UB05 and UB09 antigens (UB05‐09) to better differentiate between acquired immune protection and susceptibility to malaria. In a cross‐sectional study, recombinant UB05‐09 chimera and the constituent antigens were used to probe for specific antibodies in the plasma from children and adults resident in a malaria‐endemic zone, using the enzyme‐linked immunosorbent assay (ELISA). Anti‐UB05‐09 antibody levels doubled that of its constituent antigens, UB09 and UB05, and this correlated with protection against malaria. The presence of enhanced UB05‐09‐specific antibody correlated with the absence of fever and parasitaemia, which are the main symptoms of malaria infection. The chimera is more effective in detecting and distinguishing acquired protective immunity against malaria than any of its constituents taken alone. Online B‐cell epitope prediction tools confirmed the presence of B‐cell epitopes in the study antigens. UB05‐09 chimera is a marker of protective immunity against malaria that needs to be studied further.It has been shown that covalently linking two antigens could enhance the immunogenicity of the chimeric construct. To prioritize such a chimera for malaria vaccine development, it is necessary to demonstrate that naturally acquired antibodies against the chimera are associated with protection from malaria. Here, we probe the ability of a chimeric construct of  Although malaria has receded as a result of intensive control campaigns, it killed 438 000 persons most of them children in sub‐Saharan Africa in 2015, thereby underscoring the need to intensify control measures including the development of an effective vaccine against the disease.The use of vaccines has been shown to be an effective tool in curbing infectious diseases.Natural immunity against malaria is mediated in part by antibodies directed against the blood stage parasite, as demonstrated by passive transfer experiments of immunoglobulins.It has also been shown recently that vaccine constructs containing more than one antigen can elicit high humoral and cellular immune responses in human volunteersSero‐epidemiological studies have been used to identify most malaria vaccine candidates. In a similar study by our group that employed differential immune‐screening among other techniques, UB05 antigen was also identified, further characterized and proposed as a potential marker of protective immunity against malaria.22.1Ethical clearance for this study was obtained from the Faculty of Health Sciences‐Institutional Review Board, ref: 2013/144/UB/FHS/IRB.2.2The study was carried out in a malaria‐endemic area, Buea, Cameroon. Buea is a multi‐ethnic town with endemic malaria found along the flanks of Mount Cameroon in the South West region of the Republic of Cameroon. Malaria prevalence varies between a hyperendemic (rainy season) and meso‐endemic (dry season) zone with perennial malaria transmission.2.3A general invitation was given to the people resident in Buea and those who visited the Buea Regional Hospital for medical check‐up in order to recruit subjects for this study. After explaining the objectives of the research to voluntary individuals, informed consent was obtained from each participating subject above the age of 18 or parents/guardians of sick children below the age of 5. The medical history of each subject was recorded by a collaborating State‐Registered Medical Nurse who also assisted in completing a questionnaire form. Blood was then drawn by a Medical Laboratory Technician for the diagnosis of malaria and serology as described below.The following criteria were used to screen and admit subjects into the study; (i) subjects who were aged 18 years or older and who had resided in the study site for at least 3 years; (ii) subjects who have not had a malaria episode in the last 12 months, no parasitaemia and fever at sample collection, no prophylaxis, no use of mosquito bed net and had been exposed to mosquito bites were designated semi‐immune subjects (SIS); (iii) subjects who had parasitaemia and fever at sample collection and at least one malaria episode in the last 12 months were referred to as frequently sick subjects (FSS); and (iv) children below the age of five who had fever and parasitaemia were called sick children (SC). This cohort of individuals were highly selected and described previously.2.4Volunteers were prescreened for recruitment into any of the above groups by assessing their malaria status. Slides were prepared for staining using blood taken by finger pricking and stained with Giemsa. The slides were read under an oil immersion microscope  by two Microscopists at 1000× and 100 fields counted.5 mL of venous blood from adults and 1 mL from children were collected from recruited subjects in Vacutainer tubes containing EDTA and kept at +4°C for 1 hour. Samples were centrifuged and the plasma aliquoted and stored at −20°C until use.P. falciparum parasites in their blood in this limited longitudinal study. The body temperature of the study subjects was also noted.The SIS were followed up continuously for 2‐4 months and tested for the presence of 2.5The study antigens employed for this work were recombinant UB05 (r‐UB05), recombinant UB09 (r‐UB09) and recombinant UB05‐09 (r‐UB05‐09). These antigens were prepared as earlier described.2.6E. coli extract. After incubation with antigen, the plate wells were washed thrice with 200 μL of Wash buffer ; followed by blocking with 150 μL per well of 0.2% casein in PBS with 0.05% Tween‐20. The plates were washed and plasma added at 1:150 dilutions in Wash buffer containing 1% skimmed milk. The plate was incubated for 3 hours at room temperature. Human plasma from malaria‐naive individuals  were used as negative control plasma as well the Tag only (Fusion partner) antigen was used as a negative control antigen. The r‐UB05 antigen and soluble fraction of crude E. coli extract were used as positive controls. After incubation, the plates were washed three times and 100 μL of anti‐rabbit IgG‐HRP conjugate (Sigma) diluted at 1:10 000 in Wash buffer containing 1% skimmed milk. Incubation was done at room temperature for 1 hour. After anti‐rabbit IgG‐HRP conjugate incubation, the plates were washed and 100 μL of substrate added. Optical densities were read at 405 nm on a microplate reader . A subject was considered positive if its OD value was equal to or greater than the mean control OD +2 SD. The experiment was run in duplicates.Antibody  measurement was carried out as earlier described2.7www.immuneepitope.org).In silico prediction of antibody epitopes from UB09, UB05 and UB05‐09 was done using algorithms in IEDB Analysis Resources .33.1Table 3  were negative for malaria parasites at the time of sample collection and had no fever. Also, all of the frequently sick subjects (FSS) and the sick children retained for the study had fever and were positive for malaria parasites upon the examination of Giemsa‐stained thick blood smears, at the time of blood sample collection. Parasitaemia was mostly high and ranged from 742 to 198 000 parasites/mm3.2UB09 gene as identified in a previous studyCRA gene is made of three exons and two introns. The r‐UB09 antigen covers part of exon 2 and complete exon 3. In terms of amino acid sequence, it covers