Sri Lanka achieved the WHO certificate as a malaria free country in September 2016, thus monitoring of malaria transmission using sensitive and effective tools is an important need. Use of age-specific antibody prevalence as a serological tool to predict transmission intensity is proven to be a cost effective and reliable method under elimination settings. This paper discusses the correlation of four anti-malarial antibodies against vivax and falciparum malaria with the declining transmission intensities in two previously high malaria endemic districts i.e. Kurunegala and Moneragala of Sri Lanka.Plasmodium vivax and Plasmodium falciparum. The sero-positive samples were determined as mean OD + 3SD of the negative controls. The sero-prevalence was analyzed against the demographic characteristics of the population. A simple reversible catalytic model was fitted into sero-prevalence data to predict the sero-conversion and sero-reversion rates.Sera was collected from 1,186 individuals from the two districts and were subjected to standard ELISA together with control sera from non-immune individuals to obtain Optical Density (OD) values for four anti-malarial antibodies i.e. anti-MSP1 and anti-AMA1 for both P. vivax antibody data in that district. In Moneragala district however, all the serological markers indicated a change in transmission that has occurred approximately 15 years ago.Over 60% of the population was sero-positive for one or more antibodies except young children (<10 years). The sero-prevalence was zero in young children and very low in young adults when compared to the older age groups. The model developed for falciparum malaria that assumed the presence of a change in transmission was not significant in the Kurunegala district although significant reduction in transmission was observed when the model was used for P. vivax and P. falciparum proved to be useful indicators in predicting transmission under elimination settings as prevailed in Sri Lanka. The sero-conversion rates for the two districts studied are shown to be very low or zero indicating the absence of active and/or hidden transmission confirming a “true” state of elimination at least, in the two study districts in Sri Lanka.Assessment of MSP1 and AMA1 anti-malarial antibodies of The online version of this article (doi:10.1186/s12879-016-2164-0) contains supplementary material, which is available to authorized users. Malaria remained as a major health burden throughout history in Sri Lanka, with recurring epidemics every 3–5 years. However, countrywide malaria incidence rates started to decline during the past decade facilitated by the application of intense control programs Fig. . Sri LanMalaria was considered as an endemic disease in the dry zone of Sri Lanka, prior to year 2000, though its transmission was considered as low and seasonal. It peaked from December to March period during the northeast monsoons and smaller peaks occurred in concurrence with the southwest monsoon during June to October. The malaria endemic dry zone, as recognized traditionally, included mainly the northern, eastern and southeastern parts of the island with occasional epidemics in the intermediate zone (i.e. northwestern and western mountain slopes) . The annSri Lanka has remained free of malaria transmission for the past 3 years and was certified as a state “free-of-malaria” in September 2016 by the World Health Organization (WHO) .Standard molecular methods have failed so far to demonstrate the presence of submicroscopic levels of parasitaemias in previously endemic zones , though Serological markers are tools that have been recognized as good indicators of malaria transmission intensity under various malaria endemic settings with significant correlation with EIR estimates , 9–11. APlasmodium falciparum  at varying transmission intensities [Plasmodium vivax (P. vivax/P.v.) malaria transmission, though it is indeed the species that is more resistant to elimination efforts [Plasmodium vivax had always been the more prevalent causative agent of malaria in Sri Lanka according to past records [P. falciparum cases. The number of falciparum malaria cases were less than five during the years 2011 and 2012 whereas the total cases were 124 and 23 respectively [The modeling of age-specific antibody prevalence and fitting the reversible catalytic sero-conversion model to real data has been described for ensities , 17. How efforts . Plasmod records . Indigen−19 (Merozoite Surface Protein 119), anti-MSP2 (Merozoite Surface Protein 2) and anti-AMA1  have been widely used in predicting medium and long term variations of malaria transmission [Anti-malarial antibodies anti-MSP1smission , 10, 11.P. vivax and for P. falciparum using two anti-malarial antibodies viz. anti-MSP1 and anti-AMA1  which were proven to be sustained at high levels in Sri Lankan populations earlier [The main objective of this study was to assess the ability to predict changes in malaria transmission by fitting age-specific anti-malarial antibody prevalence to a reversible catalytic model for both  earlier .The present study was conducted during December 2013–August 2014 in two previously malaria endemic districts in Sri Lanka, namely Kurunegala  and Moneragala  located in the dry zone lowlands about 127 kilometers apart  or “estate” (1.9%) population, the latter being the resident labourers of areas with plantations Table . By 2012All the District Secretariat (DS) divisions in the two districts  were initially included in the sampling frame. Multi stage cluster sampling was used in a non- random basis  and random basis (to identify second stage clusters). Four DS divisions (out of the 11 DS divisions) from Moneragala district and 12 DS divisions (out of 30) from the Kurunegala district were selected on the basis of the presence of District/Base hospitals or Medical Officer of Health offices  was calculated after correcting the base sample size for differences in design, contingencies, laboratory errors etc. An approximate sample size of 500 also warrants a 50–75% relative length of the 95% confidence interval for estimating sero-conversion rate while studying Southeast Asia populations with entomological inoculation rate (EIR) equal to 0.01 [The base sample size was manually calculated  at a prevalence of 83% , and at to 0.01 . Further to 0.01 . HoweverThe number of individuals to be included from each DS division was calculated based on probability proportionate to the size of the populations of each DS division.Ethical clearance for this study was granted by the Ethics Review Committee, Faculty of Medicine, University of Colombo, Sri Lanka.Blood samples were collected by house to house visits, from people attending communal gathering places and local health facilities. Prior approval to collect blood samples from persons attending hospitals or from the community was obtained from the relevant administrative bodies. The required numbers of samples were collected from these selected individuals after obtaining informed consent. Blood samples from children under 12 years were collected from those who attended the local hospitals and already bled for other tests. Consent for minors was obtained from their parents/guardians.Every individual who was recruited for the study was given a unique identification number. All personal identifiers  were entered in a data collection book. Other information i.e. age, gender, history of past malaria attacks (during their lifetime) as recalled by the individual and socio-economic data was entered to the data collection sheets prepared under each individual’s unique identification number.Two milliliters (mL) of blood was drawn by a trained technician or a nurse. This was divided in to two tubes; (1 mL each) one coated with EDTA (Ethylene Di-amine Tetra-acetic Acid) and one plain tube, labeled with each individual’s identification number. The samples in EDTA tubes were stored in freezers at the end of blood collection sessions until transfer to the laboratory in Colombo, where they were stored at −70 °C for future use. Blood in the plain tube was allowed to clot for 4–5 h at 4 °C and was centrifuged at 12,000 r.p.m. (rounds per minute) to separate the serum. Serum was carefully extracted to a separate eppendorf tube labeled with the identification number and was sealed with parafilm. These serum samples were stored in the freezers until transfer to Colombo in frozen condition, in ice packs and were stored at −20 °C until further use.A standard ELISA (Enzyme Linked Immuno-Sorbent Assay) protocol described elsewhere  was used−19-Pf, MSP1−19-Pv ; AMA1-Pf or AMA1-Pv . ELISA plates  were coated with 50 micro liters (μL) of antigen with a concentration of 0.5 μg/mL. Plates were incubated at 4 °C overnight and were washed three times with Phosphate Buffered Saline with 0.05% Tween 20 (PBS/T). To each well 100 μL of diluted serum samples (1: 200 for MSP1−19 and 1: 400 for AMA1 in 2% skimmed milk in PBS/T) were added. Each ELISA plate contained 46 test samples in duplicate, a known positive control in duplicate, a known negative control and a blank well. The plates were incubated at 4 °C overnight. They were washed three times in PBS/T and 50 μL of horse radish peroxidase-conjugated rabbit anti-human IgG (Sigma-Aldrich/Cat#A8792) diluted in 1: 2000 in PBS/T was added to each well. This was incubated for 3 h at room temperature before washing three times in PBS/T. To each well 50 μL of o-phenylenediamine dihydrochloride (OPD) substrate solution (Sigma-Aldrich/Cat#P1526) (0.4 mg/mL in solvent) was added. This was left for 15 min for the colour to develop. The reaction was stopped by adding 25 μL of 2 M H2SO4 per well. Then the plates were read at 492 nm using an ELISA micro-plate reader. The Optical Density (OD) value for each well was recorded separately. Standard procedures were carried out to maintain the validity of the protocols. The same positive control was included in every ELISA plate and a chart was maintained to identify any significant deviations (+/− 2 standard deviations (SD)) of the OD value of the particular sample for normalization of the data. Plots were obtained to check cross reactivity  for each antibody was determined with OD values obtained with serum samples of 16 non-exposed, non-immune apparently healthy individuals from Matara and Colombo districts  and the cut-off values were determined (as mean) OD + 3SD i.e. 0.1115 for MSP1-Pf, 0.168 for MSP1-Pv, 0.0411 for AMA1-PF and 0.054 for AMA1-Pv) for positive and negative responses to each antigen according to previously used methods .The OD values for each test serum sample were entered into an Excel data sheet together with other information collected on the sample . The samples with an OD value exceeding the cut-off value for each antibody was considered as sero-positive for the relevant antibody. The total sero-prevalence, as well as gender and age specific sero-prevalence was determined for each district, DS division and GN division and was compared using Chi squared test with SPSS V15.0 statistical software. Reversible catalytic models were fitted to each sero-positivity data using a Binomial-product distribution for the sampling distribution and estimation methods based on maximum likelihood principle , 23. In n = 469) recruited from Kurunegala district was over three times