison of proportions of the various total IgG isotype for each antigen were analyzed using the classical and nonparametric test of hypotheses. Test with p > 0.05) except that the socio-economic status of household heads in the non-iron group was significantly higher than those in the iron group (p = 0.02)For the current objective, 871 children, aged 6–35 months were screened, from which 435 children received semi-liquid home-made meals mixed with 12.5 mg of iron daily (intervention group), and 436 received micronutrient powder without iron (placebo group) for 5 months. At baseline, there were no significant difference in the demographic and clinical characteristics between the groups (p = 0.02). . .p > 0.05). However, the female participants in the iron group had significant higher MSP3-FVO antigens antibody responses than their male counterpart (p = 0.04) (After the micronutrient powder (MNP) intervention, the IgG responses to the three recombinant malaria-specific antigens were similar between the iron-containing MNP and placebo groups ( = 0.04) .p < 0.05) (p < 0.05). However, in the placebo group, the levels of GLURP RO, GLURP R2 and MSP3 FVO in either the iron replete children or iron deficiency anaemia were similar  . The iro similar .p = 0.05) (p > 0.05) (p < 0.05). In both groups, there was a significant rise in malaria specific-antigens IgG levels from episode zero to two (p < 0.05) but reduces at episode three and rose again from episodes four or more  . However > 0.05) . In both or more . When th or more .This study for the first time assessed the impact of iron fortification on IgG responses to GLURP R0, GLURP R2 and MSP3 FVO recombinant antigens among pre-school children in malarial endemic settings. The current study found that, iron fortification did not influence IgG response to GLUP R0, GLUP R2 and MSP3 FVO. Moreover, iron fortified children with good iron status (iron replete and iron sufficient) had higher IgG response against malaria specific antigens compared to those with poor iron status (iron deficient and iron deficiency anaemics).Our findings is similar to studies by Priyanka and colleagues who conducted a randomized iron supplement controlled trial among Malawian children which compared prenatal and postnatal supplementation with multiple micronutrients, lipid-based nutrient supplementation, and observed that there iron supplementation has no effect on malaria antibody acquisition during infancy . LikewisOur study, further observed that the female participants were more protected from malaria due to differences in certain unknown exposures compared to their male counterparts irrespective of the study group but immunity was more significant among females with IgG responses against MSP3 FVO antigens compared to the males in the iron group. This observation was consistent with a study conducted in Malawi . On the Research findings in recent past have indicated the important role iron plays in the immunological developments in children especially, during proliferation and maturation of immunized cells , 31.In our study, the immunity against the malaria antigens waned as anaemia worsen among the children and even though appreciable IgG responses were observed in the iron group, the difference between the study groups were not significant.The effect of anaemia severity on immunity clearly showed a major influence on humoral mediated immunity in our study, but knowledge on its mechanisms remain a mystery. Other studies have indicated that reduced levels of malaria-specific antibodies in iron-deficient children may indicate a decline in acquiring malaria immunity and/or a reduction in malaria exposure . Though,We found that the iron-fortified children were more protected from malaria if they experienced two or more malaria episodes compared to children receiving MNP without iron. This findings corroborate with Tarun and Sachdev, who conducted systemic reviews of 28 randomized controlled trials involving 7892 children who were either receiving iron fortificants or placebo and concluded that there was no apparent detrimental effect on the general incidence of infection, especially malaria among the children, except a slight risk to diarrhea . SimilarHowever, in our study, we observed that IgG response induced by the malaria antigens among healthy  children were lower compared to malaria parasitaemic participants in the presence of iron-fortified micronutrient powder. Thereby, suggesting that iron intervention may have played a significant role in controlling parasitaemia via inhibition of the growth or invasion of malaria parasites. Consequently, it is believed that IgG antibodies that prevent blood stage malaria parasites replication are mediated by naturally adapted and induced artificial immunity in children . FurtherOur study observed progressive protection from malaria as the number of clinical malaria episodes increased among the children in the iron group compared to children from non-iron group. Reasons for these findings maybe that, the mechanisms required to offer immunity against severe ailments are different from those that provide protection against mild diseases or infections with moderate episodes –43. The Iron fortification had no impact on IgG response to GLURP R0, GLURP R2 and MSP3 FVO malaria antigens, however, IgG response to malaria specific antigens were high among children with sufficient iron status.S1 Checklist(DOC)Click here for additional data file.S1 File(PDF)Click here for additional data file.