the C‐terminal fragment of CRA, that is, r‐UB09 is 100% identical to amino acid residues 89‐162 of CRA. UB09 is therefore a fragment of the full CRA antigen.The 3.3P<.0001)  . It was observed that that the level of antigen‐specific antibody was far lower in sick children compared to adults  Figure A. As sho) Figure A. Immuno) Figure B. Also, ) Figure B.P<.0001), and unlike in r‐UB09 and r‐UB05, antibody to the chimera was significantly greater in SIS than in FSS (P<.031)  Figure .In terms of percentage of reactive study subjects, it was observed that 72.5% of SIS, 93% of FSS and 22.2% of SC recognized r‐UB09 Figure . r‐UB05‐The readouts from the naive plasma wells showed that there was no significant presence of antigen‐specific antibodies in malaria‐naive samples. Also, the fusion partner Tag negative not recognized by plasma from selected study subjects. Any background readings with the fusion partner were deducted from the test.3.4P <.0001) of antigen‐specific antibodies in the plasma of people without fever compared to those with fever for all antigens  studied.To determine the association between fever and antibody response to r‐UB09, a plot was generated showing the febrile status of the study subjects and their respective OD values for antibody response to r‐UB09. Figure 3.5P<.0001). The same trend was observed when the results for r‐UB05 (P<.0001)   Figure B and the) Figure C were an3.6Comparing the antibody levels against the study antigens in sick children showed that children with lower parasitaemia had higher OD values compared to their counterpart with a higher parasitaemia. This was done for sick children who recognized the antigens under study. It was observed that this was true for r‐UB05 and the r‐UB05‐09 chimera and but not with r‐UB09 (data not shown).3.7Using Bepipred linear epitope prediction3.8www.immuneepitope.org). Bepipred Linear Epitope PredictionTo predict the presence of B‐cell epitopes in antigen UB05, we used algorithms in the IEDB Analysis Resources (3.9The chimera UB05‐09 antigen was analysed for the presence of antibody epitopes using the algorithms at IEDB Analysis Resources. The trends found in UB05 and UB09 individual were both present in the UB05‐09 chimera using the different algorithms.4P. falciparum antigen; r‐UB09, and the previously studied r‐UB05 and also the chimera of these two antigens called r‐UB05‐09 in the plasma from people living in a malaria‐endemic region to see if the chimera improves the immunogenicity and protective property of the individual component antigens.It is now apparent that the development of a multivalent subunit vaccine with widespread application will be a welcomed innovation to tackle the malaria burden. Studies have shown that bringing two antigens into close proximity could enhance their immunogenicity and possibly their protective property and such protection should be associated with the absence of fever and malaria parasite in blood. Here, we study the humoral response of In the present study, we generated r‐UB09, r‐UB05 and a chimera thereof, called r‐UB05‐09 and observed that r‐UB09 detected greater levels of antigen‐specific antibodies in the plasma of SIS than in the plasma of FSS and this difference increased using the r‐UB05‐09 chimera. This implies there is an enhancement of the recognition by the immune system of r‐UB05 and r‐UB09 by the r‐UB05‐09 chimera. The bringing of UB05 and UB09 into a single construct could have created an antigen with a different conformation compared to the individual constituent antigens. This could lead to the presentation of more potent epitopes to the immune system, hence the increased recognition of r‐UB05‐09. If UB05‐09 was to be used in a vaccine, then the additive effect would be an advantage as there would be potential for the vaccine to invoke an immune response that target different parts of the parasite.Earlier it was shown and suggested that UB05 could be a marker of protective immunity against malaria.An association is not causation, and as such, should not be regarded in isolation. However, when taken with the fever and parasitaemia data, it suggests causation. Given the fact that higher antibody levels, low axial temperatureIn conclusion, UB09 is a marker of protective immunity to malaria and can be used in combination with another earlier detected antigen UB05 to delineate between semi‐immune and susceptible malaria subjects.DJN and VPKT conceived and designed the experiments. DJN and GSD performed the experiments. DJN and VPKT analysed the data. DJN, GSD and VPKT drafted the manuscript or revised it critically for important intellectual content. DJN and VPKT agree to be accountable for all aspects of the work. All authors read and approved the final manuscript.None.


Overall, 142/1018 (13.95%) and 83/1041 (7.97%) were G6PD deficient in Anuradhapura and Kurunegala districts respectively. The G6PD prevalence was significantly greater in Anuradhapura when compared to Kurunegala (P<0.0001). Surprisingly, females were equally affected as males in each district: 35/313 (11.18%) males and 107/705 (15.18%) females were affected in Anuradhapura (P = 0.089); 25/313 (7.99%) males and 58/728 (7.97%) females were affected in Kurunegala (P = 0.991). Prevalence was greater among females in Anuradhapura than in Kurunegala (P<0.05), while no such difference was observed between the males (P>0.05). Severe deficiency  was seen among 28/1018 (2.75%) in Anuradhapura  and 17/1041 (1.63%) in Kurunegala . Enzyme activity between 10–30% was observed among 114/1018  in Anuradhapura while it was 66/1041  in Kurunegala. Screening and educational programmes for G6PD deficiency are warranted in these high risk areas irrespective of gender for the prevention of disease states related to this condition.Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme deficiency is known to offer protection against malaria and an increased selection of mutant genes in malaria endemic regions is expected. However, anti-malarial drugs such as primaquine can cause haemolytic anaemia in persons with G6PD deficiency. We studied the extent of G6PD deficiency in selected persons attending Teaching Hospitals of Anuradhapura and Kurunegala, two previously high malaria endemic districts in Sri Lanka. A total of 2059 filter-paper blood spots collected between November 2013 and June 2014 were analysed for phenotypic G6PD deficiency using the modified WST-8/1-methoxy PMS method. Each assay was conducted with a set of controls and the colour development assessed visually as well as with a microplate reader at OD Deficiency of G6PD enzyme affects more than 400 million people worldwide and is the most common enzymopathy in humans–3. G6PD Individuals with G6PD deficiency can present with a spectrum of disorders including acute massive haemolysis, neonatal jaundice, renal failure and chronic haemolytic anaemia induced by exposure to certain drugs, infections, fava beans, chemicals and herbal medicines,6. The hP. vivax is endemic[G6PD deficiency is important in malaria since it is believed to provide partial protection against the disease, thus increasingly selecting for these mutant genes,8–11. Ths endemic,16. Lesls endemic also dems endemic, identifs endemic.The dry zone of Sri Lanka