Plasmodium falciparum or Plasmodium vivax malaria annually. In 2010, Indonesia introduced a screen and treat policy for the control of malaria in pregnancy at first antenatal visit using microscopy or rapid diagnostic tests (RDTs). A diagnostic study was conducted in Sumba, Indonesia to compare the performance of four different RDTs in predominately asymptomatic pregnant women under field condition.Malaria in pregnancy poses a major public health problem in Indonesia with an estimated six million pregnancies at risk of ®, Parascreen® and SD-Bioline®). The test results were compared with expert microscopy and nested PCR. End user experience of the RDTs in the field was assessed by questionnaire.Women were screened for malaria at antenatal visits using field microscopy and four HRP-2/pLDH combination RDTs . Compared with PCR, the overall sensitivity of the RDTs and field microscopy to detect any species was 24.6–31.1 %; specificities were >98.4 %. Relative to PCR, First-Response® had the best diagnostic accuracy (any species): sensitivity = 31.1 %, specificity = 98.9 % and diagnostic odds ratio = 39.0 (DOR). The DOR values for Carestart™, Parascreen®, SD-Bioline®, and field microscopy were 23.4, 23.7, 23.5 and 29.2, respectively. The sensitivity of Pan-pLDH bands to detect PCR confirmed P. vivax mono-infection were 8.6–13.0 %. The sensitivity of the HRP-2 band alone to detect PCR confirmed P. falciparum was 10.3–17.9 %. Pan-pLDH detected P. falciparum cases undetected by the HRP-2 band resulting in a better test performance when both bands were combined. First Response® was preferred by end-users for the overall practicality.Overall 950 were recruited and 98.7 % were asymptomatic. The prevalence of malaria was 3.0–3.4 % by RDTs, and 3.6, 5.0 and 6.6 % by field microscopy, expert microscopy and PCR, respectively. The geometric-mean parasite density was low (®, but overall, differences between the four RDTs were small and performance comparable to field microscopy. Combination RDTs are a suitable alternative to field microscopy to screen for malaria in pregnancy in rural Indonesia. The clinical relevance of low density malaria infections detected by PCR, but undetected by RDTs or microscopy needs to be determined.The diagnostic accuracy to detect malaria among mostly asymptomatic pregnant women and perceived ease of use was slightly better with First-Response Plasmodium falciparum or Plasmodium vivax malaria [Malaria in pregnancy poses a major public health problem in Indonesia, where annually an estimated 6.3 million pregnancies are potentially at risk of  malaria  and the  malaria –4. EarlyIndonesia introduced a screen and treat policy for the control of malaria in pregnancy in 2010 . It consPlasmodium species are endemic. The HRP-2 antigen is specific for P. falciparum and Pan-pLDH detects all human infecting species [The common immuno-chromatography based malaria RDTs detect the histidine rich protein-2 (HRP-2) antigen or parasite lactate dehydrogenase (pLDH) enzyme. The HRP-2/pLDH combination RDTs are commonly used in Indonesia and other Asian countries where multiple  species , 7.P. falciparum in symptomatic population in areas of varying transmissions [P. vivax in asymptomatic women [P. falciparum and P. vivax, in mostly asymptomatic pregnant women and to compare it against the performance of standard field microscopy. For this, a cross-sectional study was conducted in eastern Indonesia comparing four combination RDTs and field microscopy against expert microscopy and PCR to identify the RDT with highest diagnostic accuracy and field practicality to use as a screening test in rural antenatal clinics.RDTs are essential for the success of the malaria in pregnancy screening programme. Numerous studies have shown the ability of HRP-2 based RDTs to detect missions –13. Few missions –16 and fmissions –20, specic women –23, and P. falciparum. In the dry season (April–November) both P. falciparum and P. vivax are present in equal proportions with an overall prevalence of 4.9 % by PCR.The study was conducted between March and July 2012 in south-west Sumba district in eastern Indonesia where malaria transmission is low and seasonal and varies substantially across sub-districts . PrevalePosyandus’, which are village based community integrated activities held monthly that include provision of outreach antenatal care services provided by the primary health centres (Puskesmas). A total of 45 Posyandus were involved in the study, served by four Puskesmas in the sub-districts of Bondo-Kodi, Kori, Wallandimu and Pannengo-ede, each covering a catchment population of approximately 30,000 people and 10–11 villages.The study was conducted in ‘Posyandu for routine antenatal care were enrolled. A questionnaire consisting of socio-demographic information, obstetric history, history of fever and drug use, including malaria prevention measures was completed and the axillary temperature recorded. A finger prick blood sample was taken to simultaneously test the four RDTs, malaria microscopy and dried blood spots for PCR. Women testing positive for malaria with any RDT were treated according to the national policy which was a 3 day course of ACT (dihydroartemisinin–piperaquine) in the second and third trimester and 7 days of oral quinine in the first trimester.Pregnant women of any gravidity aged between 15 and 49 years who attended the Puskesmas who were unaware of the RDT results. Thin smears were used to identify species. Expert microscopy consisted of re-examination of all the slides by the senior microscopist at the Malaria Laboratory-1, Eijkman Institute in Jakarta, who was blinded to field microscopy and RDT results. In cases of disagreement between field and expert microscopy, PCR result was taken as final. A slide was declared negative if parasites were absent after examining 200 high power fields. Parasite density was quantified against 300 leukocytes on an assumed leukocyte count of 8000 per µL of blood. Sub-microscopic infection was defined as parasites detected by PCR, but not by expert microscopy or RDT respectively.Thick and thin smears were made on the same slide and stained with 5 % Giemsa. For the ‘field microscopy’, the smears were read by the staff microscopist at each of the four ® , SD Bioline Malaria Ag Pf/Pan® , the two RDTs used in the malaria control programme in Indonesia at the time of the study, and First Response Malaria pLDH/HRP-2 combo®  and CareStart Malaria pLDH/HRP2 combo™ , two of the best performing RDTs indicated in the WHO/FIND round 1–3 report [The four RDTs included Parascreen Rapid Test Pan/Pf® and read within 15 min; for the other three RDTs two drops of buffer were used and was read within 20 min. Malaria positivity was defined if any of the HRP-2 or pLDH bands were visible (plus the control band). If only HRP-2 band plus control band was visible it was considered as a P. falciparum infection and visibility of only Pan-pLDH band was considered a P. vivax infection. When both bands were positive simultaneously it was considered a P. falciparum infection or a mixed infection.Accordingly, four drops of buffer were used for SD BiolineP. falciparum and P. vivax. All ribosomal PCR positives samples were confirmed using mitochondrial DNA based PCR. Repeat mitochondrial PCR was performed for discordant P. vivax samples using KAPA2G™ Fast ReadyMix (2×) (KAPA Bio systems) containing all components for the PCR and the primers MitoPf-F 307 and MitoPf-R 5904 and adding 2 µL of DNA template. Species identification was done on PCR-Restriction Fragment Length Polymorphism (Rflp) using the restriction endonuclease enzyme Acl1. The amplicon was electrophoresed on 2 % agarose gel and the species specific DNA bands were viewed. Staff unaware of the microscopy and the RDT results performed PCR.Nested PCR was performed on all samples to detect malaria parasites and for speciation. A blood sample was spotted on Whatman grade-1 filter paper, air-dried, placed in a plastic bag and transported to Jakarta. DNA was extracted with a 20 % Chelex solution and stored at −20 °C. Nested PCR based on the principle described by Snounou et al.  was usedPuskesmas were monitored and recorded using Tinytag™ Data Loggers with alarms set at 40 °C, the upper limit of temperature stability for SD Bioline®. The recorded data was saved into the main database and verified fortnightly. The maximum humidity recorded was 82.1 rh % and temperature ranged between 22.8 and 28.5 °C during the study period. To follow real-life field situations, environmental conditions in storage facilities were not controlled although excessive exposure to direct sunlight was avoided during RDT transportation. Individual kits were opened only at the time of testing and were checked for the presence of the desiccant. A single batch of each RDT with same LOT number purchased directly from the manufacturer was used.Room temperature and humidity in the RDT storage rooms in the Puskesmas midwives/nurses trained for the study were given a questionnaire to assess the readability and print clarity of information on the package exterior, ease of using the blood collecting devices, transferring blood to the RDT cassette and addition of buffer drops. Each parameter was scaled from easy, moderately easy and hard and the response frequency to each category calculated.The ease of use and practicality of the study RDTs in the field situation was explored mid-way of the study. The A sample size of 940 pregnant women were estimated to be sufficient to compare RDTs with expert microscopy and PCR. Allowing for a 10 % loss of samples or unsuccessful tests a target sample size of 1045 women was estimated. An anticipated malaria prevalence of 10 % detectable by PCR was considered with reference to a previous study in the area . This asData were double entered and verified using XAMPP Windows 1.7.7. Frequencies and proportions were calculated using SPSS version 20.0. The results of the four RDTs and field microscopy were first compared against expert microscopy as the reference test. In subsequent analysis the RDTs, field and expert microscopy were compared against PCR  as the reference test, for any species  and by specific RDT bands (HRP-2 and pLDH) against the species identified by PCR. For both sets of analyses, the sensitivity, specificity, positive and negative predictive values and likelihood ratios (LR) were calculated with corresponding 95 % confidence intervals (CI) using an online calculator which uses the efficient score method to calculate 95 % CI suitable for situations where the proportion is small and prevalence is low . The diaThe study was approved by the Research and Ethical Committees of the Eijkman Institute for Molecular Biology, Indonesia and the Liverpool School of Tropical Medicine, UK. Written informed consent was obtained.Posyandus for antenatal care were enrolled between March and July 2012. Most women were indigenous to southwest Sumba (97.5 %) and lived in rural areas (100 %). Their mean age (SD) was 28.8 (6.2) years and 17.8 % were primigravidae. The proportion of women reporting ownership of long lasting insecticide treated nets (LLINs) was 29.9 % of whom 93.3 % reported to have slept under the net the previous night. A documented fever (≥37.5 °C) was present in 12 of 949 women (1.3 %) and 157 (16.5 %) reported a history of fever in the previous week of whom 31 (19.7 %) reported taking anti-malarial drugs.A total of 950 women who attended the study Overall a full set of results for the RDTs, blood smears and PCR samples were available from 934 of the 950 women. The remaining 16 women (1.7 %) were excluded from analysis because not all four RDT tests were successful or no microscopy results were available.