In the Greater Mekong Subregion (GMS), current malaria surveillance strategies rely on a network of village health volunteers (VHVs) reporting the results of rapid diagnostic tests (RDTs), known to miss many asymptomatic infections. Integration of more sensitive diagnostic molecular and serological measures into the VHV network may improve surveillance of residual malaria transmission in hard-to-reach areas in the region and inform targeted interventions and elimination responses. However, data on residual malaria transmission that would be captured by these measures in the VHV-led testing and treatment surveillance network in the GMS is unknown.Plasmodium falciparum and P. vivax infection and serological testing  to determine exposure to P. falciparum and P. vivax.A total of 114 VHVs were trained to collect dried blood spots from villagers undergoing routine RDTs as part of VHV-led active and passive case detection from April 2015 to June 2016. Samples were subjected to molecular testing (quantitative polymerase chain reaction [qPCR]) to determine n = 13,157) and serological  testing. The prevalence of molecular-detectable P. falciparum and P. vivax infection was 3.2% compared to the 0.16% prevalence of Plasmodium spp. by RDT, highlighting the large burden of infections undetected by standard surveillance. Peaks in anti-P. falciparum, but not P. vivax, merozoite IgG seroprevalence coincided with seasonal P. falciparum transmission peaks, even in those with no molecularly detectable parasites. At the individual level, antibody seropositivity was associated with reduced odds of contemporaneous P. falciparum , but not P. vivax infection  indicating a potential role of immunity in protection against molecular-detectable P. falciparum parasitaemia.Over 15 months, 114 VHVs performed 32,194 RDTs and collected samples for molecular (We demonstrated that integration and implementation of sample collection for molecular and serological surveillance into networks of VHV servicing hard-to-reach populations in the GMS is feasible, can capture significant levels of ongoing undetected seasonal malaria transmission and has the potential to supplement current routine RDT testing. Improving malaria surveillance by advancing the integration of molecular and serological techniques, through centralised testing approaches or novel point-of-contact tests, will advance progress, and tracking, towards malaria elimination goals in the GMS.The online version contains supplementary material available at 10.1186/s12916-021-01993-8. Plasmodium falciparum within the Greater Mekong Subregion (GMS) has led to the region setting elimination targets for all human malaria by 2030 ), merozoite surface protein 2 (PfMSP2 [3D7]) and circumsporozoite (PfCSP [3D7]) and P. vivax AMA1  was measured by enzyme-linked immunosorbent assay (ELISA) using a robotic liquid handling system , as previously described  RDTs per village/VHV per month); however, this varied by month, with 2628 RDTs/month in the first month of the study decreasing to 1721 RDTs/month in the last month of the study . The overall seroprevalences for PfAMA1, PfMSP2, PfCSP and PvAMA1 were 38.6 (95% CI 37.8–39.4), 37.3 (95% CI 36.5–38.1), 18.7 (95% CI 18–19.3) and 28.4% (95% CI 27.6–29.1), respectively. Both anti-P. falciparum and P. vivax IgG level and seroprevalence were associated with time , p = 0.01, anti-PfMSP2 OR 0.81 , p = 0.15, anti-PfCSP OR 0.51 , p = 0.001; Table PfMSP2 and PfCSP were associated with a 12 and 10% reduction, respectively, in the odds of P. falciparum infection , p = 0.005, anti-PfCSP OR 0.90 , p = 0.02, Table PfAMA1 IgG level was not associated with the odds of qPCR-detectable P. falciparum infection , p = 0.34, Table P. falciparum IgG level/seropositivity was not associated with qPCR-detectable P. falciparum , p = 0.89 and OR 0.98 , p = 0.75, Table P. vivax-positive participants with repeated measurements (n=68), lagged effects of IgG were not estimated.To determine the association between IgG seropositivity on P. falciparum and P. vivax infection, even in those with no detectable parasites, indicating that this surveillance approach can capture significant levels of ongoing undetected seasonal malaria transmission. Integration of molecular and serological surveillance into the extensive VHV network may be an effective tool for monitoring residual malaria transmission in hard-to-reach pre-elimination settings by supplementing an established VHV surveillance network currently dependant on RDTs. Further, the addition of this kind of supplemented surveillance could support centralised malaria control strategies such as resource allocation until more suitable field-deployable tests are available to provide VHV with real-time treatment decision-making tools. Improving malaria surveillance will advance progress, and tracking, towards malaria elimination goals in the GMS.VHVs are the cornerstone of malaria control and surveillance activities in many malaria-endemic settings. By training VHV delivering routine malaria services to integrate sample collection for molecular and serological surveillance in their village, we demonstrated that surveillance using highly sensitive molecular methods in samples collected by VHV is feasible and can detect a significant subclinical reservoir of infection undetected by standard surveillance using RDTs. Furthermore, serological data reflected monthly trends in Plasmodium spp. infections found in participating villages were missed by RDT diagnosis with ~22-fold greater prevalence of qPCR-detectable infections over the entire study period. There was significant variation in qPCR-detectable infection over time with peaks of Plasmodium spp. infection in the rainy season, where malaria transmission increases, which were not detected by RDT, nor subsequently treated. There is increasing evidence that these untreated molecular-detected infections have the potential to contribute to ongoing malaria transmission, and despite not being transmitted as effectively to mosquitoes as microscopically detected infections, the greater prevalence of subclinical compared to clinical infections in low-transmission settings is cause for concern [RDT diagnosis and reporting of malaria cases by VHVs is the key indicator for progressing towards malaria elimination goals in the GMS. Like in much of the region, a large proportion of  concern . DetectiPlasmodium spp. infection. Rather, at a population level, antimalarial antibodies may indicate recent Plasmodium spp. transmission and exposure and therefore represent a useful tool for malaria microstratification to inform resource allocation and targeted implementation of malaria control and elimination interventions by NMCPs if integrated into national surveillance. The observed peaks in anti-P. falciparum merozoite IgG seroprevalence coinciding approximately with peaks of PCR-detectable P. falciparum infection, even in parasite negative individuals, indicate that malarial serology captured ongoing malaria transmission in the population not detected by routine RDTs. However, the magnitude of antibody seroprevalence was significantly greater than the prevalence of PCR detectable infections. Caution must be exercised in estimating the prevalence of circulating infections based on serology, which may be overestimated particularly in regions where the malaria burden is transitioning from malaria control to elimination phases, as antibodies can persist for extended periods [Plasmodium spp. transmission in the study region [P. falciparum and P. vivax infections may reflect cumulative incidence in the region over this time period [Plasmodium spp. infection exposure detected by microscopy, clinical incidence and relapse with P. vivax, across diverse malaria-endemic populations [P. falciparum antigens