higher than the male participants (n = 150) but the number of males (n = 263) was comparable to the number of females (n = 286) in the Moneragala district. Gender of 18 participants from the Kurunegala district has not been recorded (Table p < 0.001).A total of 1,186 individuals were recruited from the two districts . The number of females . Mean age varied between 40.2–49.07 years in the DS divisions of Kurunegala, while in the DS divisions of Moneragala it ranged between 23.8–27.3 years. Study participants were divided into 4 age groups as follows: 1–5 years, 6–10 years, 11–20 years and age higher than 20 years. Although the number recruited for the fourth age group (age >20 years) was higher when compared to the other age groups in all the DS divisions in both districts, the deviation was not significant between any of the DS divisions. Numbers of individuals recruited in all age groups (except >20 years) were more in the Moneragala district, when compared to the Kurunegala district, (Table p < 0.001).Age of the study subjects ranged between 1–84 years  in Kurunegala and 1–85 years  in Moneragala with mean and median ages of subjects in Kurunegala being significantly higher than in Moneragala  in the past, while in the Moneragala district the percentage of a previous history of malaria was in 21.9%. Previous malaria exposure was comparable between the two districts, with relevant data not available from 102 individuals from the Kurunegala district and 56 from Moneragala district (Table p = 0.507).In the Kurunegala district 18.8% of the study population recalled (by memory) having had one or more attacks of malaria infections (either p = 0.043).Previous exposure was not significantly different between any of the DS divisions within Kurunegala district, but it was significantly higher in Kataragama DS division when compared to the other DS divisions in the Moneragala district  when compared to the other age groups, but this pattern was not observed among the participants from the Moneragala district. Previous exposure to malaria was not significantly different between males and females in either district .The number of individuals with previous malaria exposure was significantly higher in the 11–20 years and >20 years categories in the Kurunegala district   Table .Table 4Sp = 0.031) and for any Pv (p = 0.044)/any Pf (p = 0.045) antibodies (Chi square Test).Within each district, sero-prevalence did not significantly differ between any of the DS divisions in either district. Sero – prevalence of males and females in Moneragala districts were comparable. However, the sero prevalence between sexes in the Kurunegala district showed a higher prevalence in males, the differences of which were marginally significant for AMA1-Pf . In Moneragala district, this difference was even more prominent with the sero-prevalence rates being markedly low in younger age groups up to 20 years .The sero-prevalence of the subjects in age groups 1–5 years and 6–10 years were significantly lower, when compared to the values of the individuals age > 11 years (11–20 year age group and > 20 year age group) for all antibodies in Kurunegala district . However, the sero – prevalence rate for MSP1−19 antigen was relatively lower among individuals with history of previous exposure in Kurunegala district , when compared to those in Moneragala district . Though the antibody prevalence for either vivax or falciparum antigen, was high (>55%) in individuals with history of previous exposure in both districts, the association between sero-positivity and history of malaria exposure by recall was relatively poor and was not statistically significant for either district (Chi square test/Phi-Cramer’s V statistics).Over 50% with a history of previous exposure to malaria were sero-positive for AMA1 antibodies in both districts  of 0.0048-yr for MSP1−19 and a SCR of 0.0785and a SRR of 0.0608 for AMA1. Although model two indicated a significant change in transmission when P. vivax data were considered, the probable time point of such a change failed to overlap, (with time point estimated as 30 years whenMSP1-Pv data was used and 5 years with AMA1-Pv data)  of 0.0079P. falciparum approximately 10–14 years and approximately 15 years ago for P. vivax  for P. falciparum were previously reported by the authors [−19 and AMA1 for falciparum malaria have been shown through modeling experiments to be extremely low with antibodies having long half lives [Without active transmission it was a challenge to choose appropriate serological markers since there was little evidence for the presence of specific anti-malarial antibodies, except in the Moneragala district, where high levels of anti-malarial antibodies i.e. MSP1 authors . The declf lives , 25.−19is also described as a suitable immunological marker to assess endemicity/change in transmission levels even under low transmission conditions [AMA1 is a surface protein of merozoites which is highly immunogenic hence can be easily detected. MSP1nditions , 19.−19 and AMA1 were selected to model transmission intensity for P. falciparum using age-specific sero-prevalence rates. MSP1−19 and AMA1 were also selected for P. vivax since it has been the more prevalent causative species of malaria in Sri Lanka through a major part of history and is the one that persisted longer after elimination of P. falciparum a few years earlier [Therefore, both MSP1 earlier . Both th earlier .The use of NANP as a suitable serological marker to inform malaria transmission has been debated in low transmission conditions . NANP is“chena” cultivators or people who were engaged in their own small-scale businesses, and thus the male participation in the study was more or less comparable to that of females. These very reasons might also have accounted for the differences in the mean and median ages of the two districts;  as well as the differences in the number of recruits in the different age groups.The number of female participants recruited from the Kurunegala district was 3 times higher than of males. In Kurunegala the blood samples were collected mainly by visiting households during the morning hours when males have already departed for work and from people attending communal gathering places e.g. village weekly fair, where the majority of attendees were females. These reasons might have influenced the results with over representation of female participants from the Kurunegala district. On the other hand the population from the Moneragala district comprised mainly farmers, Furthermore, this difference might have had an effect on the reversible catalytic model since the number of males included in the Kurunegala district was relatively low. It appears that males tended to be sero positive more than females though this difference was not statistically significant.Past history of the disease was determined by the participants’ recall of memory. The data suggests that a history of previous exposure to malaria did not influence the sero – prevalence rates in the two districts . Kataragama, which is situated in the south of the island, is considered as a “Sacred City” with thousands of people travelling on pilgrimage each year from various parts of the country especially from the Northern areas, where malaria used to be abundant. This might have accounted for the higher transmission levels in this locality as recalled by the residents, when compared to those living in other DS divisions. These factors as well as differences in socio-economic structure in Moneragala, which is more suitable for malaria transmission could account for the higher sero-prevalence in Moneragala when compared to Kurunegala . This is further confirmed by the API indices of the two districts, with API of Moneragala being more than three times that of Kurunegala throughout the period up to year 2000 .−19 . AMA1 is considered to be highly immunogenic when compared to the MSP1−19protein [p < 0.05, Chi square test), probably due to the differences in outdoor activities and attire style between the sexes. Such a pattern could not be observed in Moneragala, however, probably due to the differences in socio-economic factors and life style when compared to Kurunegala. Sero-prevalence in younger age groups i.e. <10 years was significantly low for all antibodies  indicating low transmission levels in more recent years in both districts.Sero-prevalence for AMA1 antigen was higher than that of MSP19protein , which mP. falciparum data was concerned and the modeled data indicated a single force of infection. The sero-conversion rate calculated using P. falciparum data was extremely low (0.0026-yr/0.0045-yr). This could happen as results of very low transmission, as described by Cook et al. [P. vivax data (both MSP1−19 and AMA1) the derived change of point in transmission through the model was 5 years prior according to AMA1 data and 30 years prior according to MSP1 data. The difference in timelines indicated by the two antibodies might be due to the differences in immunogenicity of the two antigens. AMA1 is said to be more sensitive and immunogenic of the two and this might account for the differences above. Furthermore, this might also hint at a sub-microscopic transmission that may have prevailed until 5 years prior to the sample collection. Furthermore, different transmission patterns for vivax and falciparum as evidenced by data is likely to reflect the actual difference in transmission of the two species observed over the years. However, the drop in falciparum transmission may not have occurred as suddenly as that of P. vivax , subclass of antibody, half-life of the relevant antibody and polymorphisms [In the Moneragala district the change in transmission was evident through the analysis of all tested antibodies; but pointing at slightly different time points i.e. 10–14 years ago for falciparum malaria and approximately 15 years ago for vivax malaria. Furthermore, the predicted sero-conversion rates (SCR past and SCR present) and sero-reversion rates (SRR) were different for different antibody types which were observed in both districts Table −, subclasThis study reveals a clear reduction in malaria transmission in both these districts, especially in Moneragala district where a marked decline in total district malaria cases from 40,885 to 3705 in year 2000 , ecological  and interventions such as introduction of λ-Cyhalothrin in 1994, shift in IRS strategy to targeted spray in 1996–97, Roll Back Malaria initiative in 1999 and introduction of artemisinin-based combination therapy in 2007 [Information considered collectively demonstrates the usefulness of serological markers to assess the endemicity under low malaria transmission conditions and its potential for use as evidence for elimination of malaria, particularly when combined with improved technology to make this tool even more sensitive and accurate.Plasmodium species, which might be a limitation since at individual level there are likely variations in immune response to different parasite antigens. Thus there might have been individuals in the study population who did not show sero-positivity in spite of exposure to malaria. Therefore, using a higher combinations of antigens to identify sero-positive individuals might be a better approach [The study used only two recombinant antigens of each approach .A low number of sero-positive individuals in the younger age groups may have also limited the chance of detecting significant changes in transmission over time. A larger population of younger study subjects in the survey would have increased the chances of getting amore sero-positives within the younger age groups. Number of individuals that should be included in each age group was calculate (quota sampling) when calculating the sample size for each DS division to minimize this error. However, this had practical limitations due to difficulties in getting blood from young children in the community as malaria is not perceived as a threat in this community in recent years. Therefore, in this study blood samples were collected from younger children who attended government hospitals and private health facilities, and who were anyway bled to carry out other investigations, which limited the numbers included.Another possible limitation to this study may have been the inadvertent inclusion of sero-positives who acquired the disease following travel to a malaria endemic country. If the travel history was not provided during the surveys, this may have led to an over estimation of the level of transmission using the serological markers. Although unlikely, with present observations, it still remains a possibility.Toxoplasma homolog to the AMA1 antigen of the P. falciparum and similarly Gaffar in 2004 documented a protein of Babesia bovis which was homologous to P. falciparum AMA1 [Cross reactivity with other parasitic antigens in the ELISA may have also influenced the results. Donahue in 2000 reported a rum AMA1 , 29. How−19/AMA1 of P. falciparum and P. vivax species. Merozoite Surface Protein 1 (MSP1) and especially Apical Membrane Antigen 1 (AMA1) are relatively conserved between both P. vivax and P. falciparum [Plasmodia species is either unlikely or very low [Another unlikely possibility is the cross-reactivity between MSP1lciparum , 31 sevevery low , 33.Plasmodium vivax that may have prevailed until recently. A drastic decline in local transmission in the Moneragala district was evident, beginning approximately 10–15 years ago for both species and the current rates of sero-conversion are extremely low or zero, confirming the absence of active transmission.Age-specific sero – prevalence curves were proven to be a useful tool in predicting malaria transmission intensity in Sri Lanka where malaria elimination has been achieved. The data suggests a low single force of transmission in Kurunegala district for falciparum malaria but hints a decline of vivax malaria starting 30 years ago. It also suggests a possibility of a sub-microscopic level transmission of


Anemia, Leukopenia, and thrombocytopenia are commonly observed hematological abnormalities in malaria and typhoid patients. In this study, we evaluated the prevalence of cytopenias in patients with mono-infections of plasmodium parasites  or salmonella bacteria (typhoid group). Full blood counts from 79 patients (age ranging from 18 to 77 years) categorized into malaria and typhoid groups at the Tamale Central Hospital were assessed.χ2 = 5.84, p < 0.016). No association was found between cytopenias and gender in patients in the malaria group; however, the middle aged group, 36–55 years, was significantly associated with anemia . Cytopenias were not associated with gender, and with different age categories in patients in the typhoid group.Data generated were entered and analyzed using SPSS version 20 and Graphpad Prism 6. Values were observed to be significant at p < 0.05. The prevalence of cytopenias were; 29.6, 48.0% for anemia, 38.9, 12.0% for thrombocytopenia, 20.4, 12.0% for leukopenia, 13.0, 8.0% for bicytopenia and 5.6, 4.0% for pancytopenia in both malaria and typhoid groups respectively. Between the two groups of patients, thrombocytopenia was significantly associated with those in the malaria group (The online version of this article (10.1186/s13104-018-3456-9) contains supplementary material, which is available to authorized users. Cytopenias are blood cell abnormalities that result from reduction in the major hematopoietic cell lines such as red blood cells causing anemia, leukocytes causing leukopenia and thrombocytes causing thrombocytopenia. Bicytopenia occurs where there is a reduction below reference ranges in any two of the major cell lines . The simplasmodium and salmonella infections. The occurrence of Cytopenias may be attributed to bone marrow suppression and hemo-phagocytosis [Anemia –5, leukoocytosis , 17. Elsocytosis , 18, 19.Bicytopenia and pancytopenia usually result from direct or indirect decreasing effect on hematopoietic cell production in the bone marrow –22. ThesDespite many global studies reporting anemia, thrombocytopenia and leukopenia with malaria and typhoid in adults, limited studies of these cytopenias exist in Ghana. The studies in Ghana, are mostly on anemia in children –30 and iThe study was conducted in the Tamale metropolis; the Northern Regional capital of Ghana. The metropolis has 13% of the total land area of the Northern Region , and a total population of 223,252 comprising 111,109 males and 112,143 females .A hospital based cross-sectional study was conducted from February to April 2015 on patients with mono-infections of plasmodium parasites or salmonella bacteria between the ages of 18–77 years at Tamale central Hospital. Tamale Central Hospital is one of the northern regional hospitals and serves nearly 240,000 people with numerous referrals from other districts in northern Ghana.Sample size was obtained based on the number of adults that showed up with either malaria or typhoid within the period of sampling. Pregnant women and individuals diagnosed with helminthiasis, sickle cell disease, and kidney disease were excluded from this study.Demographic data (including sex and age) of each study participant was collected using a standardized questionnaire  was used to collect 3 ml of venous blood from each participant for laboratory investigation. The blood samples were collected by a trained medical laboratory scientist.A Vacutainer KMalaria was diagnosed using CareStart™ HRP-2 rapid diagnostic test . The testing, results reading and interpretations were performed by strictly following the manufacturer’s protocol. The presence of malaria parasites was confirmed by microscopy using thick blood smears which were stained with 20% Giemsa solution at pH 7.2 . The reaSalmonella typhi were added to patient’s plasma distributed on the respective plates. This was then mixed and rocked and agglutination observed and interpreted as previously reported [The slide agglutination method previously described  was perfreported .9/l and WBC count < 4.0 × 109/l were used respectively to determine thrombocytopenia and leukopenia [Hb-Hemoglobin, WBC-White blood cell and PLT- Platelets were analyzed using the automated blood cell analyzer Sysmex XS-500i . Anaemia was diagnosed by Hb < 12 g/dl for non-pregnant females and Hb < 13 g/dl for males , while Pukopenia . Bicytopχ2 test.Data were analyzed using SPSS Version 20  and Graphpad Prism 6 . All test statistics with p values < 0.05 were considered as statistically significant. Continuous variables were described using median and interquartile range (IQR), and compared by Mann–Whitney test, whilst categorical variables were presented as counts and percentages, and compared by Fisher’s exact test or Pearson’s 3/µL (IQR 142.0–287 × 103/µL) and 5.8 × 103/µL (IQR 4.5–7.0 × 103/µL) respectively.Out of the 79 individuals recruited for this study, majority, 55 (69.6%) were females Table . The medplasmodium and salmonella respectively (Table p < 0.016), with patients in the malaria group, anaemia, leukopenia, bicytopenia and pancytopenia were independent of either groups , Table The prevalence of cytopenias were; 29.6, 48.0% for anemia, 38.9, 12.0% for thrombocytopenia, 20.4, 12.0% for leukopenia, 13.0, 8.0% for bicytopenia and 5.6, 4.0% for pancytopenia in patients with mono-infections of Malaria and typhoid are quite common in Tamale because the prevailing warm and humid climate is conducive for the infection, concomitant with poor sanitary practices that promote the infections . MalariaP. vivax) is different from that in Ghana . Unlike P. vivax, P. falciparum is very virulent; it multiples rapidly in the blood and has the ability to sequester into diverse host organs [P. falciparum infected-erythrocytes can bind platelets to form platelet-mediated clumps [P. falciparum infection. Thus, differences in parasite strains could be a possible reason for the difference seen in this study; more so because [P. falciparum cases than in P. vivax cases. Our findings of a higher prevalence and significant association of thrombocytopenia with malaria adds to studies indicating that thrombocytopenia is an indicator of malaria in the adult population [In the current study, thrombocytopenia was found to be the most prevalent in the adults with plasmodium infection. This contradicts 2 studies , 19 thatt organs . It has d clumps  and postpulation .While extensive studies have been conducted on the occurrence of anemia in children and pregnant women with malaria –5, littlEven though leukopenia was the least prevalent cytopenia, its prevalence was quite significant, and could confound the estimation of malaria parasite density in adults in Tamale based on the WHO criteria of WBC count of 8000 cells/μL.The high prevalence of anemia recorded in the adults with typhoid is similar to a study by . This fiPancytopenia and bicytopenia are a common hematological problem encountered in clinical practice, which have multiple causes and the underlying pathology determines the management and prognosis of the patients. Bicytopenia and pancytopenia were observed in both infections; and the knowledge that both cytopenias are seen in quite a number of malaria and typhoid patients in Tamale is important information that would help in timely diagnosis and proper management of such cases, leading to earlier discharge of the patient. Unlike many studies that had male dominance in pancytopenia cases –51, we rThe high prevalence and association of thrombocytopenia with malaria in adults in Tamale buttresses numerous findings of low platelets count as a reliable diagnostic marker for malaria in adult populations in Africa. The association of anemia with the middle aged adults with malaria should prompt a closer look at anemia with plasmodium infection in this age category in Tamale. Our finding of pancytopenia in only females, should prompt strengthening of education on barriers preventing females from accessing healthcare delivery.This study was conducted within a relatively shorter period and the sample size was small. Since malaria endemicity is nearly constant within the metropolis, an all year investigation of these cytopenias in patients with malaria or typhoid would be important in substantiating the findings in this study.Additional file 1. Document used in the collection of socio-demographic and health data. This is a blank document which contains the questionnaire used in the collection of socio-demographic characteristics, and the health records of the study participants.Additional file 2. Data set of the subjects. This is a SPSS document containing data transribed from the questionnaire.