® (Table ® relative to Carestart™ was due to a higher number of positives by the HRP-2 band (21 versus 15). Overall, differences in results were greatest for the single Pan-pLDH band  and positivity varied from 6.6 % with SD Bioline® to 21.4 % with Carestart™.Malaria positivity by RDT (HRP2 or pLDH) ranged from 3.0 % (28 women) with Carestart™ to 3.4 % (32 women) with Parascreen® Table . The higPlasmodium malariae and 5 (10.6 %) mixed infections with P. falciparum and P. vivax.There were 34 (3.6 %) and 47 (5.0 %) malaria (any species) positives detected by field and expert microscopy respectively when both asexual and sexual stages were considered. Overall, 5 of the 34 (14.7 %) and 8 of the 47 (17.0 %) were positive for gametocytes by field and expert microscopy respectively. Expert microscopy detected 5 (10.6 %) mono infections with P. falciparum (32 mono-infections and seven mixed with P. vivax), and 23 (37.0 %) were P. vivax mono-infections. No infections with P. malariae were detected.Overall infection prevalence by PCR was 6.6 %  and 3.9 % were sub-patent infections (microscopy or RDT negative). Of the 62 positives identified by PCR, 39 (62.9 %) were with There were 31 women who had reported to have taken antimalarial drug in the previous week. Out of these, one women had all four RDTs positives, two women had a positive microscopy (6.4 %) and three women had a positive PCR (9.6 %) for any malaria.Malaria overall (any species) . The DOR of Carestart™ (24.9), Parascreen® (−25.0) and SD Bioline® (24.9) were similar. The sensitivity of field microscopy was comparable to First Response®, but the specificity and the DOR (29.1) was lower.s) Table : The senPlasmodium falciparum and single HRP-2 band . The sensitivity ranged from 18.8 % with Parascreen® to 25.0 % with First Response®. The DOR was highest for SD Bioline® followed by First Response®.nd Table : The accPlasmodium falciparum and the combined HRP-2 and Pan-pLDHbands . The sensitivities ranged between 30.7 and 41.0 %. The LR positive and DOR values ranged between 17.2 and 24.5 % for Carestart™ and 26.2 and 44.4 % for First Response®, respectively.ds Table : The accPlasmodium vivax and Pan-pLDH band . The sensitivity to detect P. vivax was lower than for P. falciparum ranging from 8.7 % with SD Bioline® to 13.0 % with the other three RDTs. The DOR values ranged from 10.8 % with SD Bioline® to 16.9 % with Parascreen®. The field microscopy indices were relatively better for P. vivax detection with higher DOR values.nd Table : The per® scoring better LR-positive and DOR values. When the ability to detect P. falciparum by HRP-2 band was assessed, First Response® had a lower false positive score reflected by higher PPV and LR-positive relative to the other RDTs  and of P. vivax was 147 parasites/µL (range 27–5733 parasites/µL). Out of the 12 PCR confirmed expert microscopy positive P. falciparum cases, there was one case of mono-infection with P. falciparum at density <100 parasite/µL and this was negative by all four RDTs. Similarly, there were 6 cases out of the 11 P. vivax PCR confirmed positives which has densities of <100 parasites/µL detected by expert microscopy; none were detected by the RDTs  was comparable between Carestart™ (6/6) and First Response® (6/6) relative to Parascreen® and SD Bioline®. The detection of low-density P. vivax infections (100–499/μL) was similar in all RDTs (33.3 %).The geometric mean parasite density of ® was the easiest to draw blood with (86.0 %)  Table , but to ®, Carestart™, First Response® and Parascreen® RDTs to screen mostly asymptomatic antenatal women for malaria were evaluated in four sub-districts of South west Sumba, Indonesia as a potential alternative to field microscopy. Overall the diagnostic accuracy, suggested that First Response® RDT had the best combined ability to detect both P. falciparum and P. vivax infections when compared against PCR. The diagnostic odds ratio (DOR) value for First Response®, as a single summary indicator of test performance, was lower than that observed for expert microscopy but higher than the 23–29 DOR values observed for the other RDTs. First Response® was also considered the easiest to use by the clinic staff. Nevertheless, the overall differences in performance of the four RDTs to detect malaria were small and importantly, comparable to field microscopy. Any of the four brands would probably be suitable as a potential alternative to field microscopy or for use in the clinics where microscopy is not available.The performance of SD BiolineP. vivax . None of the infections (any species) below 100 parasites/µL were detected by RDTs, around the threshold level of detection for many RDTs [However, of note was the low sensitivity (26–32 %) of all four RDTs (and field microscopy) relative to PCR, which detected 2–3 additional infections for each infection detected by RDT. These findings are consistent with the known limitations of this current generation of RDTs and highlight the challenge of applying them as a screening test in asymptomatic pregnant women who on average will have much lower parasite densities when infected than symptomatic malaria patients, the intended population for whom most RDTs are used. The geometric mean parasite densities in those with detectable parasites by expert microscopy were low for both species, especially for any RDTs .P. falciparum and P. vivax co-exist [The sensitivity was also low when compared against expert microscopy  and lower than reported in previous studies in asymptomatic pregnant women during pregnancy and at delivery . In addico-exist .P. vivax mono-infections  ns Table  was part density , 35.P. falciparum cases that were negative by the HRP-2 band resulting in a better test performance when results of both bands were combined  were confirmed by expert microscopy, and none of the 13 P. vivax infections detected by expert microscopy (11 of which were PCR confirmed) were detected by field microscopy.The low parasite densities for P. falciparum detection [Comparison between other RDT studies in pregnancy and this study was problematic because of the differences in transmission level, endemic species, RDT brands and analysis method used. Earlier studies have used RDTs to diagnose placental malaria at delivery , 16, 38.etection –41. Otheetection , 20.To qualify for an effective screening test used in low malaria prevalent population, the RDTs should have an acceptable balance to “rule-out” and “rule-in” infections in asymptomatic women. The RDTs demonstrated these qualities for any malaria detection and their performance overall (any species) was similar to field microscopy. The high specificity and NPV indicated that the RDTs correctly identified non-infected women, which would avoid treating women without malaria.® was favoured whereas First Response® was preferred for overall practicality including ease of blood transfer pipette and buffer usage. The loop pipette for blood transferring found in Parascreen® kit was preferred least, similar to studies elsewhere [The perceived ease of use by the field staff for the four RDTs were comparable. When the package readability was accounted for, SD Biolinelsewhere . A very Limitations of our study included the cross sectional study design, which limited determining when sub-microscopic infections may become patent and we were unable to follow women until delivery to correlate antenatal RDT findings with placental malaria. Batch quality Lot testing prior to using the RDTs was not performed. The study environment with dedicated research staff who were trained for the study and attention given to operational conditions, might not reflect the routine field situations.®, but overall differences between the four RDTs were small and performance was comparable to field microscopy. Best test performance to detect P. falciparum was achieved by combining the results of both pLDH and the HRP-2 bands. Combination RDTs are a suitable alternative to field microscopy to screen for malaria in pregnancy in rural Indonesia. However both RDTs and field microscopy missed many infections detected by PCR, especially P. vivax infections. The clinical relevance of these low density infection needs to be determined further, as it may help determine whether preventive strategies such as intermittent preventive therapy in pregnancy (IPTp) may be more effective than single or repeated screening with RDTs during pregnancy.The diagnostic accuracy to detect malaria among mostly asymptomatic pregnant women and perceived ease of use by field staff was slightly better with First Response


Plasmodiumfalciparum being the principal cause of malaria disease morbidity and mortality. Plasmodium falciparum virulence is attributed, in part, to its population-level genetic diversity—a characteristic that has yet to be studied in Rwanda. Characterizing P. falciparum molecular epidemiology in an area is needed for a better understand of malaria transmission and to inform choice of malaria control strategies.Malaria remains a public health challenge in sub-Saharan Africa with P.falciparum-infected patients identified at two sites of different malaria transmission intensities in Rwanda. Data on demographics and clinical features and finger-prick blood samples for microscopy and parasite genotyping were collected. Nested PCR was used to genotype msp-2 alleles of FC27 and 3D7.In this health-facility based survey, malaria case clinical profiles and parasite densities as well as parasite genetic diversity were compared among P.falciparum infection (MOI) was 1.73 but with mean MOI found to vary significantly between 2.13 at Ruhuha and 1.29 at Mubuga (p < 0.0001). At Ruhuha, expected heterozygosity (EH) for FC27 and 3D7 alleles were 0.62 and 0.49, respectively, whilst at Mubuga, EH for FC27 and 3D7 were 0.26 and 0.28, respectively.Patients’ variables of age group, sex, fever (both by patient report and by measured tympanic temperatures), parasite density, and bed net use were found differentially distributed between the higher endemic (Ruhuha) and lower endemic (Mubuga) sites. Overall multiplicity of p. falciparum associated parameters such as drug resistance, immunity and impact of used interventions, and in proper interpretation of malaria vaccine studies.In this study, a higher geometrical mean parasite counts, more polyclonal infections, higher MOI, and higher allelic frequency were observed at the higher malaria-endemic (Ruhuha) compared to the lower malaria-endemic (Mubuga) area. These differences in malaria risk and MOI should be considered when choosing setting-specific malaria control strategies, assessing  In spite of the significant decline in malaria cases and deaths being reported globally, malaria still accounted for about 200 million cases and over 500,000 deaths in 2014 . In spitPlasmodiumfalciparum in the past led to chloroquine being replaced with amodiaquine (AQ) + sulfadoxine–pyrimethamine (SP) in 2001 and then later, AQ + SP combination was subsequently replaced with artemether–lumefantrine (AL) in 2006, as first-line anti-malarial therapies for uncomplicated malaria. Malaria transmission levels and the associated risk of morbidity and mortality show a spatial heterogeneity even within small countries such as Rwanda , where n = sample size, Pi = allelic frequency. Odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated to evaluate the strengths of associations. Statistical significance was defined as p value ≤ 0.05  [, Kigali, Rwanda.msp-2 alleles. Stratified by site, 195 (50.3 %) cases were enrolled at Ruhuha whilst 193 (49.7 %) enrolled at Mubuga  were microscopically re-confirmed by study-trained technicians to be malaria positive. Of the 402, final data analysis was performed on 388 (96.5 %) who were successfully genotyped for the The results of group