Malaria continues to be a major burden in the endemic regions of Kenya. Health outcomes associated with case management are dependent on the use of appropriate diagnostic methods. Rapid diagnostic tests (RDTs) have provided an important tool to help implement the WHO recommended parasite-based diagnosis in regions where expert microscopy is not available. One of the questions that must be answered when implementing RDTs is whether these tests are useful in a specific endemic region, as well as the most appropriate RDT to use. Data on the sensitivity and specificity of RDT test kits is important information to help guide test selection by national malaria control programmes.This study evaluated the diagnostic performance of RDTs including First Response (FR), CareStart (CS), SD Bioline (SD), and Binax Now (BN). The performance of these malaria kits was compared to microscopy, the gold standard, for the detection of malaria parasites. The malaria RDTs were also compared to PCR which is a more sensitive reference test. Five-hundred participants were included in the study through community screening (50 %) and testing suspected malaria cases referred from health facilities.Of the 500 participants recruited, 33 % were malaria positive by microscopy while 51.2 % were positive by PCR. Compared to microscopy, the sensitivity of eight RDTs to detect malaria parasites was 90.3–94.8 %, the specificity was 73.3–79.3 %, the positive predictive value was 62.2–68.8 %, and the negative predictive value was 94.3–96.8 %. Compared to PCR, the sensitivity of the RDTs to detect malaria parasites was 71.1–75.4 %, the specificity was 80.3–84.4 %, the positive predictive value was 80.3–83.3 %, and the negative predictive value was 73.7–76.1 %. The RDTs had a moderate measure of agreement with both microscopy (>80.1 %) and PCR (>77.6 %) with a κ > 0.6.The performance of the evaluated RDTs using field samples was moderate; hence they can significantly improve the quality of malaria case management in endemic regions in Kenya by ensuring appropriate treatment of malaria positive individuals and avoiding indiscriminate use of anti-malarial drugs for parasite negative patients. Plasmodium falciparum resistance to common anti-malarial drugs, which necessitates the use of more expensive and potentially cost-prohibitive combination therapy  of the RDTs compared to Giemsa microscopy and RT-PCR as the reference methods for Plasmodium-positive cases was 33 % (166/500) by the gold standard Giemsa/microscopy, while 51 % (256/500) were positive by RT-PCR. The eight RDTs detected 44–48 % malaria positive cases and 52–56 % malaria negative cases  were mixed species of P. falciparum and Plasmodium malariae.Evaluation of BinaxNow, SD Bioline, Carestart, and First Response was based on 500 study participants recruited through the Kombewa HDSS via community screening (~50 %) and suspected malaria cases referred from health facilities. The study participants included children <12 years , and the remainder were ranged in age from 13 to 64 years . The male participants were a minority of participants  while the majority of the participants were female . Adult women who were not pregnant comprised 39 % of the female population , one was pregnant, and the remainder were children. The number of P. falciparum (HRP2-band) against microscopy was relatively similar across all the eight malaria RDTs tested. The sensitivity was shown to be >90 % while the specificity was shown to be >73 % when compared to microscopy. The SD Bioline 05FK50 kit had the highest sensitivity of 94.8 % (89.9–97.7) while First Response I16FRC kit had the lowest sensitivity of 90.4 % (84.8–94.4). The specificity of all the eight malaria kits was >73 % when compared to microscopy. The BinaxNOW 665-025 kit had the highest specificity of 79.3 % (74.6–83.6) while SD Bioline 05FK50 kit had the lowest specificity of 73.3 % (68.1–77.9)  for SD Bioline 05FK50 kit to 68.8 % (62.2–74.8) for BinaxNOW kit. The highest negative predictive value was 96.79 % (92.8–98.6) for the SD Bioline 05FK50 kit. The positive likelihood ration range was 3.5–4.4 while the negative likelihood