based on the 3D7 allelic variant. There is limited data on antigen allelic diversity in Myanmar, but previous studies have shown that the majority of circulating MSP alleles are of the 3D7 type [PfMSP2 across multiple sites in Myanmar and the Greater Mekong Subregion is minimal [Serosurveillance does not provide a diagnosis of a current  periods , 27. Thiy region . The hige period , 29. Bete period , 31. As e period . This stulations , 32–34. ulations , 36, and3D7 type  and that minimal . Further minimal , so inclP. falciparum infection in participants seropositive for P. falciparum-specific IgG. Recent longitudinal data from the GMS has shown that within infected individuals, parasite density oscillates frequently and that spontaneous clearance of PCR-detectable P. falciparum infection (i.e. in the absence of treatment) occurs regularly with the median time to resolution of infection of around 2 months for P. falciparum and 6 months for P. vivax [P. falciparum infection in seropositive individuals when sampled in the VHV network. This study provides evidence of potential protective immunity against molecular-detectable infections, which to date has only been established to reduce clinical malaria and high parasitaemia in numerous high-transmission populations [In individual-level analyses, we observed a reduction in the odds of concurrent qPCR detectable P. vivax . We hypoulations . We wereP. falciparum infection in the region is estimated to be under 3 months [The aim of this study was to investigate malaria incidence and serological data within an existing national malaria surveillance strategy, in this case, the VHV network. In the national VHV testing strategy, some individuals will be sampled multiple times and others only once, that is, malaria incidence is representative of the population that is sampled by VHV which may not be truly representative of the general population per se. Repeated sampling of a subset of individuals within the same population risks overestimating malaria incidence when the same individuals present multiple times with a single infection. In this study, which accounted for repeated measures in statistical analyses, only one participant contributing multiple samples was qPCR positive more than once, in samples collected 10 months apart. Given the estimated clearance time of sub-clinical 3 months , these lSampling at the VHV level may facilitate increased surveillance in rural and hard-to-reach areas. In this study, VHVs aimed to collect a minimum of 20 RDTs each month in accordance with programme implementation through PCD/ACD; however, data could not be stratified based on the VHV case detection method as this data was not collected. As such, we are unable to determine whether PCD alone would be sufficient to accurately capture the subclinical malaria burden or changes in serological markers reported here. The total number of RDTs declined across the study period, and this is most likely due to the declining motivation of the VHV to undertake RDTs which has been described in the region in areas approaching elimination where positive RDT results are infrequent . This waCurrent malaria surveillance strategies in the GMS rely on VHV reporting the results of insensitive RDTs that do not detect low-density infections. These undetected, untreated infections may contribute to ongoing malaria transmission and have the potential to hinder progress towards malaria elimination. We have shown that sample collection for molecular and serological surveillance can be integrated and implemented in community-delivered VHV health programmes in hard-to-reach populations and has the potential to capture ongoing malaria transmission at the individual and population levels. While molecular and serological testing in this study was undertaken after fieldwork was completed, away from the villages, data captured by sampling at the VHV level has the potential to inform programmatic decisions such as resource deployment by national malaria control programmes. With the increasing development and availability of easy-to-use, portable point-of-contact molecular and serological tests, there is future potential for VHV to undertake molecular and serological malaria surveillance and report detected cases in near real time. This will not only advance current routine VHV-led malaria surveillance, particularly in hard-to-reach areas, but will also reinforce ownership of malaria surveillance and the malaria elimination agenda to the community who will ultimately play a key role in reaching malaria elimination goals in the GMS.Additional file 1. Supplementary methodology.Additional file 2. Supplementary tables and figures.


P. falciparum predominated area. The present study found an enormous burden of Rapid Diagnostic Test negative malaria cases with P. vivax along with P. vivax and P. falciparum mixed infections during the mass surveillance from febrile and afebrile cases in dry winter months (February 2021–March 2021). Of the total 150 samples tested, 72 (48%) were positive and 78 (52%) negative for malaria by PCR. Out of the 72 positives, 6 (8.33%) were P. falciparum, 40 (55.55%) P. vivax, and 26 (36.11%) mixed infections. Out of 78 malaria negative samples, 6 (7.7%) were with symptoms, while among the total malaria positive, 72 cases 7 (9.8%) were with symptoms, and 65 (90.2%) were asymptomatic. Out of 114 samples tested by both microscopy and PCR, 42 samples turned out to be submicroscopic with 4 P. falciparum, 23 P. vivax, and 15 mixed infections. Although all P. vivax submicroscopic infections were asymptomatic, three P. falciparum cases were found to be febrile. Evidence of malaria transmission was also found in the vectors in the winter month. The study ascertained the use of molecular diagnostic techniques in detecting the actual burden of malaria, especially of P. vivax, in mass surveys. As Jhum cultivators in Tripura are at high risk, screening for the malarial reservoirs in pre-Jhum months can help with malaria control and elimination.With India aiming to achieve malaria elimination by 2030, several strategies have been put in place. With that aim, mass surveillance is now being conducted in some malaria-endemic pockets. As dry season mass surveillance has been shown to have its importance in targeting the reservoir, a study was undertaken to assess the parasite load by a sensitive molecular method during one of the mass surveys conducted in the dry winter period. It was executed in two malaria-endemic villages of Dhalai District, Tripura, in northeast India, also reported as  According to the World Malaria Report 2020, India contributed to the largest reductions in malaria cases in South East (SE) Asia from ~20 million cases in 2000 to ~5.6 million in 2019. However, it still accounted for 86% of all malaria deaths of the SE Asia region [India is aiming for malaria elimination by 2030, and a National Strategic Plan (NSP) has been taken up accordingly. According to the National Strategic Plan (NSP 2017-22), districts of India have been stratified into four distinct categories in terms of transmission intensity, which is measured by the annual parasite incidence (API). Category III Districts, the high transmission areas with API > 2, are in an intensified control stage and need concerted efforts to bring down the disease burden [P. falciparum infections persist throughout the dry season, playing the role of the reservoir infections for the transmission season, which would be several months later [Studies have shown that in the dry season, when the clinical incidence of malaria is generally low, malaria cases are found with low parasitemia and are asymptomatic. As clinical malaria is seasonal and mainly in the rainy season, subclinical hs later ,5,6.P. falciparum transmission in the subsequent