comparisons of demographic, bed net use, fever experiences, and parasite density among patients from the two study sites are shown in Table Overall, a range of one to six infection clones per sample was seen. At both sites, about 55.4 % of the infections were monoclonal, with isolates from the Mubuga site carrying a significantly higher proportion of monoclonal infections (73 %) compared to those from Ruhuha (38 %) (p < 0.0001). The numbers of strains per isolate are presented in Table P. falciparum clones were detected  between Mubuga (1.3) and Ruhuha (2.1). In this study, MOI was seen to increase proportional to age group being from 1.7 among those under 5 years old to 1.9 among those aged 6–15 years and 1.5 among those >15 years. Isolates from Ruhuha also had higher MOI compared to those from Mubuga.Results for determinants of MOI are shown in Table P.falciparum genetic diversity across two study sites. A higher geometrical mean parasite counts (2347 vs 530 parasites), more polyclonal infections, higher MOI and higher allelic frequency were observed at higher malaria-endemic Ruhuha compared to the lower malaria-endemic Mubuga area.This study reports, for the first time in Rwanda, a differential spatial distribution of patient demographics of age and sex, fever, parasite density and P.falciparum infection among younger age groups has been reported from elsewhere, particularly for severe malaria [A higher proportion of children aged <5 years was enrolled at Ruhuha compared to Mubuga while, in contrast, a higher proportion of those aged >15 years was recruited at Mubuga compared to Ruhuha. Higher malaria burden in younger age groups in settings of high malaria transmission intensity have been reported previously –28. The  malaria . The appAmong patients enrolled at Ruhuha, a significantly higher proportion were females in contrast to those recruited at Mubuga where both sexes were proportionally represented. The association between malaria risk and sex remains equivocal. In contrast to this study’s findings, at the Ruhuha site, a number of previous studies, including two conducted at the Ruhuha site, reported a bias towards higher malaria risk among males , 34, 35.In this study, the proportion of patients with a reported fever experiences and by a fever ≥37.5 °C differed across the two sites. Whilst a higher proportion of Ruhuha-recruited patients self-reported a history of fever in the last 24 h compared to those from Mubuga and, in contrast, a lower proportion of the same patients from Ruhuha were confirmed with a measured fever (tympanic temperature ≥37.5 °C) compared to Mubuga patients. Fever is a common malaria-associated symptom and a major reason for seeking care among suspected malaria in endemic settings. At the higher malaria-endemic Ruhuha site, it is plausible that residents are more likely to associate fever with malaria and hence the higher proportion of reported fevers. On the other hand, at the lower malaria-endemic Mubuga site, with presumably a lower proportion of individuals with partially protective levels of immunity, patients are more likely to have symptomatic malaria infections presenting with fever than those at Ruhuha. However, the higher proportion of children <5 years old in Ruhuha may have confounded the observed higher proportion of reported fevers in Ruhuha compared to Mubuga with malaria being associated with fever or recent history of fever in infants. In contrast, the higher malaria endemicity in Ruhuha may plausibly be associated with higher levels of protective immunity leading to a lower proportion of malaria compared to persons from the lower-endemic Mubuga site, as previously reported from the Ruhuha site , 34. ChaIn this study, mean MOI was significantly higher at the higher malaria-endemic Ruhuha site compared to the lower malaria-endemic Mubuga site. While many studies have reported comparable findings of higher MOI in higher endemic settings and correspondingly lower MOI in low endemic settings , 36, 37,In this study, MOI was positively correlated with parasite density. This finding accords with previous studies where higher MOI among individuals with higher parasite densities has been demonstrated , 43. In P.falciparummsp-2 confirmed isolates carried monoclonal  infection. By study site, a higher proportion of monoclonal infections were seen at Mubuga (73.1 %) compared to Ruhuha (38.0 %). These data are similar to other studies where higher proportions of >50 % and up to 100 % polyclonal infections have been seen in meso-endemic and holo-endemic settings [msp-1 genetic diversity marker, higher proportions of polyclonal infection have been seen in high endemicity settings, suggesting that malaria parasite polyclonality may be a useful proxy measure of level of endemicity [msp-1, similar observations of higher diversity at a holo-endemic site in Tanzania compared to hypo-endemic south-western Brazilian Amazon and meso-endemic southern Vietnam has been reported, with 3D7 reported as the most frequently circulating allele in this study [About 55 % of the settings , 44, 45.demicity . Overallis study .msp-2 FC27 alleles belonged to the 300–330-bp allele family while the most prevalent msp-2 3D7 allele belonged to the 200–300-bp allele family. Between the two sites, while the 300–330-bp allele was more frequent at Mubuga, the larger size  allelic families were more common at Ruhuha. In contrast to the FC27 gene, the 200–330-bp allele was the most frequent circulating allele at both Ruhuha and Mubuga. Of interest, findings from Mubuga of lower allelic diversity and lower frequency of circulating alleles point to a high likelihood of re-infection with the same allele. Differentiating between recrudescence and re-infection using msp-2 in a low-endemic setting such as Mubuga may be limited by the msp-2 low discriminatory power.The majority of msp-2 is a validated molecular marker of diversity, use of one marker may miss variations at other polymorphic loci and underestimate the real magnitude of diversity.A number of factors, including an adequate sample size, use of validated genetic marker for diversity and allelic frequency, use of an automated gel reader to determine allelic family base pair sizes and a comparative analysis for the two study groups drawn from settings of different malaria transmission intensities, are major strengths of this study. However, there were some limitations. Firstly, there was a lack of earlier data on transmission intensity at either study sites to delineate local malaria endemicities. Secondly, being a cross-sectional survey design, study findings can only provide a baseline comparator for current diversity and disease clinical profiles but is unable to determine the value of diversity on other disease outcomes other than parasite density as well as time and impact of used intervention related effects. Thirdly, the study was done at two sites whilst in Rwanda, malaria risk is categorized into four malaria ecologic zones. Therefore, study findings may have limited generalizability, restricted to settings of comparable transmission and malaria control tool implementation levels. Fourthly, due to cost restrictions, we used a valid but lower discriminatory power assay (agarose gel electrophoresis) compared to other assays (e.g. capillary electrophoresis) and thus findings may be of a lower accuracy. Lastly, although P.falciparum parasite density and genetic diversity as well as allelic distribution between individuals from two sites of variable malaria transmission intensities. HE and mean MOI were higher among isolates collected from the higher malaria Ruhuha site. Locally, characterizing malaria disease severity, based on clinical features and parasitaemia levels, across populations from settings of differing malaria transmission intensities is important in profiling malaria risk maps and in decision-making on which control tools may have optimal impact. The difference in diversity may have differential effects on multiple parameters including drug-resistant profiles, immunological responses to anti-malarial drug and effectiveness of vaccines tested in Rwanda in the future.This study demonstrated a differential distribution in demographics, measured temperature,


CareStart G6PD deficiency Rapid Diagnostic Test (RDT) as a point-of-care tool for screening G6PD deficiency.Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most widespread enzyme defect that can result in red cell breakdown under oxidative stress when exposed to certain medicines including antimalarials. We evaluated the diagnostic accuracy of CareStart G6PD RDT and Trinity qualitative with Trinity quantitative methods as the “gold standard”. Samples were also screened for the presence of malaria parasites. Data entry and analysis were done using Microsoft Access 2010 and Stata Software version 12. Kintampo Health Research Centre Institutional Ethics Committee granted ethical approval.A cross-sectional study was conducted among 206 randomly selected and consented participants from a group with known G6PD deficiency status between February 2013 and June 2013. A maximum of 1.6ml of capillary blood samples were used for G6PD deficiency screening using CareStart G6PD deficiency RDT was 100% and 72.1% compared to Trinity quantitative method respectively and was 98.9% and 96.2% compared to Trinity qualitative method. Malaria infection status had no significant (P=0.199) change on the performance of the G6PD RDT test kit compared to the “gold standard”.The sensitivity (SE) and specificity (SP) of CareStart G6PD deficiency RDT kit was high and it is acceptable at determining the G6PD deficiency status in a high malaria endemic area in Ghana. The RDT kit presents as an attractive tool for point-of-care G6PD deficiency for rapid testing in areas with high temperatures and less expertise. The CareStart G6PD deficiency RDT kit could be used to screen malaria patients before administration of the fixed dose primaquine with artemisinin-based combination therapy.The outcome of this study suggests that the diagnostic performance of the  Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the largest and most widespread enzyme defect affecting approximately 400 million persons worldwide. In malaria endemic areas, the prevalence of G6PD ranges from 5.0% to 23.8% . It is aPlasmodium Vivax (Pv), P. falciparum (Pf), P. ovale (Po), P. malariae (Pm), and the recently emerging P. knowlesi (Pk). The burden of malaria is heaviest in the WHO African Region, where an estimated 90% of all malaria deaths occur, and in children aged under 5 years, who account for 78% of all deaths [Malaria, the most common parasitic disease which is estimated to infect 3.2 billion people in 97 countries and territories  is causel deaths .Plasmodium falciparum infection [Pf infection [Pv  as happens in the eastern part of Africa. The prevalence of G6PD deficiency in Ghana has been reported as 6.5% [Pv relapsing malaria parasite infection [In Ghana, the entire nation is at the risk of malaria infection, particularly in the middle belt of Ghana where transmission occur all year round with about 22.8% of malaria parasitaemia of which 98.1% is as a result of nfection , 8. The nfection , 8 with  as 6.5% , the G6P as 6.5% . All repnfection . It is onfection .People with G6PD deficiency are mostly asymptomatic but exposure to oxidant drugs, such as the anti-malarial drug primaquine, may induce hemolysis. The WHO recommended the use of a single dose of primaquine to artemisinin-based combination therapy (ACT) as a component of pre-elimination and elimination programmes provided the G6PD deficiency status of patient is determined , 15.Due to the seldom availability of G6PD testing in the field the implementation of the recommendation