has been endemic for malaria from historic times with epidemics occurring at periodic intervals–20, althThe current knowledge in Sri Lanka regarding the prevalence of G6PD deficiency is extremely sparse and gathered several decades previously–25. AbeyPlasmodium vivax hypnozoites and Plasmodium falciparum gametocytes, required for radical cure of patients with malaria[P. vivax infections, while it is no longer required for the single-dose transmission-blocking use in P. falciparum[The anti-malarial drugs primaquine, dapsone and the experimental drug tafenoquine are oxidative anti-malarials and can cause haemolytic anaemia in G6PD deficiency,27–29. Ch malaria. And kilh malaria. Thus, talciparum.Several qualitative and quantitative methods are currently available for measuring the phenotypic G6PD enzyme status. The fluSince the risk of haemolysis can be reduced by the use of extended dosing schedules of primaquine without compromising on efficacy, awareneThis was a descriptive, cross sectional study carried out at the Teaching Hospitals of Anuradhapura  and Kurunegala (North Western Province). These two areas were selected because they were highly endemic for malaria from historic times until the recent past and therefore expected to have a higher prevalence of G6PD deficient hosts. Ethical clearance for the study was obtained from the Ethics Review Committee of the Faculty of Medicine, University of Colombo (EC-12-134), as well as from the Teaching Hospital, Kurunegala. Permission for sample collection was obtained from the Director General of Health Services, Ministry of Health, Sri Lanka and the Directors of the respective hospitals. Participants were enrolled for the study after obtaining informed written consent.Sample collection was carried out between November 2013 and June 2014 from persons attending the Teaching Hospitals at Anuradhapura and Kurunegala. A sample size of two thousand (one thousand from each district) was determined by assuming the prevalence of G6PD deficiency in the population as being 5% with an allowable error of 20% (precision = 0.01) at the 95% level of confidence. Patients presenting to the clinics at these two hospitals are usually referred to the ‘bleeding room’ for providing blood samples for any laboratory tests that may be required as part of their health screening/management process. We invited all patients presenting to the ‘bleeding room’ of each hospital during weekdays (morning and afternoon) to be part of the study. Blood samples were obtained from those who provided written consent until the required sample number was obtained. Two drops (~0.5mL) of blood from each person was collected on to 3MM filter papers (Whatman) and a simple form pasted on the zip-lock bag was completed giving the sample reference number with a code for each area given as a prefix, the date of collection, the address, age and gender of the participant. The blood spots were air dried and stored individually in zip-lock bags containing silica desiccant beads and refrigerated at +4°C until transported (in ice boxes) within 7 days to the Department of Parasitology, Faculty of Medicine, University of Colombo where the laboratory investigations were conducted.The WST-8/1-methoxy PMS method described initially by Tantular and Kawamoto utilizesThe method was modified by Kuwahata et al. by optim450-630nm.The G6PD enzyme activity detection assay for our study was established based on the previously validated modified WST-8/1-methoxy PMS method described by Kuwahata et al. (2010). The WSThttp://www.bensrl.it/) from a laboratory at a private hospital in Colombo. The number so tested was limited due to the necessity of having to provide 2mL of whole blood and the high cost involved per test.For further validation of our assay, 2mL of blood was collected into anticoagulant containing blood tubes from 20 persons  and tested with a commercially available kinetic UV determination test kit for G6PD  and normal reference range . Prevalence of G6PD deficiency was calculated as a percentage of the total number of positive persons from the total number tested for each district and according to gender. Chi square test was used to determine any significant difference in gender distribution of enzyme deficiency and for comparative analysis between sites. The frequencies of distribution of enzyme activity levels were determined as percentages for each gender. Enzyme concentrations were calculated as a percentage to the normal reference control using MS Excel 2007. Frequencies of colour code observations as well as discrepancies between observers for varying enzyme activity levels were calculated as percentages for each category.A total of 2059 filter paper blood samples were collected  classifiIn addition to anti malarial drugs, many analgesics, antibacterials as well as other miscellaneous drugs too can induce acute haemolysis in susceptible individuals. DapsoneVicia faba, the Broad Bean), a species of bean native to North Africa and Southwest Asia, and extensively cultivated elsewhere[Phaseolus vulgaris L. Furthermore, chemicals such as naphthalene and methylene blue, certain antifungals, henna and infectious diseases caused by viruses and bacteria have all been associated with the occurrence of haemolytic anaemias in G6PD deficient individuals[Haemolytic anaemias have also been associated with G6PD deficiency and fava beans  are under 3%. A recent median predicted allele frequency map of G6PD deficiency across malaria endemic countries predicted allele frequencies of up to 15% within Sri Lanka, especially in the northern, north western, north central and eastern parts of the country. In Sri The X-linked inheritance of the G6PD gene makes hemizygous males vulnerable to clinical manifestations in the presence of a variant, while deficiency in females is more complex. HomozygAt present, the Brewer’s test is used We conclude that screening and educational programmes for G6PD deficiency are warranted in these high risk areas irrespective of gender in order to prevent the occurrence of disease states related to G6PD deficiency.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Plasmodium reservoir circulating in the community. Further characterization and comparability of the hidden asymptomatic parasite reservoir are needed to inform on the potential impact of sampling bias. This study explores the impact of sampling strategy on molecular surveillance by comparing the genetic make-up of Plasmodium falciparum and Plasmodium vivax isolates collected by passive versus active case detection. Sympatric isolates of P. falciparum and P. vivax were collected from a large community survey and ongoing clinical surveillance studies undertaken in the hypomesoendemic setting of Mimika District . Plasmodium falciparum isolates were genotyped at nine microsatellite loci and P. vivax at eight loci. Measures of diversity and differentiation were used to compare different patient and parasitological sample groups. The results demonstrated that passively detected cases (symptomatic) had comparable population diversity to those circulating in the community (asymptomatic) in both species. In addition, asymptomatic patent infections were as diverse as subpatent infections. However, a