Malaria control intervention coverage increased nationwide in Malawi during 2000–2010. Trends in intervention coverage were assessed against trends in malaria parasite prevalence, severe anemia (hemoglobin < 8 g/dL), and all-cause mortality in children under 5 years of age (ACCM) using nationally representative household surveys. Associations between insecticide-treated net (ITN) ownership, malaria morbidity, and ACCM were also assessed. Household ITN ownership increased from 27.4%  in 2004 to 56.8% (95% CI = 55.6–58.1) in 2010. Similarly intermittent preventive treatment during pregnancy coverage increased from 28.2% (95% CI = 26.7–29.8) in 2000 to 55.0% (95% CI = 53.4–56.6) in 2010. Malaria parasite prevalence decreased significantly from 60.5% (95% CI = 53.0–68.0) in 2001 to 20.4% (95% CI = 15.7–25.1) in 2009 in children aged 6–35 months. Severe anemia prevalence decreased from 20.4% (95% CI: 17.3–24.0) in 2004 to 13.1% (95% CI = 11.0–15.4) in 2010 in children aged 6–23 months. ACCM decreased 41%, from 188.6 deaths per 1,000 live births (95% CI = 179.1–198.0) during 1996–2000, to 112.1 deaths per 1,000 live births (95% CI = 105.8–118.5) during 2006–2010. When controlling for other covariates in random effects logistic regression models, household ITN ownership was protective against malaria parasitemia in children  and severe anemia . After considering the magnitude of changes in malaria intervention coverage and nonmalaria factors, and given the contribution of malaria to all-cause mortality in malaria-endemic countries, the substantial increase in malaria control interventions likely improved child survival in Malawi during 2000–2010. Plasmodium falciparum is endemic throughout Malawi.1 Malaria transmission is stable with seasonal peaks related to the rainfall during November to April.2 The Ministry of Health (MoH) in Malawi reported that malaria accounted for 40% of all outpatient visits and 40% of hospital deaths in 2001, and was the cause of workforce losses (up to 25 days of work lost per year on average).3 Malaria was also the leading cause (39%) of hospital admissions in children less than 5 years of age.3Malaria caused by 4 The Malawi Government and other funding partners contributed funding for malaria control as well during this time.5 The Malawi Government and its national and international partners have shown a strong continued commitment to malaria control and prevention, which is reflected in three successive 5-year Malaria Strategic Plans since 2000.3,6,7There have been substantial increases in funding for malaria control in Malawi that started around 2006 and which came mainly from the Global Fund to Fight AIDS, Tuberculosis and Malaria  and the President’s Malaria Initiative, in total amounts of $42.2 and $83.1 million, respectively, from 2006 to 2010.8 and the commercial sector to deliver insecticide-treated nets (ITNs) and had the first national ITN program in sub-Saharan Africa by 2003.7 The Malawi MoH has recommended using sulfadoxine-pyrimethamine (SP) for malaria intermittent preventive treatment during pregnancy (IPTp) since 1993,9,10 long before the World Health Organization (WHO) recommended IPTp in 2002.5 Over the years, the Malawi Government changed malaria treatment policies in response to the emergence of antimalarial drug resistance. In 1993, Malawi was the first country in sub-Saharan Africa to replace chloroquine with SP as the first-line treatment of uncomplicated malaria. In 2007, Malawi changed from SP as the recommended first-line antimalarial to artemether-lumefantrine (AL), an artemisinin-based combination therapy, following significant evidence of malaria parasite resistance to SP.11 These key milestones are summarized in Malawi has been at the forefront of the introduction of malaria control interventions in sub-Saharan Africa. The country initially used social marketing Given the increased investments, changes in malaria control polices, and early adoption of interventions in Malawi, this study evaluated the impact of malaria control efforts on malaria morbidity and all-cause childhood mortality (ACCM) during 2000–2010.13 The rationale for using this evaluation design with ACCM as the primary impact measure is discussed in depth elsewhere.13–15 The RBM MERG recommended using ACCM as a primary impact measure where baseline transmission intensity is moderate or high .13 This evaluation design was strengthened by using logistic regression models to assess the strength of association between household ITN ownership and malaria parasitemia and severe anemia among child household members during the evaluation period, two malaria morbidity measures which are in the causal pathway between malaria control interventions and changes in ACCM  Partnership’s Monitoring and Evaluation Reference Group (MERG) .13 The r in ACCM . MultivaSupplemental Table 1. The Demographic and Health Survey (DHS), Multiple Indicator Cluster Survey (MICS), and Malaria Indicator Survey (MIS) use a two-stage survey design, typically with the most recent census as the sampling frame for enumeration units. The DHS Program17 and United Nations Children’s Fund MICS18 websites and survey reports provide more detailed information on sampling of these surveys. The 2010 DHS results for intervention coverage and severe anemia were used for trend analysis instead of the 2010 MIS results due to seasonal comparability with the other surveys used in this evaluation. DHS field work was conducted in the low malaria transmission season and the MIS in the high malaria transmission season.A list of population-based evaluation indicators and data sources are shown in We calculated the proportion of women with a live birth 0–2 years before the survey who received at least two doses of SP for IPTp. Data on the source of SP medications were only collected in the 2004 and 2010 DHS, so source was not included in the calculation of this indicator.19 in children aged 6–59 months who slept in a sampled household the night before the survey was calculated using DHS data. Hemoglobin values were adjusted for altitude according to the standard methodology used by the DHS.20,21Severe anemia prevalence (hemoglobin concentration < 8 g/dL)22,23 The 2001 NMS was sampled for children 6–35 months old, whereas the 2009 survey was sampled for children 6–59 months old. The 2009 survey was reanalyzed to generate estimates for children 6–35 months of age for comparison purposes. The NMS were conducted in the low transmission season. Malaria parasite prevalence (microscopy-based) data were also obtained for children 6–59 months of age from the 2010 MIS (field work March to April 2010) during the peak malaria transmission season.The National Micronutrient Surveys (NMSs) are nationally representative cross-sectional household surveys, in which data were collected on severe anemia (Hb < 8 g/dL) and parasitemia (microscopy-based) in children 6–35 months of age in 2001 and 2009.24 and used in logistic regression models to assess the association between ITN ownership and parasitemia or severe anemia. These surveys measured malaria parasitemia (by microscopy) and severe anemia in children 6–30 months of age in six districts in 2005 and 2006 and in eight districts in 2007, 2008, and 2009. These surveys were conducted in April of each year, which represents the latter portion of the rainy season and therefore the peak of the malaria transmission season. The six districts surveyed in 2005 and 2006 included four from Southern Region , and one each from Central Region (Lilongwe) and Northern Region (Rumphi). The 2006 parasitemia data were not available for this study and therefore 2006 data were not included in the logistic regression models discussed below. In 2007, 2008, and 2009, an additional district was sampled in both the Central Region (Nkhotakota) and Northern Region (Karonga).Sub-national data on severe anemia (Hb < 8 g/dL) and malaria parasite prevalence estimates were obtained from anemia and parasitemia surveys,http://data.worldbank.org/country/malawi).25Contextual factor data including information on household attributes, asset ownership, women’s education, human immunodeficiency virus (HIV) prevalence, and maternal and child health interventions were obtained from the DHS, MICS, and MIS surveys unless otherwise indicated. Gross domestic product (GDP) per capita purchasing power parity (PPP) data were obtained from the World Bank /Earth Resources Observation and Science Center, Moderate Resolution Imaging Spectroradiometer satellite.21 Mortality rates were not calculated from the 2006 MICS because these 5-year estimates overlap with the estimates from the 2004 and 2010 DHS surveys making it difficult to detect changes between the surveys. ACCM estimates were stratified by urban and rural residence (as defined by DHS) and malaria risk zones. Malaria risk zones were determined by overlaying the cluster locations from the 2010 DHS on a 2007 map of estimated prevalence rates of P. falciparum infection in children 2–10 years of age (PfPR2–10) from the Malaria Atlas Project.29 Terciles of PfPR2–10 values were then created at the cluster level. Malaria parasite prevalence ranges for the three risk categories included 0–33.7% (lower); 33.8–45.2% (medium); and 45.3–100% (higher). Disaggregated annual estimates of ACCM with 95% confidence intervals (CIs), for the 1990–2010 period were calculated from the 2000 (1991–1999) and 2010 (2000–2010) DHS data following a similar method as for the 5-year mortality rates, but limited to 1-year intervals.Five-year all-cause mortality rates were calculated from the 2000, 2004, and 2010 DHS using a synthetic cohort life table approach described in detail in the Guide to DHS Statistics.30–32Malaria control intervention coverage and severe anemia prevalence estimates were stratified by sex, residence , wealth quintiles, and mother’s education . Severe anemia prevalence was disaggregated by malaria risk terciles and by age groups (6–23 months and 24–59 months), as studies have shown the impact of malaria control interventions on malaria morbidity in these age groups to be different in malaria-endemic countries.Statistical significance for differences was determined by assessing the 95% CIs around each of the survey estimates. Nonoverlapping CIs for estimates from two survey years were deemed statistically significant. All analyses were performed using Stata 12 .33 The models included a random