rainy season. The parasite has been thought to have evolved strategies for preventing potentially fatal host pathology. It ensures persistence during periods when there are few or no mosquito vectors [Host survival during the dry months is considered essential to resume  vectors . In Suda vectors .P. vivax infections by households, are fewer and more tightly clustered during the dry season, while they expand during the wet season with the expansion of the breeding ground [P. vivax infections in the dry season, where only one symptomatic case of malaria was found by Rapid Diagnostic Test (RDT), indicating that the observed clusters are almost exclusively composed of asymptomatic carriers. It was also shown that groups of homesteads consisting of asymptomatic carriers could act as stable clusters for several years. These asymptomatic, potentially long-term infections might be sufficient for sustaining malaria transmission and therefore can be targeted for malaria control and elimination [P. falciparum and P. vivax were found, and most cases were asymptomatic [Additionally, parasite reservoirs are thought to be associated with low-density asymptomatic infections. There is a clear evolutionary advantage to the parasite for sustaining it, since asymptomatic individuals do not seek treatment and represent a parasite reservoir from which malaria vectors can become infected ,9. In sog ground . Anothermination . Hence, ptomatic .With this background, this study was planned to assess the burden of malaria infections and whether asymptomatic and submicroscopic malaria cases are present in dry post-winter months in malaria-endemic pockets of Dhalai district in Tripura. Dhansinghpara and Bidyapara villages, under the Gurudhanpara sub-center (SC), were amongst the highly malaria-endemic pockets in Dhalai and chosen by the National and State Malaria Programs for mass survey in February 2021–March 2021. The detection method used in the survey was RDT. Considering that RDT might miss asymptomatic submicroscopic cases, mostly with low parasitemia, this study was planned to use more sensitive nested PCR. A subset of samples was also screened by microscopy.A total of 150 samples, 90 from Dhansinghpara and 60 from Bidyapara, were tested by 18S nested PCR, and 72 people out of them were found positive with a total malaria case positivity of 48% , with 52.2%  in Dhansinghpara and 41.7%  in Bidyapara. Details of these infections and species distribution are given in Among the total of 150 samples tested, 13 (8.7%) were with symptoms, and 137 (91.34%) were without any symptoms. Out of those 150 tested, 72 were malaria positive, and among the positives, 65 (90.3%) were asymptomatic cases and seven (9.7%) were symptomatic cases. Out of 78 negative samples, 6 (7.7%) were with symptoms, while among the total malaria positive cases, 90.3% were asymptomatic.P. vivax, and three were P. falciparum + P. vivax. There were 65 asymptomatic cases, of which 6 were P. falciparum, 36 P. vivax, and 23 P. falciparum + P. vivax. Species wise age group distribution details with % for each age group are given in There were seven symptomatic cases, of which four were P. falciparum, while it was P. vivax by both microscopy and 18S PCR.The only case found positive by RDT was Out of 150 total samples, 65 (43.3%) were asymptomatic positive and 7 (4.7%) symptomatic positive cases, as found by PCR. Out of 78 negative samples, 6 (7.7%) were with symptoms , while aChi square analysis was conducted to test any association between symptomatic and asymptomatic cases with age. However, no such associations were found. Interestingly, asymptomatic cases occurred in younger age groups as well. However, there was a distinctly decreasing proportion of symptomatic cases from 9.09 to 6.25 to 2.67% as age groups increased from 0 to <5, 5 to <15, and 15 and above years .P. falciparum, two P. vivax, and seven mixed. Out of 114 samples tested, 42 samples were found to be submicroscopic as they were negative by microscopy, while PCR found them positive, which were 4 P. falciparum, 23 P. vivax, and 15 P. falciparum + P. vivax. In Dhansinghpara these were 3 P. falciparum, 16 P. vivax, and 3 P. falciparum + P. vivax, and in Bidyapara, 1 P. falciparum, 7 P. vivax and 9 P. falciparum + P. vivax cases.Out of a total of 150 samples, 114 samples, namely 67 from Dhansinghpara and 47 from Bidyapara, were tested by both microscopy and 18S PCR. Among them, 11 were positive by both methods: two P. falciparum + P. vivax in PCR but only P. vivax by slide; hence, they were considered as submicroscopic P. falciparum cases. One sample was detected as P. falciparum + P. vivax in PCR but only P. falciparum by slide; hence, it was considered as a submicroscopic P. vivax case. Age-group distribution of species-specific submicroscopic case details is given in P. vivax and mixed infections in the less than 5 years age group.Two samples were detected as P. falciparum, 22 P. vivax, 13 P. falciparum + P. vivax), and 3 were symptomatic .Among the submicroscopic cases, 39 were asymptomatic  and P. vivax (90–720 parasites/µL).Asexual densities calculated for some cases where microscopy could detect the parasites were in the low to moderate range for both p > 0.05) for symptomatic and asymptomatic cases was found to be associated with age groups, occupation, or long-lasting insecticidal net (LLIN) use by logistic regression analysis.No significant risk (Tectona grandis) and rubber (Hevea brasiliensis); hence, they have a closed canopy. There was less undergrowth, and both the trees are deciduous in nature. Croplands are confined only to the narrow valley area among the hilly terrain and were predominant with paddy cultivation. Cases in households showed that there were several households with more than one case, and those households were found to be located in proximity to each other and were in the vicinity of the forest areas.The ecological maps  showed tAnopheles species abundance is the number of specimens of a species obtained per the Centre for Disease Control and Prevention (CDC) miniature light trap per night.As shown in Anopheles minimus was found in these two villages during this time period (found in January). Other Anopheles found were three specimens of A. jeyporensis, six A. barbirostris, four A. Varuna and four A. Maculatus, and one A. annularis.Only a single specimen of A. minimus tested for Plasmodium sporozoite positivity by the molecular method in the dissected head and thorax part was found positive for P. vivax.P. vivax along with P. vivax and P. falciparum mixed cases among them. These were not detected by RDT during the mass surveillance from febrile and afebrile people in dry winter and spring months in two malaria-endemic villages of Dhalai District. While many cases are reported yearly in the study area, cases are very high during monsoon and post-monsoon times. A few cases are found in routine active and passive surveillance from January to March. Analysis of data of Ambassa PHC under which the study villages fall shows 2–5% of yearly cases in January to March, as evident from the data of 2018–2021 , and the majority of the cases detected by microscopy were found to have <100 parasites/µL, there were a few microscopically detected cases with a higher density, such as 600–720 parasites/µL, yet they were undetected by RDT for both parasite species. In the case of P. falciparum, these cases might have HRP II/III gene deletion, which is worthy of future exploration. In the case of P. vivax, this might indicate a lower sensitivity of RDT kits in field samples of this area, which again needs to be investigated in future. Furthermore, while the majority of these cases were asymptomatic, some of them were symptomatic as well, which were submicroscopic.In contrast, this study