significant difference in multiplicity of infection (MOI) and percentage of polyclonal infections was observed between actively and passively detected P. vivax cases . The study findings infer that, in hypomesoendemic settings, passive sampling is appropriate for molecular parasite surveillance strategies using the predominant clone in any given infection; however, the findings suggest caution when analyzing complexity of infection. Further evaluation is required in other endemic settings.The surveillance of malaria is generally undertaken on the assumption that samples passively collected at health facilities are comparable to or representative of the broader  Both of the predominant species, P. falciparum and P. vivax, are able to cause submicroscopic infections, although the relative proportion of subpatent cases tends to be higher in P. vivax than P. falciparum.7Calls for the global elimination of malaria have renewed political interest in public health interventions, reigniting control activities and placing elimination back on the agenda for many endemic countries.9 As countries approach malaria elimination, it is crucial to identify every infection to stop ongoing transmission and prevent resurgence. In this context, intense surveillance is required to monitor clinical and parasitological changes that can inform the most effective intervention strategies.Subpatent infections can be detected in a wide range of malaria-endemic settings, with the proportion tending to rise in low transmission areas.11 Furthermore, in pre-elimination settings, the low number of malaria cases in parasite population genetic studies often requires pooling of samples from patients with different disease presentations, collected using different sampling strategies and methodologies. These studies are being used increasingly to inform local trends in parasite transmission as reflected by measures of within-host and population diversity.13 A recent population genetic study of P. falciparum in a low endemic area of Zambia inferred that actively and passively detected infections formed distinct subpopulations with differing levels of diversity, but the study was partly constrained by the difference in years of collection between the actively and passively detected cases.14 Another study conducted in the higher endemic setting of Mimika District, Papua, Indonesia, using samples from both clinical and community cases revealed a subpopulation of P. falciparum isolates only observed in community cases.15 However, the latter study did not directly investigate the level of genetic differentiation or differences in population diversity and infection complexity between the actively and passively detected P. falciparum isolates. Furthermore, to date, no studies have directly compared the genetic make-up between actively and passively detected P. vivax infections, which contribute a large burden of malaria infections in the Asia-Pacific region.The majority of such surveillance efforts have been undertaken at health facilities, recruiting symptomatic individuals with patent parasitaemia seeking treatment (“passive case detection”). As malaria elimination efforts progress, more in-depth knowledge of the parasite population in asymptomatic community cases is needed, as this reservoir contributes a greater proportion of the total parasite burden. An important question is whether the genetic make-up of actively detected parasites is adequately represented by those detected passively, or whether the different sampling strategies can result in a significant bias which can affect the interpretation of a given study. The surveillance of antimalarial drug resistance could be particularly vulnerable to sampling bias, because phenotypic and molecular studies are generally applied to clinical isolates with little focus given to the asymptomatic population. In the case of ex vivo drug susceptibility testing, stringent criteria are applied which can result in even more constrained sampling.P. falciparum and P. vivax isolates obtained from a community survey and ongoing clinical surveillance studies in Mimika District, Papua, Indonesia, to compare the genetic make-up of samples collected by passive versus active case detection, and between patent and subpatent groups.This study used sympatric Plasmodium parasites can be found in the area, P. falciparum and P. vivax are the most common species causing 41% and 49% of malaria monospecies infections, respectively. Plasmodium malariae accounts for approximately 4% of malaria cases and less than 1% of cases are caused by P. ovale. Mixed-species malaria infections can be found in up to 6% of cases.16 Rumah Sakit Mitra Masyarakat (RSMM) is the main hospital in the district. RSMM is located in the capital city, Timika, and serves approximately 150,000 people, assessing up to 300 patients/day, 6 days/week. Patients with uncomplicated malaria due to any Plasmodium species are treated with dihydroartemisinin-piperaquine as per the national first-line protocol.17The study was performed in Mimika District, Papua, Indonesia. Nearly 90% of the population of approximately 182,000 people live in lowland areas where unstable malaria transmission occurs. Although all four species of human-only infecting P. vivax or P. falciparum monoinfection, a peripheral parasitaemia between 1,000 μL−1 and 80,000 μL−1, as determined by microscopic examination, and more than 70% of ring stage parasites were invited to participate in an ongoing ex vivo antimalarial susceptibility surveillance study . Between March 2015 and February 2016, patients presenting with uncomplicated P. falciparum or P. vivax malaria with peripheral parasitaemia greater than 1,000 μL−1 and 250 μL−1, respectively, but with no restriction by parasite stage, were invited to participate in an ongoing treatment efficacy study (TES). After obtaining written consent, 5 mL of venous blood was collected for all patients by venipuncture and filtered using cellulose columns to deplete the host white blood cells.18 The postfiltration packed infected red blood cells were used for DNA extraction.Samples at the RSMM were collected within the framework of two studies. Between March 2011 and August 2014, patients presenting with an uncomplicated 16 Briefly, 800 houses were selected by cluster randomized sampling. First, the four largest subdistricts were chosen purposively. Consecutively, the number of clusters per district was calculated according to their respective relative population. Finally, 20 houses were chosen randomly within each cluster, following World Health Organization recommendations. Individuals living in the same house and residing in the study area for at least 6 months were included in the study. Symptomatic patients were defined as any individual with a current fever episode  or in the last 24 hours. Venous blood from one adult volunteer and 200 µL of capillary blood in a Microtainer™ from the remaining members of each household were collected and used for blood film examination, hemoglobin measurement, and molecular analysis.Active case detection was undertaken within the framework of a cross-sectional survey conducted in Timika between April and July 2013. Details on the sampling methods of the survey can be