was limited. This challenge led to a review of the recommendation to a single dose of 0.25 mg base/kg primaquine, a dose considered unlikely to cause serious toxicity in subjects with any of the G6PD variants . DespitePhenotypic tests , unlike genetic techniques, to determine G6PD activity status are the most widely used and are the best indicators of G6PD function levels. Some of these testing products are suitable for point-of-care use in clinics because they are rapid and straightforward . HoweverCareStart G6PD RDT kit is developed to overcome some of the challenges faced when one uses the existing routine G6PD screening methods before patient management. The Kit is rapid, works in wide range of temperature [Manufacturer’s manual) and not costly compared to other commercially available G6PD test kits [A rapid and inexpensive methods to determine the G6PD status of all persons is desirable when one is considering use of drugs contraindicated in patients with G6PD deficiency. It is especially important and particularly needed in sub-Saharan Africa where G6PD deficiency is high, malaria is endemic and use of oxidative malarial drugs such as primaquine is expectedly high. One of such phenotypic biochemical tools; the perature , uses smest kits .CareStart G6PD RDT kit has not been evaluated in a malarial endemic area in an African setting as compared to what has been done largely by others elsewhere. The use of the device for point-of-care testing has been described as feasible, not labour intensive, require less training, not time involving, no special additional equipment and storage conditions required and has the ability to detect G6PD deficient individuals [To the best of our knowledge, ividuals , 16–20. CareStart G6PD deficiency RDT kit using capillary blood in a malarial endemic area in Ghana, Africa where G6PD deficiency is high and the need for RDT to rapidly screen for G6PD deficiency is urgent. This is necessary to make a tool available at point-of-care when Ghana adopts the recommendation of WHO to use fixed dose of primaquine for malaria treatment and elimination programme.We evaluated the diagnostic accuracy of the Ethical approval of the study procedures and consultations of records of potential participants with known G6PD status to recruit was granted by the Kintampo Health Research Centre Institutional Ethics Committee (KHRC-IEC). The objectives and procedures of the study were carefully explained to all potential participants to seek their written/thumb printed informed consent to participate. Care-takers gave written informed consent on behalf of their minor participants and also on behalf of their children who were between ages 12 and 17, who additionally gave their written/thumb printed informed assent to be part of the study. Participants were informed they were being contacted because of their previous involvement with the Kintampo Health Research Centre for studies that involved G6PD testing which they gave their written informed consent to be contacted anytime needful. Participants’ identity and records were anonymized prior to analysis.CareStart G6PD deficiency RDT kit among participants with known G6PD status  from previous studies carried out in the study area. This study was conducted between February 2013 and June 2013.We adopted a cross-sectional study design to evaluate the performance of the To calculate the sample size to carry out the performance evaluation, the team assumed an expected Area under ROC curve of 0.80 compared to a Null hypothesis value of 0.68  considerThe study was carried out in the Kintampo North Municipal and Kintampo South District of Ghana ; two admKintampo Health Research Centre carried out a population reference study and also a two-year cohort study. G6PD deficiency testing was one of the laboratory investigations carried out in the two studies. The database of the population of the two studies became the sampling frame to select prospective participants to evaluate the diagnostic accuracy of the G6PD RDT kit.The number of G6PD Deficient and Normal participants with known G6PD study participants with known G6PD status from the previous studies were selected from the database using MS Excel. The status of potential participants from the previous studies , 22 wereSelected individuals were initially visited at home, informed about the study and invited to participate after consenting. Individuals who were present in the community and willingly agreed to be part of the study were invited to a central point within the community to interact with the study team. A prospective participant who refused consenting to be recruited was replaced from list of prospective participants with known G6PD status. Flow chart for study participants’ selection and recruitment is presented in The process of participants’ selection was as follows. The team consulted a database from two previous studies that enrolled participants with their G6PD status established either with PCR or quantitative method. The team randomly selected the needed number of positive/deficient participants from the list of G6PD deficient individuals classified in the previous study. Same random selection was done to get the number of negative/normal individuals from the previous studies. Selected prospective participants from the previous studies were contacted in the community, consented to participate in the current evaluation study. The approach adopted in this study was not wholly community based survey and then presents the limitation of estimating prevalence parameters like the NPV and PPV. We can only describe the sensitivity, specificity and the Area under the ROC curve. We did not assign randomization codes or any special randomization technique. Simple randomization was adopted to fairly select any participant with known G6PD status in the category of interest.CareStart G6PD deficiency RDT screening kit  using capillary blood samples collected from sterile finger prick. About 1.0–1.6ml of blood was collected after G6PD and malaria RDT testing from the finger prick. The capillary blood sample collected into the EDTA microtainer test-tubes were sent to the KHRC Clinical Laboratory to perform G6PD deficiency screening tests using Trinity Biotech quantitative G6PD assay  and Trinity Biotech qualitative G6PD assay . Trinity Biotech quantitative method for G6PD was adapted and performed on The Flexor E Clinical Chemistry analyser . Quantitative and qualitative analyses were performed on the fresh blood samples within a maximum of 48 hours after the collection to determine levels of G6PD enzyme activity.G6PD deficiency test was done with Using Trinity quantitative method: The Trinity Biotech quantitative G6PD assay method was used as the gold standard . The metUnpublished data) established in the study area using the classification procedure described by Domingo et al, 2013 [The classification of G6PD deficiency status, using quantitation assay methods were based on population reference value G6PD activity of adjusted male median estimated to be 5.5 U/g Hb ), al, 2013 . The teaal, 2013 , 28.Using the Trinity Quantitative method, the G6PD deficiency status of participants were classified as either “Normal” or “Deficient” at a cut-off of 4.1 U/g Hb. All participants who measured G6PD enzyme activity <4.1 U/g Hb were classified as “Deficient” while those with enzyme activity equal to or above 4.1 U/g Hb were considered “Normal”.CareStart G6PD deficiency screening test: The CareStart G6PD deficiency RDT format test, which is a qualitative enzyme chromatographic test is based on the reduction of colourless nitro blue tetrazolium dye to dark coloured formazan. The test kit requires minimal training and no special expertise or additional equipment for use. Two microliters (2μl) of blood added into the sample well with two drops of buffer into the buffer well gave the G6PD deficiency status of an individual in 10 minutes. Samples with normal G6PD activity produce a distinct purple colour background in the result window while no colour change was observed for samples with deficient G6PD activity  (Using activity . Samplest shown) .Using Trinity Qualitative method: The Trinity Biotech qualitative G6PD assay method uses G6PD released from lysed erythrocytes to catalyze the conversion of glucose-6-phosphate to 6-phosphogluconate with reduction of NADP to NADPH. In the presence of phenazine methosulfate (PMS), NADPH reduces a blue dye to the colourless form. The rate at which the colour visually disappears in the reaction mixture is proportional to the G6PD content of red cells. Disappearance of blue colour of the dye within 1 hour while incubating reaction mixture at 37°C denoted participant had NORMAL G6PD enzyme activity. DEFICIENT was when there was no change in the blue colour .Full Blood Count was determined using a validated ABX Micros 60 analyzers  to obtain the haemoglobin concentration, Red Blood Cells and White Blood Cell count levels of participants were used in the calculation of the G6PD activity in the erythrocytes and malaria parasite density.CareStart Malaria RDT ) were used to screen for malaria simultaneously with the G6PD RDT. Samples collected were transported to the KHRC laboratory to prepare blood slides to examine for malaria parasites. Microscopic examinations of thick and thin blood films were conducted to confirm malaria diagnosis. Thin and thick peripheral blood smears prepared were read by two independent microscopists, and by a third microscopist if the results were discordant on positivity or on parasite density, as described by Adu-Gyasi et al, 2012 [Malaria RDT test kits . The performance of G6PD deficiency RDT was expressed by calculating the sensitivity (SE), specificity (SP) and the Area under the Receiver Operator Characteristics (ROC) curve for G6PD deficiency status using the Trinity quantitative results as gold standard . DiffereCareStart G6PD deficiency RDT and Trinity Quantitative methods were controlled with two levels of commercially prepared G6PD quality control (QC)  samples from Randox.All 5 personnel involved in the various tests were trained before the start of the study. The training included classifying a qualitative test outcome as ‘‘positive” or ‘‘negative”  as described by the manufacturer’s instructions. Qualitative tests were performed before the quantitative analysis. Personnel who performed the tests were blinded to the results of each method and stage of testing. Quality control checks were performed for the Full Blood Count on the automated haematology analyser with commercially available controls. The A total of 206 participants were recruited. About 57.8% (119/206) of the participants were males and 42.2% (87/206) were females. The median age of the participants was 27 years (range 2–85 years); 21.4% (44/206) of participants were less than or equal to ten years.CareStart G6PD deficiency RDT method were 100% and 72.1% using Trinity Biotech quantitative method as a gold standard. The ROC was 0.86  and Trinity biotech quantitative (P = 0.123) method were not significantly affected by gender (The diagnostic accuracy of y gender .CareStart G6PD deficiency RDT was not affected significantly (P = 0.199) when we considered participants that had malaria parasites infection compared to those that had no malaria parasite  had positive malaria parasites using microscopy. The performance of parasite . The difEarly diagnosis of Glucose-6-phosphate dehydrogenase deficiency could avert the potentially harmful effect of the deficiency encountered during patient management with primaquine , 31 whicPlasmodium species infections. Adoption of the WHO recommendation would mean deploying treatment with primaquine and ACTs in most malaria endemic regions including Ghana