intercept for each enumeration area. The models assessed the odds of malaria parasitemia or severe anemia for children aged 6–30 months after controlling for survey year and child, household, region, and meteorological factors. Differences in meteorological factors (at the district level) preceding each survey  were controlled for by including anomalies from the 5-year means for February and March rainfall and minimum temperature. Districts were collapsed into regional categories . An interaction term for survey year and region was included in the models. Malaria infection  was included as a covariate in the severe anemia model.Separate multivariable, random effects logistic regression models from the sub-national survey data predicting the outcomes of malaria parasitemia and severe anemia were conducted using household ITN ownership, defined as at least one ITN in the household, as the primary ITN exposure variable. Although the proportion of children sleeping under an ITN might have been used as an indicator of ITN exposure, it has been shown that the pooled effect of children sleeping under an ITN the previous night or household ITN ownership on the prevalence of parasitemia are of similar magnitude and not significantly different.Supplemental Figure 1 for maps of district-level household ITN ownership).Household ownership of at least one ITN rose significantly from 27.4% (95% CI = 25.7–29.0) in 2004 to 56.8% (95% CI = 55.6–58.1) in 2010 . HousehoSupplemental Tables 2 and 3). ITN use also improved among the total population, increasing from 13.8% (95% CI = 12.6–15.1) in 2004 to 29.0% (95% CI = 28.0–30.1) in 2010  increased substantially between 2000 and 2010 . ITN use in 2010 .Indoor residual spraying (IRS) was implemented sub-nationally in Malawi. Starting as a pilot in 2007, it was then expanded to seven districts along Lake Malawi and in the Shire Valley. At the time of the 2010 DHS, only households in Nkhotakota District had been sprayed during the year, resulting in 59.4% of the households in Nkhotakota District having been sprayed with IRS in the past 12 months .Supplemental Table 4).The proportion of women with a live birth in the 2 years preceding the survey who received at least two doses of SP during pregnancy increased from 28.2% (95% CI = 26.7–29.8) to 55.0% (95% CI = 53.4–56.6) between 2000 and 2010 . IPTp coSupplemental Table 5). Of all children less than 5 years of age who experienced fever in the prior 2 weeks, use of any antimalarial increased over the study period from 27.1% (95% CI = 25.3–28.9) in 2000 to 43.4% (95% CI = 41.4–45.4) in 2010. The proportion of febrile children treated with a recommended (first-line) antimalarial drug did not change between 2000 (23.3% [95% CI = 21.6–25.0]) and 2006 (20.2% [95% CI = 18.7–21.8]); however, an increase occurred between 2006 and 2010 (36.4% [95% CI = 34.4–38.4]) . SP was the recommended first-line antimalarial in 2000, 2004, and 2006 and ACTs (AL) in 2010.Although overall care seeking for fever in children under five has increased over the decade from 35.3% (95% CI = 33.1–37.5) to 65.4% (95% CI = 63.6–67.1), timely care seeking (within 24 hours of fever onset) has increased only slightly, albeit a statistically significant change (26.4% [95% CI = 24.6–28.3] in 2000 and 32.3% [95% CI = 30.7–34.0] in 2010) . Care se4–38.4]) . There wIn the Malawi NMSs, conducted during the low transmission season, malaria parasite prevalence in children 6–35 months of age decreased from 60.5% (95% CI = 53.0–68.0) in 2001 to 20.4% (95% CI = 15.7–25.1) in 2009 . MalariaSupplemental Table 7). Declines in severe anemia were also seen in the NMS, where severe anemia in children 6–35 months declined significantly from 17.7% (95% CI = 13.5–21.8) in 2001 to 7.3% (95% CI = 4.8–9.8) in 2009 .The prevalence of severe anemia did not decrease significantly between 2004 (10.6% [95% CI = 9.1–12.4]) and 2010 (8.7% [95% CI = 7.6–10.0]) in children aged 6–59 months using the DHS surveys. However, significant decreases were evident in subgroups of younger children, who were at higher risk for severe anemia than older children. Severe anemia prevalence in children 6–23 months of age declined from 20.4% (95% CI = 17.3–24.0) in 2004 to 13.1% (95% CI = 11.0–15.4) in 2010, a relative change of 36% , whereasSupplemental Table 8), with the smallest and largest reductions in the neonatal (26% reduction) and child (47% reduction) mortality rates, respectively. Significant declines were observed in ACCM among children from rural areas, but not urban areas  were examined. The GDP per capita (PPP) remained relatively constant between 2000 and 2006 , but then rose from 634 in 2006 to 749 in 2009, an 18.1% relative increase.25 Significant improvements were seen in access to an improved water source, increasing from 65.2% (95% CI = 62.1–68.2) in 2000 to 79.7% (95% CI = 77.7–81.5) in 2010; households with an improved roof, increasing from 25.8% (95% CI = 24.5–27.3) in 2006 to 35.0% (95% CI = 32.9–37.0) in 2010; electricity, increasing from 4.8% (95% CI = 3.6–6.4) in 2000 to 8.7% (95% CI = 7.6–9.9) in 2010; and telephones, increasing from 5.1% (95% CI = 3.8–6.9) in 2004 to 39.3% (95% CI = 37.6–41.1) in 2010  in 2000 to 68.9% (95% CI = 67.8–70.0) in 2010 and the percentage of births in a health facility also increased significantly from 55.3% (95% CI = 52.7–57.9) in 2000 to 73.2% (95% CI = 71.3–74.9) in 2010 . HoweverSupplemental Table 9). Early initiation of breastfeeding worsened over the evaluation period declining from 72.1% (95% CI = 70.7–73.5) in 2000 to 67.0% (95% CI = 66.3–67.7) in 2010, whereas exclusive breastfeeding increased from 44.2% (95% CI = 40.7–47.7) in 2000 to 71.4% (95% CI = 68.0–74.6) in 2010 and Vitamin A supplementation improved from 70.6% (95% CI = 69.0–72.1) in 2000 to 85.5% (95% CI = 84.6–86.4) in 2010 . Treatment seeking for both ARI (from 26.7% [95% CI = 24.4–29.1] to 65.4% [95% CI = 62.9–67.9]) and diarrhea (from 28.3% [95% CI = 25.7–31.2] to 62.4% [95% CI = 60.0–64.7]) improved  from 2000 to 2010  significantly improved from 70.1% (95% CI = 67.2–72.8) to 80.9% (95% CI = 78.9–82.8) between 2000 and 2010  and prevention of mother-to-child transmission (PMTCT) coverage both increased during the evaluation period. HIV-positive pregnant women receiving ARVs increased from 2,179 to 33,200 between 2004 and 2009.38 According to UNAIDS, PMTCT coverage during pregnancy and delivery was 24% in 2009 and 53% in 2011.39Among women aged 15–49 years, HIV prevalence did not change between 2004 and 2010 with an estimated 13% infected according to the DHS. Similarly, new child HIV infections remained relatively stable at 20,000 cases in 2000 and 19,000 cases in 2010.4,28 Additional meteorological analyses are presented in the accompanying article by Thomson and others.28Meteorological data for rainfall and temperature were assessed over the evaluation period. Rainfall and temperature patterns do not suggest that any long-term climate differences existed over the period of malaria control scale-up.In logistic regression models, lower household wealth status (quintile), older age (in months), not having an ITN in the home, and survey year by region  interaction were all significant predictors of malaria parasite prevalence . Mean raITN ownership was similarly protective against severe anemia in children 6–30 months of age. Lower household wealth status (quintile), younger age (in months), and not having an ITN in the home were all significant predictors of severe anemia . ChildreThis evaluation found a 41% reduction in ACCM in Malawi during 2000–2010, substantial increases in household ITN ownership and use in children under five, reductions in malaria parasitemia and severe anemia, and insufficient changes in socioeconomic factors and other determinants known to influence child health to fully explain the decline in child mortality. Consequently, the evaluation indicates that malaria interventions contributed to the decline in ACCM in Malawi during 2000–2010. The association of individual ITN ownership with reduced odds of parasitemia and severe anemia further strengthens this conclusion.Supplemental Figure 3 for estimates from the United Nations Interagency Group for Child Mortality Estimation34 and the Institute for Health Metrics and Evaluation).35 The magnitude of ACCM decline was greater in the medium and higher malaria risk areas compared with the lower malaria risk areas, which is consistent with what would be expected if part of the declines in ACCM were malaria-related given children in these areas had the greatest potential to benefit from increased malaria intervention coverage.40,41 Similarly, the relative decline in ACCM was greater in rural areas (43%) than urban areas (10%). Rural areas are where most of the malaria transmission occurs and most (84%)42 of the population lives. However, the largest decline  took place between 1996–2000 and 2000–2004, a period preceding the largest increase in malaria control intervention coverage in Malawi. However, malaria control intervention coverage was low but not insignificant between 2000 and 2004. For example, an ITN program was underway in all districts (household ownership of at least one ITN was 27% in 2004) as well as IPTp roll out (two doses of SP was 28% in 2000 and 47% in 2004). These early declines in ACCM are therefore likely due in part to the initial scaling up of malaria control interventions, along with increases in other child survival interventions and socioeconomic improvements. In the 2004–2010 period, when malaria control was intensified, mortality continued to decline.The 41% decline in ACCM observed in our evaluation during 2000 and 2010 is comparable to the mortality declines over this period shown by other investigators using different mortality estimation methods  population use of ITNs can achieve community benefits (change in EIR) of ITN use beyond individual protection alone.45 Thus, the increase in coverage of ITNs provided individual protection to the user and likely community protection as well. Malawi used IRS as another vector control measure in addition to ITNs; however, in this national-level impact analysis, IRS was unlikely to play a significant role given only a 2% national coverage in 2010.46 IRS could have had important sub-national effects due to the high coverage in targeted districts.Malaria control intervention coverage increased nationally during 2000–2010. ITNs as the primary