found elsewhere.Plasmodium species was assessed initially by microscopy using Giemsa-stained thick and thin blood films. Peripheral parasitaemia was determined from the number of parasites per 200 white blood cells, assuming a white cell count of 7,300 cells/µL. Asexual stage composition was determined by classifying 100 parasites into ring, trophozoite, or schizont stage on examination of a thick smear.Plasmodium species confirmation was undertaken in duplicate with 2 µL gDNA template using a nested PCR protocol as described elsewhere.19Plasmodium falciparum, P. vivax, P. malariae, and P. ovale small-subunit rRNA DNA clones  were used as positive controls.For actively detected cases, genomic DNA (gDNA) was extracted from 50 µL packed red blood cell (RBC) pellets using the QIAamp 96 DNA Blood Kit . For passively detected cases, 2 mL of packed RBCs were extracted using the QIAamp DNA Midi Kit . Plasmodium falciparum genotyping was conducted using nine previously described short tandem repeat (STR) markers , following the protocol described by Anderson et al.20 For P. vivax genotyping, nine previously described STR markers selected as a consensus panel by the Asia Pacific Malaria Elimination Network Vivax Working Group  were used following previously described protocols.22 The fluorescently labeled PCR products were separated by capillary electrophoresis on an ABI 3100 Genetic Analyser with GeneScan LIZ-600 size standard . The resulting electrophoretograms were analyzed using VivaxGEN version 1.0 and verified manually.23 To reduce potential artifacts from background noise, an arbitrary fluorescent intensity threshold of 50 relative fluorescence units was used. Only samples with information in at least 50% of the loci were considered as successfully genotyped.Rs). Rs was calculated by quantifying the number of alleles and normalizing the value to the sample size, using the hierfstat package in R.24 The expected heterozygosity (HE), which is defined as the probability of finding a different allele at a specific locus when randomly selecting a pair of samples from the same population, was measured as an additional index of population diversity. The pairwise FST metric was used to determine the genetic distance between populations and interpreted according to the classification of Balloux and Lugon-Moulin25 . Arlequin software (version 3.5) was used to calculate HE, FST, and the standardized measure (F’ST), which adjusts for high marker diversity. Significance values for the FST were derived using 10,000 permutations of the data in Arlequin.Diversity was estimated by means of allelic richness (IAS). First, multilocus genotypes (MLGs) were reconstructed from the predominant allele at each locus in isolates with no missing data. Then, using the MLGs, IAS was measured using the web-based LIAN 3.5 software, assessing the significance of the estimates using 10,000 permutations of the data.26 The analysis was also performed using low-complexity infections  and with each unique MLG represented just once.To assess changes in transmission over time and to further characterize the parasites constituting the different subgroups, multilocus LD was measured using the standardized index of association  and polyclonality estimates between parasite populations with differing DNA quality or quantities.P < 0.05.28 Subgroup analysis was performed according to 1) patient recruitment method (active or passive) and 2) infection patency  using the χ2 test and Fisher’s Exact Test. Differences in MOI and HE between subgroups and species were assessed using the Mann–Whitney U or Kruskal–Wallis test.R and SPSS software were used for statistical analysis with a significance level at Ethical approval for this study has been obtained from the Eijkman Institute Research Ethics Commission, Eijkman Institute for Molecular Biology, Jakarta, Indonesia , and the Human Research Ethics Committee of the Northern Territory Department of Health & Families and Menzies School of Health Research, Darwin, Australia (HREC 2010-1396).P. falciparum and P. vivax markers in all genotyped samples is presented in Supplemental Table 1. Moderate to high diversity was found for all P. falciparum markers except for TA42 and TA109, particularly in the TES and ESS samples . By contrast, high diversity was found in all P. vivax markers across all sample sets. However, the MS8 assay presented artifacts that affected the reliability of the allele calling and this locus was, therefore, excluded from further analysis. Genotype information for 50% of the marker set  was obtained from 84.5% (414/492) P. falciparum samples and from 70.7% (390/552) of P. vivax samples. Most of the failed samples were asymptomatic P. vivax or P. falciparum infections .A summary of the diversity and genotyping success rate for each of the P. falciparum  or P. vivax . Furthermore, given that the TES and ESS samples were collected across multiple years, an assessment of temporal differences in MOI, polyclonality, LD, HE, and Rs was undertaken with grouping by year of collection. Over the 6 years of sample collection, the proportion of polyclonal infections for P. falciparum varied from 0% to 31%, (P = 0.085) and from 23% to 67% (P = 0.104) for P. vivax, whilst the mean MOI ranged from 1.0 to 1.3 for P. falciparum, and from 1.2 to 2.0 for P. vivax . Population diversity (HE) values ranged from 0.448 to 0.608 for P. falciparum (P = 0.648) and from 0.840 to 0.881 for P. vivax (P = 0.932). Furthermore, there was no difference between Rs for P. falciparum  or P. vivax . LD demonstrated modest fluctuations between the study years, with a slight increase observed in the P. falciparum cases in 2015 . Nonetheless, because there were no significant differences in diversity or complexity of infection between the study years, all TES and ESS samples were pooled into a single group defined as the passively detected patent sample group (PP) for subsequent analyses.All passively detected cases presented with microscopically confirmed parasitaemia. The comparability of the isolates collected from the clinical and ex vivo studies were assessed before pooling. No difference was observed between the median parasitaemia of the TES and ESS samples either in N = 5 P. vivax and N = 13 P. falciparum). The small sample size did not allow comparisons with other subgroups and, therefore, these were excluded from further analyses. Because venous blood samples were only taken from adults, MOI, polyclonality, Rs, and HE were compared between adults (≥ 15 years) and children (< 15 years), but no significant differences were observed in any of these measures . Likewise, there were no significant differences in MOI, polyclonality, Rs, and HE between isolates from capillary or venous samples . Samples were, therefore, pooled into the following two subgroups: actively detected patent infections (AP) and actively detected subpatent infections (AS). In summary, AP comprises asymptomatic cases with a slide positive for P. falciparum or P. vivax monoinfection, whereas AS comprises asymptomatic cases with a slide negative for malaria, but PCR positive for either P. falciparum or P. vivax monoinfection.Only 18 of the isolates collected by active case detection were symptomatic (P = 0.482), but