showed a considerable number of cases with a very high prevalence of ~48%, especially of P. vivax and mixed infections found in this study were asymptomatic in all the age groups, i.e., in below 15 years and more than 15 years groups. While there was no significant association of the symptomatic infections with the age group, the proportion was higher in the <5 age group  in the survey in an unbiased manner. The survey was attempted to include all the people who were available and willing to give blood, irrespective of their febrile status.Unusually high P. vivax, and the importance of screening in the dry season, which can assist in determining the course for control and elimination of malaria. Previously, a study conducted during winter in Tripura after the epidemic had shown a high prevalence of low-density asymptomatic cases (unpublished data).This study thus warrants the use of molecular techniques in the mass survey for detecting parasite positives, especially P. vivax as clusters, raises the possibility of these infections serving as the infectious reservoir. More direct proof of transmission in cold, dry months comes from our finding on the presence of parasites in A. minimus in January. Although traditionally it has been thought that temperatures below 18 °C would not be conducive to parasite development in the vectors, recent studies are challenging this notion. Waite et al. (2019) [Although the infectivity of the asymptomatic and submicroscopic cases was not tested in this study, several reports ,9,10 hav. (2019)  showed tP. falciparum and P. vivax [P. falciparum and none of P. vivax, which were not seen by RDT [P. falciparum, 29% P. vivax, and 14% P. falciparum + P. vivax.There have been reports of some mass surveillance studies from other parts of India. One study  in ChennP. vivax . A surven by RDT . A crossn by RDT  that of However, there have been few such studies in NE India, which has multiple malaria-endemic pockets. A previous study in 2014 in dry winter months (January–February) in the Missamari area of Assam, situated in northeast India, found no positive cases by RDT, microscopy, or PCR . AlthougP. vivax and mixed infections was also much higher, and that too in the dry post-winter month, which is not typical malaria season.This study found a considerably higher positivity rate in RDT negative samples than all other studies reported in India. Among them, the proportion of P. falciparum and P. vivax species. High proportions of P. vivax cases (60–80%) were seen in Arunachal Pradesh and Nagaland in the north with the alpine environment, 42–67% in Manipur, whereas in Assam, they varied from 23 to 31% with a subtropical and tropical climate. On the other hand, Tripura, with a warm and humid tropical climate and large stretches of tropical evergreen, semi-evergreen and moist deciduous forest and Meghalaya and Mizoram, had the lowest proportion of P. vivax cases [The NE states were shown to be co-endemic for ax cases .P. falciparum has been consistently the dominating species as per the clinical cases reported by the national malaria program [P. vivax malaria cases have been reported as very low by NVBDCP for the whole of Tripura, where the results are primarily RDT-based and only to some extent on microscopic examination while being performed exclusively on the symptomatic febrile cases. The same can be seen from the data from 2008 to 2019  and about 12 km south of the town of Ambassa, which is the district headquarters. About 40% of the area is under forest cover, while 45% is under scrubland. Agriculture land use covers about 0.13%, while about 6% of the site is under shifting cultivation.A door-to-door and health camp-based mass survey was carried out in Gurudhan subcenter under the Ambassa Primary Health Centre (PHC) in Dhalai District, from the third week of February 2021 to the first week of April 2021, by state health workers and volunteers. As these two villages are high endemic villages in the area , these tHealth camp-based mass surveys are those where health camps are organized by the health department in the village premises, and villagers are informed beforehand to come and get tested, irrespective of fever status. Regarding the choice of household or individuals, the aim of the survey, as decided by Health Department and Malaria Program was to cover as many villagers as possible, if not all. Thus, whoever was available for testing and willing to get tested was tested and treated.The total population in the two villages was 1009, namely 626 in Dhansingh and 383 in Bidyapara. Anyone available during the survey time and willing to give blood, irrespective of the presence of symptoms, was recruited for taking blood. There were some villagers who were not present in the village for a more extended period, either for a job or study or other reasons. They were not considered for the mass survey. Total such temporary absentees were 330: 203 in Dhansinghpara and 127 in Bidyapara. Hence, a total of 679 people was considered for the mass survey. During our survey in this period, among the available people, a total of 104 refused, namely 71 in Dhansinghpara and 33 in Bidyapara. Thus, 373 people (~55% of the considered population) could be covered, and blood was taken from them to test RDT. The rest of the people who live in the villages but were not found during the survey as they had gone out to the forest for Jhum cultivation or some other work were termed as unavailable. This year, due to elections in this area and other unrest, evening time visits were not possible, which are usually undertaken while conducting a mass survey to reach people who are unavailable during the daytime. However, as it could not be done this year, there was a high number of unavailable people. Questions on age, relations to HH head, fever history, travel history, forest exposure history, symptoms, and vector control measures were asked during the mass survey. In the case of febrile patients, detailed questionnaires were filled up.Finger-prick blood samples were collected from individuals irrespective of the febrile status. Finger-prick blood samples were used for microscopic slide preparation and collected in small tubes with EDTA powder for molecular technique. Written informed consent from the participant or the guardians in case of minors was obtained. Blood spots were also taken on filter paper, air-dried, and stored at 4 °C for future molecular analysis.All the 373 samples collected were tested by RDT by the Malaria Program. Blood slides and blood spots on filter papers were taken, and also whole blood was taken from the same finger prick in a tube containing EDTA, wherever available. For only 150 samples could whole blood be collected in the tubes. From the others, finger-prick blood could not be collected in tubes, as sufficient blood was not there. Therefore, in this study, we conducted PCR on these 150 whole blood samples collected in the tubes, not on the dried blood spots.P. falciparum and P. vivax parasites using an RDT Malarigen kit (Aspen Laboratories) supplied by NVBDCP, India, according to the manufacturer’s protocol.An RDT was performed only on febrile cases (people complaining of fever in the last seven days). Blood samples were collected to detect the presence of Plasmodium parasite [Both thick and thin blood smears were examined. Thick smears were hemolyzed, and thin smears were fixed with methanol and stained with 10% Giemsa stain. Stained blood smears were examined under 100X oil immersion using a Zeiss microscope by three experienced technicians to confirm the presence of the parasite .Parasite DNA was extracted from the blood samples that were collected in 0.5 mL microcentrifuge tubes containing EDTA. Briefly, 200 µL 1× phosphate-buffered saline (PBS) was added to the tube, followed