especially with the target of eliminating Plasmodium falciparum gametocytes[CareStart G6PD RDT which has high and acceptable diagnostic performance as assessed in this study could make G6PD testing available at point-of-care and alleviate this challenge.Ghana does not use primaquine in the management of patients with malaria  since itmetocytes, 15. Impmetocytes. The CarCareStart G6PD deficiency RDT compared to results from Trinity quantitative method used as the “gold standard” in a high malaria endemic area in Africa was higher than what was recorded in the evaluation study by Kim et al, 2011 [The SE and SP of the al, 2011 . This dial, 2011 , 20, 32.al, 2011 , 18 estaal, 2011  as pointSimilarly, the BinaxNow qualitative method has a good performance but the range of temperatures (18°C to 25°C)  requiredObviously, most of the methods available and accepted for routine screening of G6PD status of patients are not suitable to be employed to screen for G6PD status of patients at point-of-care in malaria endemic regions in the tropics where screening for G6PD is most needed. These rapid methods need sophisticated equipment and temperature regulation. Again, unlike the available routine test methods that require a minimum of 5μl to 3000μl of blood depending on the method to use, this screening kit requires a volume of 2ul of capillary blood to determine the G6PD deficiency status of an individual in 10mins at a temperature range of 18°C to 32°C . This alManufacturer’s manual). It would have been preferable to have this in the design to control each test that is performed with the kit. Analyzing each batch of samples with controls might not be cost effective. Also, the CareStart G6PD deficiency RDT kit produce a purple colour to differentiate between normal and deficient subjects . Borderline results also produce a pale purple colour. This can result in operator-to-operator variations due to the subjectivity of interpretation for qualitative colormetric results. The development of a technique to semi-quantitate the results of the rapid test would be helpful to minimise misinterpretation of RDT results as mentioned by others.None of the test cassettes had control window  and positive predictive values (PPV). The team could only describe the sensitivity, specificity and the Area under the ROC curve to assess the diagnostic performance of the kit.CareStart G6PD RDT is acceptable at determining the G6PD deficiency status in a high malaria endemic area in Ghana, Africa. The CareStart G6PD RDT kit presents as an attractive tool for point-of-care G6PD deficiency testing in areas with high temperatures and requires significantly less time and training to implement. The G6PD RDT kit could be used to screen malaria patients before administration of the fixed dose primaquine with ACT as recommended by the WHO.The results from this study suggest that the diagnostic performance of the


Plasmodium species distribution. Here, we characterized malaria in febrile children in Franceville, Gabon through a cross-sectional study at the pediatric unit of the Franceville Regional Hospital. We registered 940 febrile children. Their general condition was markedly altered in 11.7% of cases (n = 89/760); among them 19 (21.4%) had a severely altered condition. Malaria was the second most frequent etiology , after respiratory tract infections . Children with malaria (63 ± 39 months) were older than children without malaria (40 ± 37 months) (p = 0.0013). Hemoglobin, red blood cell, white blood cell, and platelet values were lower in children with malaria than in those without malaria (p < 0.0001). Anemia was the most common feature of severe malaria , followed by neurological involvement . The prevalence of malaria was significantly higher in children older than 60 months than in younger children . Plasmodium falciparum accounted for 97.5% of cases (158/162), followed by Plasmodium malariae . Bed net use was high  and contributed to malaria prevention (p = 0.001). Good basic knowledge of malaria also had a preventive effect (p < 0.0001). The prevalence of malaria in children in Franceville did not decrease significantly from 2009 to 2012, remaining at about 20%, highlighting that preventive measures should be reinforced.Recently, major progress has been made in controlling malaria in Africa. However, in Gabon, little information is available on the role of malaria in childhood febrile syndromes, the use and efficacy of preventive measures, and  Febrile syndromes are one of the main reasons for pediatric consultations in developing countries and may be due to various viral, bacterial, and parasitic infections. Malaria is the most widespread febrile disease, present in 99 countries and territories. One-third of the world population is at risk, and malaria causes about one-fifth of all childhood deaths worldwide . In 2013Plasmodium species infect humans, namely falciparum, vivax, malariae, ovale  and 2012 [21.1% (n = 81/384)] (p = 0.5). The temporal distribution of the parasite infection with the seasons shows that overall prevalence of Plasmodium infection was not the same depending on the season in 2011–2012 (p = 0.02) (Plasmodium infection was significantly lower during the long rainy season in 2011 (15.8%), and during the long rainy season (14%) and long dry season (13.2%) in 2012. It was highest during the short rainy seasons (29.8% and 26.4%) in 2012 and 2011 (p < 0.04).The overall prevalence of  = 0.02) . The prePlasmodium-infected children, 8.1% (17/209) had one sign of severe malaria. Severe anemia was the most prevalent sign [70.6% (12/17)], followed by neurological involvement [23.5% (4/17)]. There was one case of prostration (5.9%).Among the Plasmodium-infected children was 172 months. The prevalence was significantly lower in children under 60 months of age [15.5% (105/676)] than in children over 60 months [40% (102/255)] (p = 2.4 × 10−15) (p < 0.00001). The prevalence was similar in children over 108 months of age (36.5%) and in those between 37 and 108 months . The prevalence was significantly lower in the 13–36 months age group [17.5% (55/315)] than in older age groups (p < 0.05), and significantly lower from 37 to 60 months [27.6% (45/163)] than from 61 to 84 months [40.8% (42/103)] and from 85 to 108 months [43.3% (29/67)] (p = 0.02). Mean parasitemia was significantly lower in children aged 37–60 months  than in those aged 61–84 months , (p = 0.04). Parasitemia was also significantly higher in children aged from 61 to 84 months, and lowest in children over 108 months , (p = 0.01). Finally, we observed a significant difference in mean parasitemia between children aged 85 and 108 months  and those over 108 months , (p = 0.02).The maximum age among the × 10−15) . The preCytb gene. Speciation was based on enzymatic digestion (RFLP) of PCR products. Plasmodium falciparum was characterized by two DNA fragments (640 and 159 bp), and P. malariae by three fragments . Only 162 PCR products could be digested, because of insufficient yield and the low sensitivity of the primer set used. We found that 158 (97.5%) infections were due to P. falciparum, and 4 (2.5%) to P. malariae. No mixed infections and no P. ovale, or P. vivax infections were found.Molecular diagnosis was based on amplification of an 816 bp fragment of the n = 561/729] than among children with malaria , (p = 0.001). The use of window nets  had no preventive effect (p = 0.4), and neither did the use of insecticide house spraying  in children without and with malaria (p = 0.06). Insecticide sprays were used in 28.1% of homes (n = 263/936). The parents/guardians of children with and without malaria were aware of essential information on malaria in, respectively, 51% (n = 105/207) and 71.3% (n = 519/728) of cases (p = 4.8 × 10−8).Finally, we analyzed the patients’ sociodemographic characteristics and use of preventive measures . We founP. falciparum antigens, such as RSP2/RAP2, could be transferred to the surface of uninfected and infected red blood cells, reducing their deformability, inducing their sequestration by the spleen, accelerating both complement-mediated lysis as well as macrophage uptake, leading to anemia [Plasmodium falciparum infection also modulates the cytokine balance, leading to erythrocyte clearance [Elimination or eradication of malaria is a new major challenge in some endemic areas. Herein, we assessed the epidemiology of malaria in febrile children in South-East Gabon (Franceville). More than three-fourth of febrile children reporting to the Franceville Regional Hospital during the study period had an altered global health state, probably linked to a high prevalence of anemia, as indicated by the large proportion of children with conjunctival pallor. Similar results have been obtained in Tanzania . We confo anemia . Plasmodlearance . Howeverlearance , 44.In addition, we confirm that malaria is the second leading cause of fever among children in Franceville, after respiratory tract infections , 17. GasOur findings confirm that malaria mainly affects older febrile children in Gabon . HoweverWe found that children under seven years of age tended to have lower parasite loads. The same result has been reported after the increased coverage of insecticide-treated bed nets and the decrease of malaria transmission in some countries , 43. HowPlasmodium. Surprisingly, the data indicate that malaria prevalence was highest in the short rainy seasons. Indeed, breeding sites in this season are maintained while they are cleaned in long rainy seasons.The low frequency of malaria in children less than 12 months is consistent with maternal immune protection through breast-feeding. The higher prevalence of malaria among febrile children over 5 years is consistent with reports from Rwanda , where tWe confirm the decrease in the malaria burden in Franceville in recent years, despite its stability since 2009 . In contPlasmodium-infected children than in other febrile children. This is consistent with red blood cell lysis and platelet sequestration induced by P. falciparum, as recently reported in India [Hematological values were lower in in India . We confin India . Severe in India .Plasmodium falciparum was responsible for the majority of cases of malaria in this study, as reported elsewhere in Gabon [P. malariae and P. ovale accounted for, respectively, 47.6% and 9.9% of cases [P. falciparum to the detriment of other species during acute malaria. Another possible explanation is the use of different diagnostic methods. Delicat-Loembet et al used 454 sequencing to identify Plasmodium species, while we and other authors used less sensitive methods . Despite the non-detection of P. ovale in our study, this species is occasionally found in Franceville, and we have diagnosed one case of P. ovale infection since the end of this study. The low sensitivity of the PCR can be explained by insufficient yield and the low sensitivity of the primer set used. We confirm that P. vivax does not circulate in humans in Franceville, and neither does P. knowlesi, a primate species that causes some episodes of severe malaria in humans [in Gabon , 31. Thiof cases . This con humans .We found that 66.7% of parents/guardians had a good knowledge of malaria, and confirm that this knowledge is associated with a lower risk of childhood malaria. Surprisingly, the use of window nets and insecticide sprays was not associated with a lower risk of malaria, possibly because few parents/guardians used these two measures.We confirm the stability of the malaria burden in Franceville, Gabon, implying that stronger preventive measures are needed to reduce it further. Studies are also needed to evaluate the malaria burden throughout Gabon.The authors declare no conflict of interest in relation with this paper.