significantly different between active and passively detected cases for both species (P < 0.0001).The demographic details of all subgroups are presented in P. falciparum infections almost all comprised predominantly ring stages in both actively detected patent  and passively detected cases . By contrast, the percentage of synchronous ring stage P. vivax infections was 11.1% (9/81) in AP compared with 41.3% (19/46) in PP samples .Isolates for ex vivo susceptibility testing (ESS) were only selected if the ring stage composition was ≥ 70% and thus, the potential confounding of parasite staging was assessed. After excluding the ESS and AS samples, P. falciparum infections showed similar complexity between synchronous  and asynchronous infections , with polyclonal infections present in 23% (19/84) and 30% (3/10) of the cases, respectively (P = 0.694). Similarly, P. vivax infections showed comparable complexity of infection between synchronous and asynchronous infections: MOIS = 1.78 (SD ± 1.1) versus MOIA = 1.75 , as well as proportion of polyclonal infections: 44% (4/9) and 54% (39/72), respectively (P = 0.728). Likewise, comparison between synchronous and asynchronous passively detected P. vivax cases (ESS and TES) revealed no significant difference in complexity of infection (MOIS = 1.39 (±0.6) versus MOIA = 1.52 (±0.8); P = 0.463), or percentage of polyclonal infections (34% (42/123) versus 41% (11/27), P = 0.516; P. falciparum infection among the passively detected cases, no further comparisons could be performed. However, when the three datasets were combined, the results remained similar , but not for P. falciparum . However, the correlation was not apparent after excluding the ESS samples for neither P. vivax , nor P. falciparum .Assessment of MOI and parasitaemia in patent infections revealed a weak, but significantly negative correlation between MOI and parasitaemia for P. falciparum Rs between AP and AS (P = 0.635), between AP and PP (P = 0.170), and between PP and AS , between AP and PP (P = 0.809), and between PP and AS , 23%; and MOISP = 1.2 (±0.39), 16%; P = 0.199 and P = 0.189, respectively). Similar MOI and polyclonality rates were also observed between AP and AS infections in P. vivax .As summarized in P. falciparum cases showed a higher mean MOI and a higher proportion of polyclonal infections compared with the passively detected infections, the difference was only statistically significant between the proportion of polyclonal infections in the AP (23%) compared with the PP cases  compared with the passively detected infections , and a higher proportion of polyclonal infections . The actively detected P. falciparum infections presented a lower average MLOCI than passively detected infections but the results were not statistically significant .The number of multiallelic loci (MLOCI) in the polyclonal infections in each of the P. falciparum and P. vivax. Knowledge regarding potential sampling bias is vital for the effective design and interpretation of surveillance studies of malaria, particularly passive sampling strategies. The current study highlights the challenges that are associated with the interpretation of molecular data generated from different sampling sources and the key issues to consider for sampling strategies.This study presents a comprehensive comparative genetic analysis of actively versus passively detected cases of P. falciparum population, with a small subpopulation of infections found in actively detected cases only.15 On further investigation of the genetic differences between Plasmodium populations collected by active versus passive sampling strategies conducted in the same population, the current study revealed that there was no evidence of any significant differences in genetic diversity between the passively and actively detected subgroups in either P. falciparum or P. vivax. Furthermore, low differentiation between the subgroups in both P. falciparum and P. vivax confirmed that the different subgroups appear to comprise the same general pool of parasite strains. The highest levels of genetic differentiation were observed in comparisons of actively versus passively detected cases in P. falciparum , likely reflecting the aforementioned small subpopulation of asymptomatic infections and possibly also inflated by modest temporal changes in LD. Indeed, LD analysis revealed higher allelic association among the actively detected P. falciparum cases in comparison to the passively detected cases. Nonetheless, the FST and F’ST values observed between the P. falciparum subgroups were all markedly lower than those reported by Noviyanti et al.29 between different Indonesian islands .In a previous study conducted in Mimika district, genetic substructure was observed in the 11 the results of drug susceptibility testing are likely to be representative of the parasites circulating in the broader population. Therefore, if parasites with resistance-conferring mutations were arising in the community, they would be likely to be detected in the ex vivo surveillance study.The equivalence in population diversity and low differentiation between the ESS and the other sample subgroups suggests that despite the application of strict sample selection,30 Our results suggest that passive surveillance studies are a reliable “index” of the parasite strains circulating in the broader population. However, direct comparison of known resistance genotypes in patent versus subpatent infections will be required to confirm these findings.31The results of the study have important implications for our understanding of the molecular epidemiology of the parasites beneath the surface of the “malaria-iceberg.” This is especially relevant in the context of mass drug administration campaigns, as one of the greatest concerns of this practice is the selection of resistant parasites circulating in the community at very low frequencies.32 who explored the genetic background of P. falciparum parasites in cases of uncomplicated versus cerebral malaria, finding no evidence of closer relatedness  among the cerebral cases. In a genetic study of P. falciparum and P. vivax in Colombia, Pacheco et al.33 also found no evidence of differences in the circulating genotypes between severe and uncomplicated cases in either species. However, further studies need to be performed to support this hypothesis.Our study also suggests that parasites derived from passively detected symptomatic infections had similar genotypes to actively detected asymptomatic infections. These findings suggest that the development of clinical symptoms may be more attributable to host factors such as immunity and RBC polymorphisms, than to intrinsic parasite factors resulting in a higher multiplication rate. Similar results were reported by Ferreira et al.P. falciparum and P. vivax. The similar estimates of diversity and low differentiation in comparisons of different blood withdrawal methods (capillary versus venous samples), disease presentation (symptomatic/passively detected versus asymptomatic/actively detected), and concentration of DNA (patent versus subpatent) in both species, indicate that diversity estimates are not affected by any of these conditions. Therefore, population diversity and