malaria prevention tool were implemented in Malawi using a variety of delivery methods, including social marketing, commercial sector, routine distribution at ANC and EPI clinics, and mass distribution campaigns .4 A soci5,47 Thus, given the protective effect of IPTp on low birth weight,48 increasing IPTp coverage could have improved birth outcomes leading to reductions in neonatal mortality before the study period and continued to contribute to reductions in these outcomes over the evaluation period. However, given the minimal improvements in neonatal mortality seen here  and the increases observed in the proportion of babies born with low birth weight , the effect of IPTp may have been mitigated by other contextual factors, for example a reduction in women receiving four ANC visits between 2000 and 2010 (19% relative reduction). In addition, there was an increase in the prevalence of P. falciparum mutations that reduce the effectiveness of SP used for IPTp in Malawi.49At the time of this evaluation in 2010, Malawi had one of the highest IPTp coverage (55%) levels in sub-Saharan Africa, up from 28% in 2000.50,51 Studies of ACTs have demonstrated higher rates of parasite clearance and better treatment outcomes compared with SP and cholorquine.52,53 Among children less than 5 years of age with fever, treatment with a first-line antimalarial increased from 23% in 2000 to 36% in 2010 and treatment with a first-line antimalarial within 24 hours increased slightly from 19% in 2000 to 24% in 2010. There are limitations to using household survey data to assess malaria case management, including problems with recall by the mothers or caretakers. In addition, the scaling up of diagnostics could have blunted the true coverage estimates of appropriate malaria case management; however, the RDT roll out occurred after the end of the evaluation period. Overall, it can be concluded that treatment of fevers increased and there was increased use of more effective antimalarial treatments over the evaluation period, which may have contributed to a decline in ACCM.Care seeking for fevers from formal health providers in children less than 5 years of age increased (35% in 2000 to 65% in 2010). The majority (80%) of this increase in care seeking for fever occurred in the later part of the decade between 2004 and 2010. ACTs replaced SP as the first-line antimalarial drug in 2007, midway through the evaluation period.54,55 The 2012 and 2014 estimates were in the high malaria transmission season (rainy season) or just following and were lower than the 2010 MIS estimate, also conducted in the high transmission season. The prevalence in the NMS and MIS are not directly comparable due to survey design and fieldwork being conducted in different malaria transmission seasons, which poses challenges in interpreting trends in malaria parasite prevalence during and beyond the evaluation period. As a result, we included the logistic regression models to further explore the impact of malaria interventions  on malaria parasitemia and severe anemia.Declines were observed in malaria parasitemia and severe anemia, which lie along the causal pathway between malaria control intervention increases and reductions in malaria mortality . The ana56 Thus, the impact of malaria control interventions on malaria-related syndromes would be expected to be larger in younger age groups (under 2 years of age) compared with those 4 or 5 years of age.18,57 Severe anemia in children in the subgroup 6–23 months of age decreased significantly (36% relative reduction) between 2004 and 2010 based on DHS data, whereas there was no decline in severe anemia in children 24–59 months of age. Children 6–23 months of age are at a higher risk of severe malaria-related outcomes, such as anemia and mortality, than children 24–59 months of age.30,31 In addition, the relative contribution of malaria to the overall anemia burden in young children would be expected to be greater in areas of medium to higher malaria transmission; thus, control of parasite transmission would be expected to reduce anemia in these populations.19 In line with these expectations, declines in severe anemia in the 6- to 23-month age group varied based on malaria risk zone; severe anemia declined by 46% in higher risk areas and by 44% in medium risk areas as compared with 12% in lower risk areas. Thus, the observed trends are consistent with expected changes in anemia due to malaria control interventions with respect to both age and malaria endemicity.In settings of high malaria endemicity, the burden of disease and the impact of malaria control are largely concentrated in infancy and early childhood.37 particularly for evaluations that were conducted when rapid changes were under way in many other aspects of health services.36 Numerous socioeconomic factors and maternal and child health interventions were assessed for their potential impact on ACCM. Relative increases were observed in many of these factors  including GDP (18.1% relative increase), care seeking for diarrhea (120%) and acute respiratory illness (145%), breastfeeding (61.5%), and deliveries in a health facility (32.4%), which likely contributed to reductions in ACCM. Nutritional improvements were also seen with decreases in stunting (13%), underweight (38%), and wasting (37%). Sustained high coverage of BCG, measles, DPT3, and polio3 immunizations were observed during the evaluation period with coverage at levels exceeding 80% throughout the period. Coverage with these vaccines undoubtedly saved many lives over the evaluation period, but the further increases in coverage of these vaccines are unlikely to have had a large impact on the change in mortality in children less than 5 years of age. One exception may be the increase in measles immunization coverage (83% in 2000 to 93% in 2010); measles is highly contagious, so immunization coverage levels of at least 90% need to be maintained to confer population-level protection.58 Also, Haemophilus influenza (b) and Hepatitis B immunizations were added to the EPI package, as a component of the pentavalent vaccine DPT3-HBV-Hib, during the evaluation period (2002); thus coverage of these two vaccinations rose from near-zero in 2000 to 93% in 2010, and the implementation of the Hib vaccine in particular likely contributed to the declines in ACCM. Hib is the main cause of nonepidemic bacterial meningitis in children less than 12 months of age and is also a major cause of pneumonia.59 It is estimated that three doses of the Hib vaccine can prevent 38–43% of the childhood meningitis mortality.59Appropriate considerations of contextual factors are essential for ensuring the internal and external validity of evaluations of large-scale health programs,4,28 Rainfall and temperature patterns do not suggest that any long-term climate differences existed over the period of malaria control scale-up that would have independently led to substantially different patterns of malaria morbidity and mortality at the end of the decade versus the start.4 However, analysis of meteorological data in Malawi was limited to a national level and may have missed the sub-national variation in climate factors.28Meteorological factors were considered over the evaluation period.6 Given that changes in fundamental determinants often act through the proximate determinants (examined here) to impact childhood morbidity and mortality, the focus here is on the proximate determinants.In addition to changes in the contextual factors highlighted here, there were improvements to the overall health system, including health financing, which could have impacted ACCM. For example, the Government of Malawi developed a Sector Wide Approach program, which included the provision of an essential healthcare package.To mitigate limitations inherent in plausibility based evaluation designs , multivariable models were used to assess the association of one malaria intervention (ITN ownership) with malaria parasitemia, severe anemia, and child survival. In sub-national district analysis between 2005 and 2009, household ownership of ITNs was protective against malaria parasitemia and severe anemia in children 6–30 months of age, controlling for meteorological factors, geographic location, household wealth, and child’s age; odds of parasitemia were 19% lower and odds of severe anemia were 18% lower in children living in households with at least one ITN compared with those without ITNs.16 It should be noted that the Cox proportional hazards model assesses individual level associations, which could be influenced by residual confounding and may not translate directly to population level impact. However, given there are numerous controlled studies that have assessed the association between ITNs and malaria morbidity and mortality,8,44 it was important to assess the association in Malawi under programmatic conditions where distribution of ITNs was not under the control of a study, but relied on the routine distribution systems operating in country .We also found that household ITN ownership was associated with increased child survival using a Cox proportional hazards model and a district-level platform model presented in the accompanying paper by Florey and others.16 In addition, ITN ownership was found to be protective against malaria morbidity measures (severe anemia and parasitemia in children 6–30 months of age). Taken together this evidence suggests that malaria control interventions in Malawi likely contributed to reductions in ACCM during 2000–2010. Reductions in ACCM will likely continue as intervention coverage intensifies and the overall malaria control program increases efficiencies in service delivery.Malawi has made progress in improving coverage of malaria prevention and treatment interventions during the evaluation period. Part of the decline in all-cause mortality, particularly the declines earlier in the evaluation period could be due to the improvements in the coverage of socioeconomic factors and other child health interventions as well as the initial expansion of malaria control interventions. In addition, it is likely that the decline in all-cause mortality among children less than 5 years of age was in part due to a reduction in malaria-specific mortality, particularly later in the evaluation period. Multivariable models support this claim; ITN ownership is associated with improved child survival in Malawi.Supplemental Tables.