Plasmodium falciparum malaria in the Peruvian Amazon Region.Focal screening and treatment (FSAT) of malaria infections has recently been introduced in Peru to overcome the inherent limitations of passive case detection (PCD) and further decrease the malaria burden. Here, we used a relatively straightforward mathematical model to assess the potential of FSAT as elimination strategy for P. falciparum malaria transmission in a low endemic area of the Peruvian Amazon. The model was then adjusted to simulate intervention scenarios for predicting the long term additional impact of FSAT on P. falciparum malaria prevalence and incidence. Model parameterization was done using data from a cohort study in a rural Amazonian community as well as published transmission parameters from previous studies in similar areas. The effect of FSAT timing and frequency, using either microscopy or a supposed field PCR, was assessed on both predicted incidence and prevalence rates.A baseline model was developed to simulate a scenario with seasonal malaria transmission and the effect of PCD and treatment of symptomatic infections on the P. falciparum incidence and prevalence. The strongest reduction was observed when three consecutive FSAT were implemented at the beginning of the low transmission season, and if malaria diagnosis was done with PCR. Repeated interventions for consecutive years (10 years with microscopy or 5 years with PCR), would allow reaching near to zero incidence and prevalence rates.The intervention model indicated that the addition of FSAT to PCD significantly reduced the predicted P. falciparum elimination levels in low endemic areas of the Amazon Region, yet the progression rates to those levels may vary substantially according to the operational criteria used for the intervention.The addition of FSAT interventions to PCD may enable to reach The online version of this article (doi:10.1186/s13071-015-0868-4) contains supplementary material, which is available to authorized users. P. vivax [Despite several decades of intense control efforts, malaria remains an important public health problem in Peru, especially in the Amazon region where most (85%) of the malaria cases occur, mainly due to P. vivax . IncreasP. vivax . During P. vivax .In contrast to the classical statement that the development of acquired malaria immunity requires long-term exposure and intense transmission, recent studies in endemic countries of South America have reported a large proportion of asymptomatic malaria infections -12 suggeNational malaria surveillance in the Amazon Region relies on passive case detection (PCD) with microscopy . PatientP. falciparum infections are treated with an ACT -mefloquine and artesunate (MQ-AS)-, and P. vivax infections with chloroquine (CQ) and primaquine (PQ) [Active case detection (ACD) has recently been recommended in low transmission and malaria-elimination settings to overcome the inherent limitations of PCD and target the asymptomatic parasite reservoir . Accordiine (PQ) . Howeverine (PQ) ,22, the P. falciparum malaria transmission intensity (MIT) in a low endemic area of the Peruvian Amazon Region. Retrospective data from a cohort study in a rural community of the Peruvian Amazon were used to estimate infection rates and a time-variant seasonal vector parameter, and then to evaluate the model. Other malaria-transmission parameters were obtained from previously published studies in the Amazon region. Eventually, the model was used to predict the potential reduction in malaria transmission following the addition of FSAT to the ongoing PCD, allowing for a simulation of the effect size according to the diagnostic tools used, as well as the timing and frequency of FSAT interventions.Mathematical models of various levels of complexity have been used for understanding malaria transmission dynamics and informing decision-making in malaria control and elimination efforts -26. HereP. falciparum infections , the human feeding rate per mosquito (a), the infectiousness of the mosquito population (V/M), and the susceptibility of an individual after being bitten by an infectious mosquito (b). Individuals progress to the latent state (E), from which they develop gametocytes in the blood and become infectious after a latent period (l). Two consecutive sub-states are distinguished in infected latent individuals (E1 and E2) thus dividing the latent period into two time intervals: between sporozoite inoculation and development of detectable asexual stage parasites in blood (l1), and between asexual parasite infection and development of gametocytes (l2). Then, the number of individuals who move from E1 to E2 at time t is equal to the number of individuals who entered E1 at time t-l1 adjusted by the factor e-l1*h, which incorporates the human mortality rate (h) and the average time interval for developing asexual blood parasites (l1). On the other hand, infected individuals leave sub-state E2 at a rate (1/l2) which is the reciprocal of average duration of the gametocytogenesis process.An extension of the Anderson-and-May’s malaria transmission model  and the y) of symptomatic infections of all newly infected individuals is routinely detected by PCD and treated with an ACT of known effectiveness (ε) within the treated state (T), the remaining fraction (1-y*ε), including asymptomatic individuals and previously treated individuals with parasitological treatment failure, remains infectious in an untreated state (I) until the “spontaneous cure” of the disease  unless they are actively detected and treated. Once recovering from compartments T and I, individuals return to the susceptible state (S). While recovery rate for treated individuals (1/r1) is the reciprocal of average human infectious period (i.e. length of time gametocytes remain in the bloodstream) with effective treatment, recovery for untreated individuals requires they pass through two sub-states before becoming recovered: those who have not yet (I1) and those who have spontaneously cleared the asexual blood stage parasites (I2). Then, the rate of movement of individuals from I1 to I2 (1/r2a), and from I2 to S (1/r2b) are the reciprocal of the average time for the spontaneous clearance of asexual parasites from blood (r2a), and the remaining average infectious period after the clearance of blood asexual parasites (r2b), respectively in untreated individuals.While a constant fraction (a), fraction of infectious individuals in the human population ((T + I)/N), and the mosquito susceptibility to infection after biting an infectious individual (c). Infected mosquitoes remain in a latent state (L) for a specific time period before developing sporozoites and becoming infectious (V). The estimation of surviving mosquitoes by the end of the latent period requires an adjustment by the factor e-g*n which incorporates a constant mosquito mortality rate (g) and the average mosquito latent period (n). The model assumes that mosquitoes remain infectious until they die.Regarding the mosquito population (M), the model includes an initial susceptible state (U) for all mosquitoes, becoming infected at a rate determined by the following three parameters: human feeding rate per mosquito (The main assumptions of this model are the following: i) human and mosquito populations remain constant ; ii) malaria-attributable deaths are not explicitly incorporated ; iii) no individual differences in transmission parameters between subjects; iv) super-infections do not occur ; v) all P. vivax and P. falciparum infections occurring at a ratio 4:1. Previous reports from this area indicated that more than two thirds of P. falciparum infections were asymptomatic with low parasite density [Anopheles darlingi is the major malaria vector with a strong anthropophilic behavior [P. falciparum infections was conducted, sampling each month a new group of individuals living within a 100-m radius of houses where at least one P. falciparum infection was detected during the previous month (index houses), either through the surveys, ACD or the routine PCD at health facilities [P. falciparum infections by microscopy in Ninarumi was measured at 6.5% and 1.1%, respectively in January and August 2004 , 91 (65.4%) of them being symptomatic either at the time of sampling or during the following week. Of the additional 48 infections which remained asymptomatic during the follow-up, 19 were confirmed only by retrospective PCR analysis on blood samples collected on filter paper from individuals during weekly ACDs. The detailed procedures for the PCR analysis are described elsewhere [The model was fine-tuned by using retrospective data from a cohort study carried out between 2003 and 2008 in four rural communities of San Juan district, south of Iquitos city in the Peruvian Amazon ,28,29. Mbehavior ,31. The Considering days as the time unit, the model parameterization was done in several steps, i.e. using first the available cohort data for the estimation of important local parameters, and in a second step, using published data to improve the first step estimates or to set values for the remaining transmission parameters. A systematic literature search was undertaken in different databases  prioritizing published studies on malaria in the Amazon Region in English and Spanish language, especially those from the Iquitos area when available. Additional searches were conducted in Google and through reference tracking. Third, when parameters could not be identified in previous steps, they were estimated by fitting the model to the observed clinical malaria incidence in Ninarumi. Table 2, I1 and I2 to the treated state (T) , the sensitivity of the diagnostic test used for malaria screening (s), and the effectiveness of treatment in confirmed infections (ε).In the scenario with FSAT interventions, the baseline model was improved by allowing movements from sub-states EA. darlingi sporozoite rates in the area [P. falciparum infections recorded for 20 more simulated years. Then, the model was run for an additional year (year 0) after which interventions based on FSAT (intervention scenario) were added to the routine PCD (baseline scenario). A first intervention scenario was modeled with the following assumptions: one FSAT during the peak of the high transmission season (only in February of year 1); FSAT covers 100% of population (∆ = 1) in one day (v = 1); microscopy used in FSAT allows for the detection of 50% of asexual blood-stage infections by P. falciparum (sub-states E2 and I1), and 10% of infections with only sexual stages (gametocytes) in blood (sub-state I2) [P. falciparum infections are treated with a 98% effectiveness (ε = 0.98).After setting initial conditions for human and mosquito compartments based on the average observed prevalence in the 2004 Ninarumi surveys and the reported the area , the bastate I2) ; and allP. falciparum infections. Besides microscopy, a field PCR was also considered as diagnostic test during malaria screening, assuming sensitivities of 95%, 95% and 80% for detecting infections in sub-states E2, I1, and I2, respectively. The predicted cumulative incidence (i.e. cumulative number of new symptomatic and asymptomatic infections) was recorded over one-year and five-year periods from the start of the first FSAT in each intervention scenario, and was compared with the cumulative incidence found with the baseline PCD scenario. The predicted impact of each intervention was determined as the percentage difference between the cumulative number of new P. falciparum infections with baseline and with the respective intervention scenarios (cumulative number of infections averted). In addition, minimum and maximum predicted prevalence were determined during both the first and the fifth year after the onset of FSAT interventions. Finally, operational criteria for implementing FSAT interventions during only one year which had the strongest impact were used again to assess the effect of the same interventions if implemented during several consecutive years.Additional intervention scenarios were modeled to assess the impact of different operational criteria for FSAT implemented during only one year  on the predicted cumulative incidence and prevalence of P. falciparum incidence and prevalence from the cohort study. A Wilcoxon matched-pairs signed-ranks test was used to test differences between observed and predicted monthly incidence, with p < 0.05 indicating statistical significance. Different initial values for the A. darlingi sporozoite rate (V/M) were tested and did not affect the results. Moreover, a one-at-a-time sensitivity analysis was conducted to determine the effect of the following parameters on the baseline model predictions [m), the human feeding rate (a), the fraction of symptomatic infections (y), and the treatment effectiveness (ε). Changes (increase and decrease) of 20% in parameter values were considered in the analysis. Finally, only human parameters  were used later to assess their impact on the predictions of the intervention scenarios with FSAT.Malaria transmission scenarios were simulated in R software version 2.15.3 (function “dede” in deSolve package) allowing to solve delay differential equations to take into account latent periods for infected individuals and mosquitoes before being infectious  in 2004, the baseline model predicted 99 symptomatic and 43 asymptomatic infections . Furthermore, predicted prevalence curves accurately reflected the seasonal dynamics of malaria infections , malaria transmission decreased progressively until almost undetectable levels. In addition, seasonal malaria transmission persisted only when mosquito density (m) and mean expected number of bites on humans per mosquito (a) are higher than 67 and 0.080, respectively. Increasing monthly values of human feeding rate (a) by 20% had by far the strongest effect on the annual incidence rate, with more than