other population genetic measures derived from predominant allele calls can be reliably estimated on pooled samples and effectively compared between different sample types.These observations are particularly relevant when considering the sampling strategy for population genetic studies of 34 In contrast to the population-level estimates of diversity, which are based on frequencies of the major alleles in a given infection, assessments of the deeper complexity within infections revealed several differences between sampling groups. Actively detected cases exhibited a higher MOI than passively detected cases of both species, albeit only reaching statistical significance in P. vivax, but not in P. falciparum. This finding has important implications for sampling strategies in population genetic studies of P. vivax investigating within-host diversity. The contrast between the species could reflect interspecies differences in the persistence of subpatent infections resulting from new infections or relapses. In a recent longitudinal survey of individuals with asymptomatic malaria parasitaemia in a low endemic setting in Cambodia where P. vivax predominates, Tripura et al.35 showed that 35% of individuals with P. vivax infection remained parasitaemic for up to 11 months, whereas only 13% of individuals with P. falciparum infection remained parasitaemic for up to 4 months. The persistence of asymptomatic parasitaemia might enhance the opportunity for multiple inoculations of P. vivax by different mosquitoes before the onset of clinical symptoms or parasite clearance.Population genetic studies routinely use estimates of complexity of infection as indicators of transmission intensity.36 These challenges mainly relate to the presence of stutter peaks or nonspecific PCR products that can interfere with the discrimination of artifacts from true minor alleles. Moreover, higher DNA concentrations can increase the detection of minor alleles.37 To counter potential differences in sample quality or quantity in population genetic studies of Plasmodium, minimum threshold values of 25% or 33% for calling additional alleles relative to the major allele are commonly applied.36 The negative correlation between parasitaemia and MOI in the current study suggests that applying a relative threshold such as 33% is not as effective in P. vivax samples as it was in P. falciparum samples when pooling samples from the community with those attending health facilities. Consequently, these results suggest caution when inferring transmission intensity from MOI and percentage of polyclonal infections from pooled samples.Several previous studies have highlighted the challenges of characterizing multiple-clone infections using STR typing.14 who found that actively detected P. falciparum infections collected in a low endemic setting in Zambia were significantly divergent from passively detected cases, not only in terms of complexity of infection, but also in their genetic background. Key differences between the two studies that may explain the observed discrepancy are the greater temporal heterogeneity in sampling of active versus passively detected cases combined with greater bottlenecking in the P. falciparum population in the Zambian study. The Searle et al.14 study shows evidence of increasingly unstable P. falciparum transmission over time, including increased inbreeding, consistent with rapidly declining transmission. These temporal genetic changes could account for the differences found between the beginning of the sampling period (when most of the actively detected cases were enrolled) and the end of the sampling period (when most of the passively detected cases were enrolled) as observed in other temporal genetic studies of P. falciparum.39 By contrast, the current study comprises samples collected in a period during which there was no marked change in malaria transmission and, with the actively detected cases enrolled within the sampling period of the passively detected cases. Differing from the situation in P. falciparum, reports on P. vivax population diversity remaining relatively resilient to changes in the intensity of transmission suggest that,34 except in areas with extreme bottlenecking such as Malaysia,40 passively detected cases should remain representative of the broader P. vivax population.The results of our study differ from those reported by Searle et al.3 may have identified parasites at an even lower level of the “malaria-iceberg” that were not identified with the conventional PCR approach used here. However, it is also highly likely that these very low-density infections would be refractory to STR genotyping.3 Lastly, given the multiple factors affecting the dynamics of asymptomatic malaria and the influence of malaria transmission on parasite diversity and parasite differentiation, the results of the current study may not necessarily be extrapolated to areas with different malaria epidemiology.42 Indeed, available evidence inferred that the minor subpopulation of asymptomatic P. falciparum infections observed in Mimika district might reflect infections imported from other parts of Indonesia.16 As local transmission declines, the impact of imported cases on the parasite population diversity and structure may become more pronounced.40A limitation of this study is the large proportion of adult males missed from sampling during the cross-sectional study, which constitutes a bias per se that needs to be considered. The study also presents a potential bias reflecting the omission of very low-density infections that exhibited low genotyping success. Furthermore, the recently described high-volume, ultrasensitive PCR approach by Imwong et al.In conclusion, asymptomatic malaria infections constitute a major challenge in pre-elimination areas where their prevalence may force the need to modify ongoing case detection strategies for surveillance. Many studies continue to use passively detected samples as an index of diversity and drug resistance in the broader parasite population. Our results suggest that passively detected samples provide effective representation of the ‘strains’ circulating in the broader community including the hidden asymptomatic reservoir. However, passive sampling strategies may not be effective for investigation of within-host infection complexity in the asymptomatic reservoir. These results highlight critical considerations when undertaking molecular surveillance of malaria.Supplemental Tables.