by a short spin. DNA extraction was carried out using QIAamp DNA blood mini kit as per the manufacturer’s protocol . Then, DNA was eluted in 50 µL elution buffer and extracted DNA was stored at −20 °C for further molecular analysis.Plasmodium species targeting the 18S rRNA as described by Siwal et al. (2018) [2, and 5 µL of DNA template. Nest-2 PCR was carried out in a final volume of 10 µL reaction containing 1× Promega master mix, 0.5 µM primer, 2 mM MgCl2, and 2.8 µL of nest-1 PCR product as template. Laboratory adapted Dd2 and 3D7 Plasmodium falciparum, and previously confirmed positive P. vivax DNA samples were included as positive controls for every set of PCR. Nuclease free water and DNA of healthy individuals were also included as negative controls for the PCR assays. The amplified products were then visualized in 2% agarose gel stained with 0.5 µg/mL ethidium bromide under BioRad XR UV transilluminator.Species-specific nested PCR was performed to confirm the presence of . (2018) . For eacHost seeking adult female mosquitoes were collected using the CDC miniature light trap fitted in human dwellings of Dhansinghpara and Bidyapara villages. Light traps were fitted in the verandah, an outdoor porch with a roof outside the house. Collections were made monthly using five light traps in each village with two index houses fixed per month and three randomly rotated. Based on the baseline data, collections were made from two fixed houses every month and a few randomly selected houses, often those households which reported malaria cases. Traps were usually placed adjacent to the bed.Anopheles mosquitoes collected in the field were brought to the laboratory and identified morphologically using the keys of Nagpal and Sharma 1991 [rma 1991 . Morpholrma 1991 ,40,41.Plasmodium cytochrome b gene as described by Canier et al. (2013) in Plasmodium blood stages was performed to detect Plasmodium species with some modifications [Anopheles minimus and Anopheles baimaii [Anopheles specimen for each reaction. As we obtained little DNA from the head and thorax part of a single mosquito, we used the more sensitive technique of detection by nested quantitative PCR (nqPCR) for cytochrome b gene, which has more copy numbers, and this method is more sensitive in detecting a low amount. For all the real-time assays, in a 10 µL reaction volume, Promega 2× SYBR green master mix was used with carboxy-X-rhodamine (CXR) passive dye where 2.5 µL DNA was used as a template, and for every sample, the reaction was carried out in duplicate.The very sensitive nested real-time PCR method targeting the ications  in the k baimaii ,38. For The study used rainfall data from National Aeronautics and Space Administration’s (NASA) GPM  project from January to March 2021. The dataset used is the Integrated Multi-Satellite Retrievals for GPM (IMERG) Late Daily 10 × 10 km Level-3 Integrated Multi-Satellite Retrievals for GPM Daily Late Run (GPM 3IMERGDL) of GPM Level 3, derived from the dataset of the half-hourly 3IMERGHL. The result is an accelerated late estimate of the accumulated daily precipitation. The temperature and relative humidity data were taken from NASA’s MERRA-2  model. The used products are the daily maximum (T2MMAX), minimum (T2MMIN), average temperature (T2MMEAN), and relative humidity on a spatial resolution of 50 km × 62.5 km. The datasets were downloaded in NetCDF formats, which were further extracted using software codes.Land use land cover mapping was prepared using orthorectified Indian Remote Sensing satellite data, Cartosat-1 (2.5 m) and LISS-IV (5.8 m), employing on-screen visual interpretation techniques in the GIS  platform. Major land use and land cover categories and subcategories were delineated and updated using the latest data (2019) on the spatial layer initially prepared under NRSC/ISRO .P. vivax positive households were collected during the survey and plotted on the map for analyses of the proximity of the households to the Jhum fields/plantations/open forests/deep forests/croplands.Space-based Information Support for Decentralized Planning at the Panchayat level was used. The mapping was done at 1:10,000 scale. Field verifications were made by the project team to check the accuracy of the interpreted data. Geolocations of IBM SPSS 20 and Epi InfoTM version 7, CDC  were used for statistical analysis.Malaria yearly cases, API, and cases from January to March for Bidyapara and Dhansingh villages were compiled from the reports and registers of health workers and volunteers, which include routine active/passive surveillance and mass surveillance data. Data on annual malaria cases were taken from the reports of the Malaria Program, which include routine active/passive surveillance and mass surveillance data.


Plasmodium parasite. Altogether, 52 blood samples collected from patients with acute febrile illness were tested by microscopy, RDT, and real-time PCR. The results were compared in terms of sensitivity and specificity. Microscopy detected the malaria parasite in 5.8% of the blood samples whereas 13.5% were detected by the RDT and 27% by real-time PCR. Considering real-time PCR as the gold standard method, microscopy had a sensitivity of 21.4% and a specificity of 100%, and the RDT had a sensitivity of 28.6% and a specificity of 92.1%. Microscopy together with the RDT successfully detected malaria positive cases in blood samples of Ct value below 20, but both were unable to detect malaria cases between 26–40 Ct value ranges amplified by real-time PCR. Despite various diagnostic tools being available, microscopy still remains the method of choice for diagnosis, while the RDT is user-friendly when applied at the point of care. However, our preliminary results emphasize the need to implement the test with higher sensitivity and specificity in the context of a malaria elimination programme. Such programmes can be a crucial opportunity to understand the species prevalent in a low-endemic region. However, these results should be further verified with a large cohort study to document the submicroscopic infection.For ongoing malaria elimination programmes, available methods such as microscopy and rapid diagnostic tests (RDTs) cannot detect all malaria cases in acute febrile illness. These methods are entirely dependent on the course of infection, parasite load, and skilled technical resources. Our study objectives were to estimate the performance of light microscopy and a RDT as well as real-time PCR for the detection of the  Plasmodium that cause malaria fall under the phylum Apicomplexa. Five Plasmodium species infect humans: Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi . The tests were performed using rapid diagnostic kits, following the manufacturer's instructions [The histidine-rich protein-2 and ructions .