three times the baseline number of P. falciparum infections (~225% more infections)  had the highest impact on the predicted incidence of asymptomatic infections (increase of baseline incidence by ~250%)  Figure A. On the) Figure B. Moreov) Figure C and D. P. falciparum incidence after the addition of different FSAT interventions according to timing and number of interventions. Conducting one FSAT intervention with microscopy alone between July and September during only one year would have a larger impact on the P. falciparum incidence than conducting it during any other month, with a reduction of about 20% and 15%, respectively at 1- and 5 years post intervention. However, the magnitude of this effect would be twice as high if PCR is used instead of microscopy, reaching reductions of more than 40% and 30% respectively at 1- and 5 years after intervention. Similarly, the impact on P. falciparum prevalence was the highest when FSAT was added in LTS (in August), instead of in HTS (in February) during only one year  on the P. falciparum malaria incidence for interventions implemented during only one year was observed with three consecutive FSAT starting at the beginning of LTS and separated by time intervals of 7 to 60 days  on the P. falciparum prevalence would be smaller, hence delaying the time to reach zero prevalence levels only by one or two years (6 or 7 years after onset of interventions)  Figure B.Figure P. falciparum malaria in low transmission areas of the Peruvian Amazon. The strongest reduction was observed if three consecutive FSAT were implemented annually at the beginning of low transmission season, and if malaria diagnosis was done with a highly sensitive field PCR.Using a relatively straightforward mathematical model, our analysis showed that targeting the asymptomatic parasite reservoir with FSAT interventions, in addition to routine PCD, would significantly reduce P. falciparum infections which were asymptomatic at the time of detection (either by microscopy or by retrospective PCR) developed malaria symptoms (reported and/or measured fever) after one week of follow-up [Modelling the effects of interventions that target the asymptomatic malaria reservoir requires a good knowledge of the burden of asymptomatic infections and their transmission dynamics. Cross-sectional surveys in the Peruvian Amazon reported that more than two thirds of malaria infections were asymptomatic ,4; howevollow-up .P. falciparum infections in the area, and these seasonal dynamics were well reproduced by the baseline model. In addition, the model predicted not only a 2–3 times higher prevalence of infections during the peak of HTS than during the LTS, but also temporal differences in the proportion of asymptomatic infections, which accounted for about 60% of all prevalent infections during the peak of HTS while they represented almost all infections in the middle of LTS. Moreover, the proportion of prevalent infections detectable by microscopy (“patent infections”) varied also with season, i.e. from almost 50%  in the HTS to 20%  in the LTS. Those predictions suggest that in areas of low and seasonal transmission such as the Peruvian Amazon, non-detectable asymptomatic P. falciparum infections are able to persist during the LTS being potentially infectious to mosquitoes emerging after the start of the rains, as previously described in other areas with markedly seasonal transmission [The cohort study also allowed for an accurate measurement of seasonal variations in the incidence of symptomatic and asymptomatic smission ,37.P. falciparum infections are more likely to be identified and treated during PCD, and the duration of treated infections is considerably shorter than untreated infections; it is anticipated that potential changes in the fraction of symptomatic infections as well as in the effectiveness of case management, may significantly impact the malaria prevalence as shown by the sensitivity analysis. Indeed, simulations showed that a decrease in the fraction of symptomatic infections from 0.65 to 0.5 or a decrease in the treatment effectiveness from 0.98 to 0.8 resulted in similar effects as changes in entomological parameters  which are known as being important determinants for the vectorial capacity and the malaria transmission [P. falciparum parasites and the lack of systems that assure the quality of both the antimalarial drugs and the case management procedures  [Since symptomatic smission . Asymptosmission -12,21, eegimens) ,39.P. falciparum incidence and prevalence in the short term, but also decrease the progression rates of these indicators to the initial levels. Furthermore, the effect of FSAT would be maximal if three successive FSAT at time intervals of 7–60 days, would be implemented at the start of the LTS. Kern et al. found a similar impact on the P. falciparum transmission with a single FSAT with microscopy during the dry season using a seasonally-forced malaria model which incorporated periodically mosquito birth rates through a sinusoidal function and different levels of infectiousness for gametocyte carriers; however, while in areas of simulated low disease burden  < 10 infective bites/person/year) such as the Peruvian Amazon, the effect was sustained for over three years after the intervention, in areas of high endemicity (EIR > 200) the incidence rates of infection returned to their initial level in the subsequent year [In contrast to the current strategy of the National Malaria Control Program consisting of adding FSAT with microscopy to PCD during HTS in prioritized localities of the Peruvian Amazon , our siment year . MoreoveP. falciparum prevalence and incidence and eventually reach elimination levels. Long-term effects of FSAT has been previously described by Griffin et al. using a complex modelling approach  fed with published transmission parameters and local epidemiological data of different endemic areas of Africa [Beside timing and frequency of a given intervention, the other important determinant of impact is the duration (number of years) the intervention needs to be applied to reach sustainable elimination levels. Our model suggested that three successive FSAT with microscopy for ten consecutive years, would result in a sustained declining f Africa . The resf Africa .P. falciparum malaria transmission , but would also enable to sustain the impact despite eventual reductions in the proportion of symptomatic infections, treatment effectiveness and/or coverage of FSAT. Although these results support the importance of detecting and treating sub-microscopic infections at point of care for successful elimination strategies [Plasmodium parasites.As a consequence of increased use of molecular tests for detecting submicroscopic malaria infections in both low and high-malaria transmission settings, as well as of recent studies reporting failures of FSAT to reduce transmission when using microscopy or RDTs , it has rategies ,46, implrategies . Recentlrategies ; these mP. falciparum malaria transmission cycle . Further research is still needed to define to what extend asymptomatic and submicroscopic malaria infections contribute to the infectious reservoir [P. vivax transmission using data of a recent cohort carried in the Peruvian Amazon [Like with other mathematical models for malaria transmission, our predictions should be interpreted taking into account the underlying assumptions. Some assumptions were intended to simplify the complex malaria dynamics , while others attempted to fill the knowledge gap in the eservoir ; this inn Amazon , it willP. falciparum malaria transmission, trying to reach the maximum population coverage (avoiding areas with highly mobile populations). Alternatively to reach the areas of mobile populations, resources could be invested to implement a “smart surveillance system” based on reactive case detection guided by information from the PCD.Our model suggested that FSAT combined to PCD enables to reach elimination levels in an area of low transmission of the Amazon Region, but the progression rates to those levels vary substantially according to the operational criteria used for the intervention  and the sensitivity of the diagnostic test used for malaria screening. Despite its limitations, the modelling approach can contribute to design new field studies in the Peruvian Amazon to assess the effectiveness of FSAT and other strategies to further decrease malaria transmission. Even before easy-to-use molecular tests will be available at the point of care, as well as reliable evidence of the effectiveness of FSAT, the current strategy applied by the NMCP in Peru should be revised in the light of our results in order to be more cost effective. Programmatic FSAT interventions with microscopy (minimum of two successive FSAT) can be conducted at the beginning of the LTS in areas with known high risk of


Background. Malaria is a leading cause of morbidity and mortality worldwide. Rapid and accurate diagnosis of malaria would improve control measures and reduce morbidity and mortality. Objective. The aim of this study was to assess the prevalence of malaria in high risk foci in Egypt and the effectiveness of rapid diagnostic tests in diagnosis and subsequently control of malaria. Methodology. A total number of 600 cases of both sexes with different ages were included in the present study. Cases were included in 2 groups; first group (500 cases) were randomly selected from households in Fayoum Governorate and second group (100 cases) were admitted to Fayoum Fever Hospital with signs suggestive of malaria. Cases were subjected to detailed history taking, clinical examination, microscopic examination of thin and thick blood films, and immunological test to detect plasmodial antigens. Results. A total of 3 positive cases were detected by rapid diagnostic tests (RDTs). Out of these 3 cases, one case was positive for malaria parasite by microscopic examination of blood films. All positive cases in the study had history of travel to malaria endemic areas. Conclusion. RDTs are simple and effective for rapid diagnosis of malaria to help in implication of control measures in different localities. Plasmodium which affects about 3.4 billion people  which allows detection of malaria antigen in blood flowing along a membrane containing specific anti-malaria antibodies and enables differentiation of Plasmodium falciparum infection was indicated by development of pf band in addition to C band  and 29.3% of cases were males and 70.7% were females. The mean ages of cases in group 1 and group 2 were 23.30 ± 17.7 and 25.89 ± 18.7 years, respectively. All cases were residents of Fayoum Governorate in Egypt and 14.8% of them gave history of travel to El Khartoum in Sudan.As part of malaria control program in Egypt, 20.5% of cases received antimalarial drugs. The administered drugs included Chloroquine, Coartem® (a combination of artemether and lumefantrine), and Larum® (mefloquine) in 16.7%, 0.3%, and 0.8% of cases, respectively.Detailed history taking and clinical examination of all cases revealed a variety of clinical manifestations  with ele Plasmodium falciparum in one case  , aiming  P. falciparum [In the present study, a large number of cases presenting with fever or history of fever (64.7%) did not have malaria. Diagnosis based on signs and symptoms has varied sensitivity and specificity depending on clinical features and transmission-associated acquired immunity . In accolciparum . In thislciparum . Plasmodium species in Giemsa-stained thick blood films for screening and thin blood films for species' confirmation remain the gold standard for laboratory diagnosis [ Plasmodium falciparum in one case. The expected sensitivity that can be achieved by examination of the thick blood film procedure is about 50 parasites/μL of blood which is equivalent to 0.001% of RBC infected [Microscopic detection and identification ofiagnosis . In our infected . falciparum malaria from cases of vivax malaria [Rapid diagnostic tests for malaria could improve the targeting of antimalarials to true cases of malaria and to distinguish cases of malaria . Plasmodium falciparum, or parasite specific lactate dehydrogenase produced by all four species [RDTs for malaria are based on the detection of either histidine-rich protein 2, produced only by species . Plasmodium falciparum antigen in 3 cases (2 cases of them were missed by microscopy). The highest sensitivity of any RDT for P. falciparum was 98% and lowest sensitivity was 76% [ P. falciparum infection, tests detecting PfHRP2 showed high and similar sensitivity (96%). One-step malaria pf/pv detected plasmodial antigen in 2 samples that were missed by aldolase based detection tests [In the present study, malaria one-step rapid test pf/pan detected was 76% . For theon tests . Plasmodium falciparum infections missed by microscopy and were detected by RDT probably due to low parasitemia in tested samples or drug intake that clears parasitemia with persistence of antigenemia [In the present study, there were 2igenemia . Anotherigenemia . P. falciparum than different rapid diagnostic tests.In agreement with our results, Leslie et al.  reported P. falciparum diagnosis by RDTs and microscopy found RDTs to be more cost-effective [ P. vivax makes microscopy most cost-effective [Studies comparing the cost offfective . On the ffective  found miffective . Plasmodium falciparum which coincides with high level of Plasmodium falciparum activity in Sudan [In the present study, fever was a constant symptom in malaria positive cases and this was in accordance with the universal screening symptom for malaria in research studies . All posin Sudan .In conclusion, RDTs were found simple and effective for rapid diagnosis of malaria which might enforce the control measures in Egypt against imported malaria that represents a potential risk of reintroduction of malaria in Egypt.