Background. The World Health Organization recommends that all malaria management be based on parasitological identification. We monitored performance of trained community health workers (CHWs) in adhering to this recommendation to restrict artemisinin-based combination therapies (ACTs) to positive rapid diagnostic test (RDT)–confirmed cases in children in 3 malaria-endemic sub-Saharan African countries.Methods. In 33 villages in Burkina Faso, 45 villages in Nigeria, and 84 villages in Uganda, 265 CHWs were trained over a minimum of 3 days to diagnose malaria using RDTs  and treat RDT-confirmed uncomplicated malaria cases with ACTs. In Nigeria, CHWs were also taught to obtain a thick blood smear. Spent RDT kits and prepared blood slides were collected and interpreted independently in Burkina Faso and Nigeria to confirm CHWs' diagnoses. Interviews were held with 12 of 17 CHWs who prescribed ACTs for patients with RDT-negative test results, and with 16 of 29 caregivers to determine factors related to noncompliance.Results. Of 12 656 patients treated with ACTs in the participating countries , 29 patients  were RDT negative. The small number of RDT-negative ACT-treated cases limits statistical analysis. Only a few CHWs were involved, and they were more likely to be traders rather than farmers . RDT-negative children who were treated with ACTs had a significantly higher probability of residing in a village other than that of the CHW . Parental pressure was identified in interviews with parents.Conclusions. Noncompliance with results of RDT tests is relatively rare when CHWs are trained and well supervised.Clinical Trials Registration. ISRCTN13858170. Despite improvements in control of malaria, the disease remains a leading cause of death in children in Africa . AccuratMalaria microscopy remains the reference method for malaria parasite diagnosis, but its value is often undermined because it needs equipment and skilled laboratory technologists and cannot therefore be done quickly, at the point of care. Antigen-based malaria rapid diagnostic tests (RDTs), introduced in the 1990s, have increased from a few products to >250 tests, which are submitted annually for evaluation and inclusion in procurement lists used by countries . The avaPlasmodium falciparum RDT strips use immunochromatographic methods to capture P. falciparum–specific antigens in lysed blood. A dye-labeled antibody binds to the parasite antigen and the resultant complex is captured on a nitrocellulose strip by a band of bound antibody, forming a visible “capture” or “test” line. The HRP2-based tests can pose problems as the antigens can remain in the blood for some time after successful treatment, confounding diagnosis when transmission is high and patients present with successive fevers within a short timeframe ) prescribed ACTs to RDT-negative patients. Separating CHWs who did not treat an RDT-negative case from those who did, noncompliant CHWs had substantial enrollment rates, and therefore experience (Supplementary Table 1). RDT-compliant CHWs had an average of 49.9 treated cases per CHW compared with an average of 64.6 treated cases for the noncompliant CHWs; the average is higher for every participating country as well as overall . The total number of erroneous treatments per CHW is low  reflecting the low overall error rate. The mean overall error rate for the 17 noncompliant CHWs was 2.6% .In Supplementary Table 1).To check whether “noncompliance” occurred in new CHW recruits and immediately after training, when CHWs had less experience and might yield to parental pressure, we calculated the time from their first treatment to their first error. The mean number of days to their first error was 51.9 overall, 47.8 for Burkina Faso , 60.3 for Nigeria , and 14.5 for Uganda . Consequently, we cannot confirm a hypothesis that errors are higher in new CHW recruits  indicated they would use another drug to treat the child instead of not treating, and 6 of 7 CHWs said that they would refer the child to nearest health facility. They also indicated (5/8) that they would use ACTs for their own children if the child was RDT negative, as they had been using ACTs without RDTs in the recent past:What I do is to give paracetamol and if there is no improvement I will refer to health centre … because they have better knowledge. Not only malaria presents with fever but people believe when a child is feverish, it is malaria. — CHW at Kupalo village, Nigeria, during KIIOnly 1 CHW responded that the appropriate action was to treat the child with paracetamol (acetaminophen) or refer him to the health facility:… My child was weak and looked very sick, I explained to the [CHW], and he told me that it is malaria, and told me that he is going to give her medicine for malaria … and indeed after 2 days there was a change, but I know if the child is given medicine that does not treat the disease the child can become worse and can be taken on a drip in the facility … My child got better … as long as my child gets better, the [CHW] healed my child, the [CHW] did good. — Mother, Busaana village, Uganda, during KIIWhen I took the child to the [CHW], she asked me if the child was playing, and eating; She got something that is like a watch and counted the breathing rates, then after she explained to me why she needed to prick the child and take blood then test it for malaria. We waited for some time and she told me my child did not have malaria but according to me it seemed like he did because he was not eating and his body heat was high. I requested for the medicine though she was hesitant to give it to me … I requested and pleaded with her to give me the medicine because I felt the child was sick and maybe the testing had a problem and could not detect malaria and after some days under medication, he became fine that means he actually had malaria.— Mother, Migamba village, Uganda, during KIIA few (3) mothers/parents of the children were interviewed in Uganda, 9 in Nigeria, and 4 in Burkina Faso. The results confirmed the CHW's statement and there was no sense of guilt, especially if the child improved:In approximately 16 000 patients diagnosed with an RDT by trained CHWs in their malaria endemic areas , 12 627 Only 3 prior studies carried out between 2006 and 2010 are known to have monitored community treatment and compliance with RDT results involving a total of 7328 patients assessed by trained CHWs in their communities in Uganda, Ghana, and Burkina Faso , 24. Micand severe malaria, and more expensive, there is increased focus on targeting treatment to malaria-positive cases through the use of more expensive RDTs, which become cost-effective when treatment is based on the result [Until recently, the policy for treating malaria had assumed that most febrile illnesses were malaria, and that the risk of not treating with an effective antimalarial was greater than missing a true case. This approach probably saved many lives, but led to overuse of antimalarial drugs. When the cost of antimalarial drugs was lower than the cost of ACTs, microscopy was the primary method of diagnosis ; antimalarial overprescription was tolerated, despite preventing other causes of fatal illnesses from being diagnosed and treated. With highly effective artemisinin drugs being the key component of ACTs and essential for treatment for both uncomplicated e result . Multiple result . IntroduThere were some limitations to this study. We found both false-negative and false-positive results in patients on re-reading RDT test cassettes and comparing them with microscopy, supporting a legitimate concern that some children should have been treated in the face of negative RDT cassette results. The levels of overdiagnosis by CHWs who had received training were very small, but the constant presence of research staff in the communities, even where the study was being conducted by the Ministry of Health in Uganda, may have altered practice and compliance with RDT results.Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.