μL of blood samples using QIAamp DNA blood minikits  following the manufacturer's instructions in the QIAamp DNA mini-handbook [Blood samples, preserved in 0.5 mg EDTA vials, were stored at −70°C to prevent DNA degradation. Samples were left at room temperature for 30 minutes before the extraction process was carried out. DNA was extracted from 200 handbook .Plasmodium species designed by the manufacturer. Firstly, a master mix solution was prepared using the following scheme: primer/probe: 2 μL, enzyme/reaction mix: 6 μL, nuclease-free water: 12 μL, and template: 5 μL. The thermal cycler was programmed as phase 1 at 95°C for 2 min to implement one cycle, phase 2 at 60°C for 20 seconds to produce 30 cycles, and phase 3 at 68°C for 1 minute per kb to generate 30 cycles on the basis of manufacturer's instructions. Fluorescence intensity was measured using the Rotor gene Q software, and the graph of fluorescence intensity vs. the number of cycles was plotted [Extracted DNA was amplified using a Qiagen Rotor-Gene Q system using abTes™ Malaria 5 qPCR commercially available kit  using a set of specific primers for each  plotted .Plasmodium DNA was amplified. Five fluorescent dyes, i.e., FAM, HEX, ROX, Cy5, and Quasar 705, were used to detect five different species of Plasmodium [Plasmodium falciparum,  FAM (green)-Plasmodium ovale  Cy5 (red)-Plasmodium malariae,  HEX (yellow)-Plasmodium vivax  ROX (orange)-Plasmodium knowlesi  Quasar 705(crimson)-where positive control = all malaria-positive control available in the commercial kit and negative control = phosphate buffer saline.Real-time PCR-based TaqMan assay was performed to identify the species and compare the results with microscopy and the RDT. asmodium , i.e.:  Blood samples were considered positive for malaria based on fluorescence intensities which were greater than the threshold or cutoff values.p values were calculated by using the chi-square test by using SPSS [The collected data were entered into an excel spreadsheet, coding was done for different variables, and the data was transferred to SPSS version 11.5. The comparison between different diagnostic tools was done using real-time PCR as the gold standard assay. Applying the chi-square test by constructing 2 × 2 table, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the diagnostic tests were calculated. The level of agreement between microscopy and the RDT were estimated by using the interrelated kappa coefficient, and ing SPSS .n = 26) of men and women (n = 12) age group. The fewest participants were in the age group 70–79.In our study, 52 individuals were involved, with an equal number (nd women . The hign = 3 (5.8%) samples were positive by microscopic examination, and n = 49 (94%) samples were negative by microscopic findings. Schizonts of Plasmodium vivax  blood samples were positive for malaria where n = 3 (5.8%) were found to be Plasmodium falciparum, n = 4 (7.69%) were found to be species other than Plasmodium falciparum, and n = 45(87%) cases were negative using immunochromatography tests.Out of the total 52 blood samples, n = 14(27%) blood samples were positive for the detection of Plasmodium species and n = 38(73%) blood samples were negative for Plasmodium species using real-time PCR assay. Amplification of Plasmodium DNA from 52 blood samples of patients with acute febrile illness is shown in Figures Out of the total 52 whole blood samples, n = 3 (5.8%) were falsely diagnosed by the RDT for malaria.The detection of malaria from whole blood samples were implemented by using three diagnostic tools such as light microscopy, RDT, and real-time PCR in our study. The prevalence of cases stratified by microscopy was 5.8%, by the RDT was 13.5%, and by real-time PCR was 27%. Large proportions of cases were missed by light microscopy (94%) and the RDT (86.5%). Unfortunately, p value <0.05).Using real-time PCR as a reference method, 14 (27%) participants were positive for malaria while 38 (73%) participants were negative . The RDTp < 0.05) in mean Ct value was observed when comparison was done individually by applying Student's t-test as summarized below in Microscopy and the RDT had a similar rate of detection as PCR for malaria-positive cases with a Ct value below 20. Microscopy diagnosis reduced to 50% as it failed to detect one positive malaria case between 20 and 25 Ct value . HoweverAccurate estimation of malaria burden is essential for malaria management following a prompt treatment regimen. The diagnosis of malaria has been a remarkable challenge, especially in poor regions, due to the availability of limited resources, equipment, and methods. Laboratory diagnostic methods have been mandatory for the detection of cases and the timely initiation of appropriate therapy. The study was conducted to determine the proportion of malaria cases missed by microscopy and the RDT but detected by real-time PCR. Microscopy and rapid diagnostic tests are routinely used diagnostic tools. Research in other countries suggests a fivefold difference of estimated prevalence using light microscopy and PCR , 18. In μl of blood [μL between expert and field microscopists. This implies that the negative results of microscopy does not exclude Plasmodium infection. The RDT missed 71% of real-time PCR-positive infections. However, the sensitivity of malaria detection using the RDT is greater than that of microscopy, which is consistent with the study conducted in Kenya [μL [Plasmodium falciparum. This was due to the merged pLDH panels for different Plasmodium species. RDT's sensitivity was 28.6% with a specificity of 92% in our experiment. Other studies showed RDT sensitivities ranging from 60 to 80% and specificities between 80 and 90% based on the detection of different Plasmodium species [Plasmodium falciparum with Plasmodium vivax that was later confirmed by PCR [Other studies in Southeast Asia have reported similar results where light microscopy has detected a low level of malaria , 18. Theof blood , 24, 25.in Kenya . Limitatenya [μL , 25, 27.enya [μL , 27. RDT species . Our stu species –32. Few d by PCR .Plasmodium species conducted in the US showed real-time PCR was a reliable confirmatory tool for the diagnosis of malaria to resolve conflicting doubts between light microscopy and RDT [Real-time PCR performance showed better sensitivity and specificity than conventional PCR in several past research studies , 34, 35. and RDT . False d and RDT , 38.Our PCR data helped us to understand the difference between light microscopy and RDT results. The LOD of the RDT was roughly comparable to light microscopy in the field survey, although the newer generation of RDTs showed a higher sensitivity than the previous ones , 6, 39. Light microscopy efficiency seems inadequate for surveillance of parasite infections in Nepal in areas where the intensity of transmission rate is shifting from high to low. The RDT test has the advantage of allowing empirical treatment for symptomatic individuals with positive RDT results. Our preliminary results emphasized the need to implement the test with the higher sensitivity and specificity in the context of a malaria elimination programme, which provides an opportunity to understand the parasite circulation in a low-endemic region. However, these results should be further verified with a large cohort study to document the submicroscopic infection.On examining the cost analysis of each methodology applied in our study, one of the major challenges in a low-income country for the diagnosis of malaria and conducting a large cohort study is a cost barrier in different applied diagnostic methodology. In addition, the RDT costs (11.25 USD per single test) are more expensive than the routine investigation of peripheral blood smear microscopy . DNA extraction kits per box (500 USD) cost approximately 60000 NPR (500 USD) and master mix reagent kits also cost approximately 500 USD per box (50 test) in Nepal  . The cosAlthough light microscopy remains a standard method for detecting malaria parasites, it has many shortcomings despite the use of experienced microscopists, such as the quality control of reagents, effects of self-medication, and the inability to detect low levels of parasitemia. This implies that the presence of a large proportion of submicroscopic parasitemia at tertiary care hospitals has been vastly underestimated. The application of PCR adds to our understanding of the malaria transmission level, in the sense that they can determine whether recently cleared infections are treated or not and are no longer detectable by a light microscope and the RDT. Thus, applying all available diagnostic tools  together was able to detect actual parasitemia and recent infections and may provide the most precise information to decide on the best malaria control strategies.