Plasmodium falciparum escapes the human host immune system by switching expression of variant surface antigens (VSA). Piecemeal acquisition of variant specific antibody responses to these antigens as a result of exposure to multiple re-infections has been proposed to play a role in acquisition of naturally acquired immunity.During intra-erythrocytic replication P. falciparum infections.Immunofluorescence was used to explore the dynamics of anti-VSA IgG responses generated by children to (i) primary malaria episodes and (ii) recurrent Consistent with previous studies on anti-VSA responses, sera from each child taken at the time of recovery from their respective primary infection tended to recognize their own secondary parasites poorly. Additionally, compared to patients with reinfections by parasites of new merozoite surface protein 2 (MSP2) genotypes, baseline sera sampled from patients with persistent infections (recrudescence) tended to have higher recognition of heterologous parasites. This is consistent with the prediction that anti-VSA IgG responses may play a role in promoting chronic asymptomatic infections.This pilot study validates the utility of recurrent natural malaria infections as a functional readout for examining the incremental acquisition of immunity to malaria.The online version of this article (10.1186/s12936-017-2097-0) contains supplementary material, which is available to authorized users. Children who grow up in malaria endemic areas develop naturally acquired immunity to severe forms of the disease. This immunity takes several years to build up, even in hyperendemic areas, and probably never confers sterile immunity. Thus, many individuals in malarious areas carry asymptomatic infections. The transition from malaria to asymptomatic infection is still poorly understood. The staggered age distribution of severe malaria, mild malaria and asymptomatic infection suggests distinctive immunological mechanisms that (i) confer protection against pathogenic features of infection (ii) limit parasite replication leading to control of parasite burden. The role of antibodies is demonstrated through studies in which purified IgG from immune adults was used to bring about rapid reduction in parasitaemia and symptoms of malaria , 2. One A major target for protective antibodies against malaria are the variant surface antigens (VSA) that are anchored in the cell membrane of infected erythrocytes. These antigens mediate adhesive parasite-host interactions thought to be essential for evading innate immune clearance mechanisms in the spleen. Variant specific anti-VSA immune responses are thought to play an important role in the development of naturally acquired immunity to malaria –6. FolloPlasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1), encoded by the large multigene var family is thought to be the major VSA [var genes, and expression is mutually exclusive at the individual parasite level (reviewed in [var genes with age [ajor VSA . Each haiewed in ). Immunewith age , 14, andwith age .Here, to explore the transition from malaria disease to asymptomatic infection and the relationship between recrudescence and immunity to VSA, anti-VSA antibody acquisition was studied in children within the age bracket where the transition from symptomatic to asymptomatic infections occurs, who presented to hospital with mild malaria, and who returned within 84 days with recurrent parasitaemia. The kinetics of antibody acquisition to the baseline and recurrent isolates was studied. The anti-VSA antibody repertoire of sera from patients that carried parasites that recrudesced was compared to the repertoire of those that experienced reinfection by new genotypes only.P. falciparum isolates) were collected at any time the children were parasitaemic according to microscopic examination as described [In this study, samples from children presenting with mild malaria who were included in a Phase III, randomized, non-inferiority trial was used to assess the efficacy and safety of dihydroartemisinin-piperaquine (Artekin) in comparison with artemether–lumefantrine (Coartem©) in Pingilikani, Kenya, with a follow up period of 84 days. This study has been described in detail elsewhere . Childreescribed . Serum wDuring the follow up period recurrent parasitaemias were screened for recrudescences using MSP2 genotyping. Initial screening was done using the RFLP method described by Felger et al.  and isolSera from these 7 patients, taken at day of recruitment and at follow-up visits , were used to study antibody acquisition towards the respective infecting parasites in each of these patients over time. Whenever a serum is tested against an isolate from the same patient, they are referred to as homologous serum and isolate.To test heterologous recognition of the isolates, i.e., testing serum derived from different patients than the parasite isolate tested, a panel of heterologous sera Table  from 14 Improved MSP2 analysis using capillary electrophoresis  was perfg for 5 min) cells were incubated in 30 µl of PBS (0.1% BSA) containing antihuman-CD45-PC7 (1:30 v/v) , antihuman IgG-FITC (1:1 v/v)  and antihuman IgM-SPRD (1:30 v/v) . After washing three times  samples were resuspended in PBS (0.1% BSA) and analysed using an FC500 Flow Cytometer (Beckman Coulter).Live isolates were removed from liquid nitrogen, washed with ice cold 3.5% NaCl, then washed with culture medium , and transferred into culture flasks where the isolates were allowed to stabilize for 18–24 h under standard culture conditions  until trFlowJo and Excel were used for the initial flow cytometry analysis, where first nucleus containing cells stained by ethidium bromide were included, i.e. leukocytes and infected red blood cells (irbcs) at the trophozoite stage. At this stage ring stage parasites were excluded by low ethidium bromide staining. Next CD45-containing leukocytes were excluded and finally the IgG recognition measured as mean fluorescence intensity (MFI).The flow cytometer was set to count 1000 irbcs. Samples where less than 200 irbcs  had been measured were excluded from analysis. When the volume of cells was smaller than expected (pipetting errors on semi-packed cell pellets), the last cells measured would give unusually many counts with high MFI values. To exclude these erroneous measurements, samples where the whole population of measured cells (mostly noninfected red blood cells) had more than 2.5% IgG positive cells, defined by a cut-off at MFI arbitrary units of 12, were excluded from analysis. Manual analysis confirmed that no samples with high parasitaemia were excluded by this process. All flow cytometry results are presented in this study as the MFI for each isolate-serum pair, with the individual “no serum” sample MFI subtracted. The “no serum” value for each parasite isolate was subtracted rather than the value of the negative control pool to allow comparison of the data with the negative control group. The MFI values for each measurement together with parasite isolate and serum information is presented in the Additional file If the no serum sample was missing (excluded by reasons explained above) or differing by more than 90% from the median of all no serum samples, the whole series, i.e. all data from the corresponding isolate, was excluded.To ensure that the measured fluorescence was not simply an effect of higher parasitaemia in the sample, i.e. an experimental artefact, linear regression analysis was performed for fluorescence vs. parasitaemia but no correlation was found .All flow cytometry results used for analysis is presented in Additional file ® 16th Edition were used for analysis and graphical presentation. A mixed-effects linear regression model using a REstricted Maximum Likelihood (REML) [Graphpad Prism 6.0e for MAC OS X and Genstatd (REML)  approache (MFI + 0.145) to make the smallest observation positive and to normalize the distribution. Fixed effects for response type (originating from a patient that responded to its parasite isolate vs. a patient that did not) and serum day (day 0 or 14), together with their interaction were then tested in the model.To test for intrinsic differences in the ability of the parasites to be recognized, the data were initially transformed by natural logarithm, logThe same analysis was performed to test for a difference between recognition of baseline vs. recurrent isolates. In this case the fixed effects were isolate type (baseline or recurrent) and serum day (day 0 or 14).e (MFI + 0.062) to make the smallest observation positive. Fixed effects tested in the model were serum type (homologous recognition of recurrent isolates vs. recognition of the same recurrent isolates by heterologous sera) and serum day (day 0 or 14).The ability of homologous sera taken before recurrence to recognize the recurrent parasites was also compared to that of their overall recognition by heterologous sera with a linear mixed effect regression model. In this analysis, only sera taken before the recurrent infection were used. The data were transformed by loge (MFI + 0.145) to make the smallest observation positive. Fixed effects for serum type (originating from a patient that eliminated its parasite infection vs. a patient that did not) and serum day (day 0 or 14) were fitted.To compare the anti-VSA antibody repertoire of children who eliminated the baseline parasites to that of those children who did not, a similar linear mixed effect regression model was used. The data were transformed by logHistograms or residuals, fitted-value plots and normal plots were performed to test the suitability of the transformed data in the linear mixed effect regression models.Plasmodium infections transit from causing disease to being asymptomatic. Table For this study, samples from children presenting with mild malaria who were included in a dihydroartemisinin-piperaquine vs. artemether–lumefantrine drug trial were used . In totaSera derived from children in the same study were used to test antibody recognition of the isolates described above. Homologous sera (sera derived from the same patient as the parasite isolate tested) were used to test acquisition of