The decreased notification-fatality rate is likely associated with improved use of intravenous artesunate for severe malaria. Plasmodium knowlesi malaria have been linked to delayed parenteral treatment. In Malaysia, early intravenous artesunate is now recommended for all severe malaria cases. We describe P. knowlesi fatalities in Sabah, Malaysia, during 2012–2014 and report species-specific fatality rates based on 2010–2014 case notifications. Sixteen malaria-associated deaths  were reported during 2012–2014. Six patients with severe P. knowlesi malaria received intravenous artesunate at hospital admission. For persons >15 years of age, overall fatality rates during 2010–2014 were 3.4, 4.2, and 1.0 deaths/1,000 P. knowlesi, P. falciparum, and P. vivax notifications, respectively; P. knowlesi–associated fatality rates fell from 9.2 to1.6 deaths/1,000 notifications. No P. knowlesi–associated deaths occurred among children, despite 373 notified cases. Although P. knowlesi malaria incidence is rising, the notification-fatality rate has decreased, likely due to improved use of intravenous artesunate.Deaths from  Plasmodium knowlesi is the most common cause of malaria in East Malaysia, and the incidence of disease is increasing despite intensive control efforts that have substantially reduced the incidence of P. falciparum and P. vivax malaria in Malaysia  with artesunate ; the other 4 received chloroquine or sulfadoxine/pyrimethamine  global guidelines  among notified cases for each species . PCR-confirmed P. malariae infection is rare in Sabah, accounting for <1% of clinical samples diagnosed by microscopy as P. malariae or P. knowlesi ; or P. vivax (1 case). Details of 1 P. knowlesi case (case 2) were previously reported ; 4 were women. Six of these cases had been misdiagnosed by microscopy as P. malariae (4), P. falciparum (1), or P. vivax (1) infections. Severe malaria was recognized in 5 patients when they sought medical care; all received intravenous artesunate within 90 (median 30) minutes of diagnosis. Severity criteria at admission for these patients were jaundice (4 patients), acute kidney injury (3), metabolic acidosis (4), hyperparasitemia (2), respiratory distress (2), and coma (1)  μmol/L; bilirubin, lactate, and bicarbonate results were not available. Within 12 hours, she became hypotensive and tachypneic; chest radiographs showed diffuse opacities in both lung fields. She was intubated and started on intravenous artesunate but died within 23 hours of admission. Subsequent reexamination of her initial blood slide showed 263,772 parasites/μL. The other patient  μmol/L, and her creatinine level was 143 μmol/L. She was treated with 1 dose of intravenous artesunate followed by chloroquine and primaquine. Blood film results the next day indicated P. knowlesi infection with 20,000 parasites/μL. Acute respiratory distress syndrome (ARDS) and metabolic acidosis developed, and the patient died on day 3, despite recommencement of intravenous artesunate. Postmortem reexamination of her initial blood slide showed 55,111 P. knowlesi parasites/μL.Two patients with fatal  case 5,  Table 1 P. knowlesi malaria. He was unresponsive when brought into a health clinic by relatives, who reported a 1-day history of weakness and drowsiness and a 3-day history of fever, chills, arthralgia, and myalgia. At hospital referral, his blood pressure was 85/60 mm Hg, pulse rate 150 beats/min, and oxygen saturation 81% on room air. He had a Glasgow Coma Scale score of 6/15. Pupils were reactive but asymmetric . Meningism was not present, and neurologic examination showed normal tone, symmetrically reduced reflexes, and downgoing plantar reflexes. Blood investigations showed 6,471 P. knowlesi parasites/μL and metabolic acidosis. Computed tomography brain scan and lumbar puncture were not performed. The patient was intubated and begun on intravenous artesunate and ceftriaxone but died 41 hours after admission. Blood cultures for bacterial infections were negative.One patient, a 56-year-old man, was comatose, a condition not previously reported in P. vivax malaria. At admission, he had a 7-day history of fever, rigors, myalgia, nonproductive cough, and abdominal pain. Physical examination results were unremarkable. Thrombocytopenia was present, and his bilirubin level was 21.5 μmol/L; creatinine and hemoglobin levels were normal. A blood film result was reported as 2,090 P. vivax parasites/μL; oral chloroquine and primaquine treatment were begun. The next day, the parasite count was 890 parasites/μL of blood, but the patient became hypotensive, and ARDS developed; a postintubation chest radiograph showed bilateral opacities. Intravenous artesunate and antibiotic drugs were initiated, and hemodialysis was performed for acute kidney injury (creatinine level 316 μmol/L), but the patient died 4 days after admission. Blood cultures for bacterial infections were negative.One person, a 53-year-old man, died from P. falciparum malaria–associated deaths, 2 (29%) occurred in children  and 5 (71%) occurred in adults . At the initial examination, all patients met WHO criteria for severe malaria: jaundice (5 patients), cerebral malaria (4 patients), renal failure (3 patients), respiratory distress (3 patients), and anemia (2 patients). Within 2 hours of malaria diagnosis, 1 patient was given oral artesunate/mefloquine and all others were given intravenous artesunate. All patients were intubated and ventilated, 6 received inotropes, and dialysis was performed on 3. All patients died within 2–9 days of admission. Blood cultures for 1 child and 1 adult were positive for Klebsiella pneumoniae and coagulase-negative staphylococci, respectively; the latter was thought to represent contamination.Of the 7 P. knowlesi malaria was 3.08 deaths/1,000 cases, compared with 4.83 and 0.87 deaths/1,000 cases of P. falciparum and P. vivax malaria, respectively (>15 years of age), notified CFRs were 3.37, 4.17, and 1.02 deaths/1,000 cases for P. knowlesi, P. falciparum, and P. vivax malaria, respectively. Despite 373 notifications of P. knowlesi malaria and 611 notifications of P. vivax malaria among children (persons <15 years of age) during 2010–2014, no deaths from either species were reported. However, children with P. falciparum malaria had a notified CFR of 6.7 deaths/1,000 cases. Notified CFRs among adults with P. knowlesi malaria declined from 9.2 to 1.6 deaths/1,000 notifications in 2010 and 2014, respectively .The overall notified CFR for ectively . Among aP. malariae/P. knowlesi notifications, but they accounted for 6 (46%) of the 13 fatal P. knowlesi malaria cases. Thus, the notified CFR for P. knowlesi malaria was 7.66 deaths/1,000 cases for female patients, compared with 2.04 deaths/1,000 cases for male patients . However, this difference was not significant in a multivariate logistic regression model adjusting for age . For P. falciparum and P. vivax malaria, no difference was seen in the number of notified CFRs for male and female patients.During 2010–2014, female patients accounted for only 783 (19%) of the 4,217 P. knowlesi infections, management of severe malaria in Sabah appears to have improved; all patients recognized to have severe P. knowlesi malaria on admission received intravenous artesunate as initial therapy. Although our findings clearly demonstrate the ability of P. knowlesi to cause fatal malaria despite optimal therapy, P. knowlesi notified CFRs in Sabah have fallen over the past 5 years in association with the increased early use of artesunate documented in this and other reports , which is inconsistent with a diagnosis of P. malariae infection but highly suggestive of P. knowlesi infection. Malaysia’s malaria guidelines recommend that blood films with parasites resembling P. malariae be reported as P. knowlesi/P. malariae . Postmortem reexamination of these patients’ initial blood films showed a parasite count substantially higher than initially reported . Parasitemia has been shown to be a major risk factor for severe P. knowlesi malaria: in a recent prospective study, severity criteria were present in >50% of patients with >20,000 parasites/μL of blood and >80% of patients with >100,000 parasites/μL of blood  of 16 patients treated with intravenous quinine died. In contrast, at the same hospital during 2010–2011, none of the severe P. knowlesi malaria patients treated with early intravenous artesunate died  of patients with fatal P. falciparum malaria than fatal P. knowlesi malaria, and it is possible that a delay in seeking care at a healthcare facility may be a confounding factor in comparing CFRs for malaria caused by these Plasmodium spp.This study had several limitations. First, the retrospective design of the case series resulted in unavoidably incomplete laboratory and clinical data. In particular, alternative diagnoses cannot be excluded in the case of possible P. knowlesi malaria cases in Sabah, the number of fatal cases has not increased. The reduction in notified CFRs may be associated with the increased recognition of the ability of P. knowlesi to cause severe and fatal malaria and improved use of intravenous artesunate for severe malaria caused by any Plasmodium spp, as per recent policy changes (P. knowlesi to cause fatal malarial disease in adults, despite optimal therapy, and that P. knowlesi remains the most common cause of fatal malaria in adults in Sabah. In contrast, the study shows a notable absence of deaths among children with P. knowlesi malaria.In conclusion, our findings show that despite increasing notifications of Plasmodium knowlesi malaria and the fatal P. vivax and microscopy-diagnosed P. malariae cases identified in Sabah, Malaysia, 2010–2014.Detailed descriptions of the 7 fatal cases of