The results demonstrated that the malaria RDTs currently commercially available might cause FN results in practice.Despite the widespread use of malaria rapid diagnostic test (RDT) in clinical practice, there are a lot of challenges. We conducted a secondary analysis of 129 malaria RDT data from rounds 5–8 of the World Health Organization (WHO) product testing summary and discuss the causes of false-negative (FN) results with a focus on low parasite density, improper RDT storage, operation and interpretation, and The online version contains supplementary material available at 10.1186/s40779-021-00345-0. Dear Editor,Malaria rapid diagnostic test (RDT) is becoming the most-used method to diagnose malaria worldwide, especially in sub-Saharan Africa because malaria RDTs are easier to utilize than microscopy and have been shown to have comparable detection capability in the field . DespitePlasmodium falciparum (Pf) with a pfhrp2/3 gene deletion.There were 129 RDT products enrolled into the product assessment on malaria RDT performance in this summary . The sumPlasmodium vivax  strip. The storage of RDTs for prolonged periods in hot or humid conditions may impair the diagnostic ability. This analysis showed that the positive rate of some products at low parasite density was impaired as the incubation temperature increased. Malaria is endemic in some geographic areas, such as sub-Saharan Africa and Southeast Asia, which are characterized by a hot, humid climate and poor storage infrastructures. As a result, RDTs were usually exposed to temperatures and humidity above the product recommended limits in these endemic areas, which may result in poor test performance.RDTs are considered to be an ideal way for parasite-based diagnosis, particularly in remote and resource-limited areas, mainly due to the simplicity of the test. The results of this analysis showed that approximately 20–27% of products need to be improved for blood safety and instruction quality, 30–35% of RDT test bands against Pf were minimally visible, and that the RDT test bands against Pv returned a higher proportion . Table S2. Percentage distribution of test band intensity score against wild-type Pv in 200 parasites/μl (%).


In non-endemic settings, transfusion-transmitted malaria (TTM) is rare but potentially fatal and becoming more common with globalization. We present two pediatric cases that demonstrate donor screening using questionnaires is subject to error and that TTM should be considered with fever following numerous transfusions in children, particularly sickle cell patients. In non-endemic settings, malaria is mostly associated with travel or emigration from endemic areas. Less commonly, it is acquired via placental transfer, organ transplantation, or transfusions . We presA 14-month-old premature male was admitted with multiple respiratory viruses leading to respiratory failure requiring emergent veno-venous extracorporeal membrane oxygenation (ECMO). ECMO decannulation was complicated by right atrial perforation, hemoperitoneum, and disseminated intravascular coagulation requiring a total of 113 transfusions, including 48 aliquots of packed red blood cells (PRBCs).Plasmodium parasites were incidentally identified. A thin smear revealed 0.3% parasitemia with morphology consistent with P. vivax or P. ovale  at ARUP Laboratories and confirmed by the US Centers for Disease Control and Prevention (CDC). Further subspecies identification by CDC identified P. ovale curtisi. Given the lack of travel to a malaria-endemic area, a diagnosis of TTM was made.Nine days after his last transfusion , he developed fever, and empiric antibiotics were initiated. Bacterial and viral infectious workup was negative. His examination and imaging were significant for splenomegaly without evidence of pneumonia, intracardiac vegetations, or intra-abdominal abscess. Six days later, a 48-hour cycle to his fevers became apparent, and he developed pancytopenia and elevated inflammatory markers. A peripheral blood smear was requested to evaluate for hemophagocytic lymphohistiocytosis. P. ovale . The patP. ovale and P. falciparum, suggesting prior infection with both species, but relapsing P. ovale as the source [The source investigation focused on 30 red cell donors, of which three reported travel or emigration from malaria-endemic areas. Only one had positive malaria testing. This donor mistakenly reported travel, rather than emigration from Cameroon 2 years prior, and was not appropriately deferred  . Also, te source , 6.Mycoplasma pneumoniae by respiratory PCR panel, and azithromycin was prescribed. On day 7 of illness, he was admitted for acute hypoxic respiratory failure requiring noninvasive ventilation secondary to pulmonary edema without acute chest syndrome.A 13-year-old male with transfusion-dependent Hemoglobin (Hgb) SS sickle cell anemia repeatedly presented to emergency departments with fever, chills, headache, body aches, abdominal pain, emesis, and diarrhea over a week. Infectious workup detected only P. falciparum  associated with TTM is likely due to delays in diagnosis and treatment as well as comorbidities and compromised host defense among transfusion recipients , 7. SickIn non-endemic settings, blood donor screening policies balance the risks of TTM against the costs of excluding healthy donors , 5. In EBetween 2000 and 2017, all reported TTM cases in the United States (n = 11) were from asymptomatically infected donors that emigrated from malaria-endemic countries , 6. ConsAs TTM is rare, our pediatric institution having two cases in 3 years is notable. We hypothesize a few reasons. First, TTM may be underrecognized due to limited experience by practitioners and laboratory technologists. Incidental detection in our cases can be attributed to our hospital having highly skilled laboratory technologists. Second, children versus adults may be more likely to be symptomatic, which prompts additional workup leading to diagnosis. Third, among US cities, the Dallas-Fort Worth-Arlington Metroplex has among the highest concentration of immigrants from sub-Saharan Africa, thus increasing the likelihood of asymptomatically infected donors . We addiOur cases illustrate that blood donor screening using questionnaires is subject to error, despite recent changes in deferral criteria. In an increasingly globalized world, TTM should be considered with fever following numerous transfusions in children, particularly sickle cell patients. Awareness by pediatricians, infectious diseases specialists, and laboratory technologists can facilitate early diagnosis and treatment to prevent potentially fatal outcomes.