antibodies to the infecting isolates of the respective patients. Sera from all available time points during the study follow up were used.Heterologous sera (sera derived from other patients than the tested isolate) were used to test general recognition of the isolates in the population. For this a panel of sera from in total 18 patients in the same study were chosen to characterize heterologous recognition of the parasite isolates Table . Sera frAntibodies to the infected red blood cell (irbc) surface were measured by immunofluorescence and flow cytometry. To explore the kinetics of antibody responses to irbc sampled at baseline (day 0) and at the time of recurrent parasite infections each parasite isolate was tested against homologous sera (sera derived from the same patient as the parasite isolate tested) sampled at different time points. Figure As shown in Fig. All sera used to test heterologous recognition of the isolates described above were derived from patients who had also experienced a recurrent, i.e. secondary infection during follow-up. Those patients who were unable to clear their parasites in spite of treatment, resulting in a recrudescent infection are of specific interest. Therefore, MSP2 genotyping analysis was performed on the parasite DNA isolated from these patients to differentiate between recrudescence (persistent blood stage infection) and re-infection (new infection emerging from the liver). A recrudescent infection is defined by the presence of at least one genotype during follow-up also detected before treatment. Thus, 6/18 patients whose sera were part of the heterologous serum panel were confirmed to have recrudescent infections within the follow-up period Table .50 values for the long-acting drugs lumefantrine and piperaquine. A recrudescence is not determined by the sensitivity of the baseline isolate to the long-acting drugs used in the study , may themselves boost antibodies. However, the anti-VSA antibody repertoire in children who later had recrudescent parasites was higher than in those who later had recurrent infections at day 0 Fig. . This suAntibodies that bind parasite-encoded antigens on the surface of infected red blood cells  have been implicated in alleviating specific pathogenic interactions between parasite ligands and endothelial host receptors , but farP. falciparum infections that trigger disease to asymptomatic infections is well documented in populations with high re-infection rates, but the exact nature of the host-parasite interactions that orchestrate this survival strategy of the parasite has been difficult to study, and recurrent P. falciparum infections in paediatric patients within an age bracket that encompasses the switch from symptomatic to asymptomatic infections could provide a useful readout.The transition from Children who received treatment for malaria were recruited and were then followed-up to detect recurrence of infections. Stratification of recurrent infections in recrudescent primary infections or new secondary infections gave us a functional in vivo readout to assess the effects of anti-VSA responses determined at baseline.The pattern of antibody responses relating to malaria and recurrent infections were consistent with previous studies showing that episodes of disease are associated with specific gaps in the anti-VSA repertoire –16. TherPersistent primary infections were detected as mild or asymptomatic recrudescences in patients with higher than average heterologous recognition capacity measured at day 0. This is consistent with a potential role of anti-VSA antibodies maintaining chronic infections , 23–25. A model in which anti-VSA antibodies help sustain chronic infections helps to explain previous observations of higher than average risk of recrudescent infections in young children , 27. ThiThis pilot study is limited by a small sample size. Recrudescences are rarely observed after highly efficacious artemisinin-combination therapy, and the definition of a recrudescent infection was based on a repeat polymorphism of a single, if highly variable, gene (MSP2). This method is reliable at a population level, but has limitations at the individual level because there will always be a bias toward majority genotypes. Studies in children also constrain the amount of sample volume, here only one measurement per isolate-serum pair tested was performed. Future larger studies will be required to confirm the hypotheses set out in this study.var genes expressed at different time points was observed. Specific studies to analyse such differences in transcriptome between isogenic parasites at different points during the establishment of chronic infections would be of great value in understanding of how these infections become established.It would be very interesting to make a detailed comparison of expressed VSA from recrudescent parasites in comparison with those that are isolated at day 0. It was not possible to do this accurately in this study because blood for which white blood cells had not been removed was used. Standard conditions for DBL-alpha tag amplification cannot be used in the presence of high levels of human RNA. A preliminary analysis using amplification at higher temperature was performed and no major changes in the profile of In short, here data is presented that support the “hole in the wall” model, i.e. a framework for explaining how the parasite exploits gaps in the anti-VSA repertoire of the host. It is suggested that this model may be key to understanding how chronic infections are established in humans. First data is provided to support the hypothesis that a more complete anti-VSA antibody repertoire acquired after repeated infections could favour the transition from an uncomplicated malaria episode to an asymptomatic carrier status. These findings must be confirmed in larger studies, but it opens up to new interpretations of immunological data, and to the usage of recrudescence as an alternative, possibly more useful resource to measure immunity to malaria.Additional file 1: Table S1. Samples and flow cytometry data used for analysis in this article.Additional file 2: Figure S1. Heat map showing heterologous recognition of each individual isolate-serum pair (and homologous recognition highlighted by bold boxes). Mean fluorescence intensity is shown for each isolate-serum pair, and is colour graded in a scale from light green , to dark green .Additional file 3: Table S2. A recrudescence is not determined by the drug sensitivity of the baseline isolate.


The battle for iron between host and pathogen in children living in malaria-endemic areas is fierce, and new evidence presented by Brickley and othersHepatic production of hepcidin is known to have multiple drivers, including dietary iron sufficiency, inflammation, and slowed erythropoietic rate.The question arises as to whether higher cord blood hepcidin, seemingly reflecting maternal inflammation and predictive of future childhood anemia but protection from subsequent malaria, is also a marker of chronic early childhood functional iron deficiency. Serial measurements of hepcidin, ferritin, and CRP throughout early childhood could confirm such a mechanism. In a birth cohort, this functional iron deficiency would manifest as high concentrations of hepcidin, ferritin, and CRP concurrent with a low hemoglobin concentration, and would reflect a state in which body iron was in reticuloendothelial stores and unavailable to invading pathogens, but also unavailable for red blood cells and the developing brain. Giving iron supplements in such a setting may at best be futile, as high hepcidin concentrations would limit dietary iron absorption,The study by Brickley and othersThe findings from the study by Brickley and others and the identification of high cord blood hepcidin as a promising biomarker of future risk of childhood infection and mortality raise important questions for future research and programmatic interventions concerning iron in malaria-endemic areas. The results underscore the necessity of effective malaria-control efforts through all stages of pregnancy and childhood as a prerequisite for safe attainment of sufficient iron status. However, they also raise the question of the relative contributions of dietary iron deficiency versus maldistribution of body iron to childhood iron deficiency in malaria-endemic areas. Could alleviation of inflammation and resultant release of iron from reticuloendothelial stores be sufficient for erythropoiesis and brain development, while obviating the need for iron supplementation? Would the resulting “iron sufficiency” still place the infants at higher risk for malaria and death in early childhood? Conversely, should children with low cord blood hepcidin concentration be put on intermittent preventive treatment of malaria and then be provided iron supplementation? These questions need to be answered in well-designed randomized clinical trials with both short-term morbidity and long-term neurodevelopmental outcomes. Assessment of the latter outcomes will help to answer the additional question of the extent to which iron supplementation in infants in malaria-endemic areas improves neurodevelopment, and which forms of iron supplementation most effectively improve child neurodevelopment. Although it is unlikely that iron deficiency is good for a child's developing brain in any part of the world, the effectiveness of iron supplementationto prevent or reduce child neurodevelopmental impairment in malaria-endemic areas has not been studied, largely because of concerns about the general safety of iron supplementation.The study by Brickley and others sheds an important and clarifying light on links between iron and malaria dynamics in pregnancy and early childhood, and identifies cord blood hepcidin as a promising biomarker for prediction of key child health outcomes in children born to mothers with malaria-induced and possibly other infection-induced inflammation. If confirmed by other malaria-in-pregnancy studies, these findings set the stage for future pathogenesis studies and clinical trials that aim to save children's lives while preserving their developmental potential.