ratio range was 0.07–0.12 (Table The RDTs showed a moderate measure of agreement (>80 %) when compared to microscopy, the gold standard for malaria diagnosis Table . The FDAP. falciparum in patient with high levels of the parasite  <200 P/µL (n = 9), 200–500 P/µL (n = 29), 500–2000 P/µL (n = 42), 2000–5000 P/µL (n = 29), >5000 P/µL n = 38). At <200 P/µL (n = 9), the sensitivity ranged between 72 and 89 % and increased to >93 % at 200–500 P/µL. At very high parasite densities >5000 P/µL, all the RDTs had 100 % sensitivity indicating their ability to correctly detect 8. At <20P. falciparum when compared to RT-PCR was similar across all the kits but quite different from microscopy. The sensitivity ranged from 71.1 % (65.1–76.6 %) for First Response I16FRC to 75.4 % (69.6–80.5 %) for SD Bioline 05FK60 and CareStart G0181. The specificity of all the kits was >80 % when compared to PCR results. The highest specificity was 84.8 % (79.7–89.1 %) for BinaxNOW while lowest specificity was 80.8 % (75.2–85.6 %) for SD Bioline 05FK50. Unlike when compared to microscopy, the positive predictive values for the eight RDTs was >83.3 % while the negative predictive value was low at >73.6 %. The positive predictive values ranged from 80.3 % (74.6–85.2 %) for SD Bioline 05FK50 to 83.3 % (77.7–87.9 %) for BinaxNOW. The negative predictive values ranged from 73.6 % (68.0–78.6 %) for First Response I16FRC to 76.1 % (70.5–81.2 %) for SD Bioline 05FK60. The positive likelihood ratio ranged between 3.9 and 4.7 while the negative likelihood ratio was at 0.3. The RDTs showed a moderate measure of agreement (>77 %) when compared to RT-PCR as a more sensitive reference method. CareStart had the higher agreement value of 78.8 % while First Response had the lower agreement of 77.6 %. All the RDTs had a relative agreement with PCR at κ > 0.6  was evaluated using 500 samples from febrile patients (n = 500). This study reproduces the actual performance these RDTs would be expected to provide under real-world field conditions, complexities and physical stresses. Sensitivities of these kits was shown to be >90 %, specificities were shown to be >73 %, PPV was shown to be >62 %, and NPV was shown to be >94 %, and >80 % agreement with microscopy was shown by these RDT assays. The data from this study are consistent with other published work when RDTs are compared to microscopy. For example, a study in Central Africa Republic showed that SD Bioline antigen (HRP2) had a sensitivity range of 88.1–95.4 %, specificity range of 76.2–87.2 %, PPV of 0.8–89.8, and a NPV of 85.0–94.1 . A studyP. falciparum [Field and laboratory evaluations in India for the First Response malaria antigen HRP2/pan kit showed a sensitivity ranging from 89.5 to 97.7 % and specificity ranging from 63.6 to 75.6 % for detection of lciparum . A studylciparum .P. falciparum, however, stratified P. falciparum sensitivity calculations indicated a sensitivity of 93 % , 89 % , and 54 %  [The sensitivity of RDTs in this study was shown to increase with increase in parasite density. This implies the inability of RDTs to reliably detect malaria parasites at very low parasites densities. Performance of BinaxNow in a large field trial by Gasser et al. showed an overall sensitivity of 95 % for detection of –100/µL) . An eval–100/µL) .The WHO recommends that RDTs should have a sensitivity of >95 % and a specificity of >90 % , and theThe relatively high sensitivity demonstrated in this study decreased the likelihood of RDTs incorrectly identifying a patient as a false negative, hence showing a substantive ability to identify patients who truly had malaria. A cross-sectional study conducted in Blantyre District in Malawi assessed the performance of HRP2 RDTs  and overall, there were 44/633 (7 %) false-negative RDT results compared to microscopy .P. falciparum mono-infection was done after anti-malarial treatments using ICT malaria and optimal-IT assays; 82.1 % of the patients demonstrated persistent antigenaemia for the HRP2 antigen up to 14 days of follow-up [The results showed a lower specificity as compared to WHO-recommended specificity of an effective RDT, and this is probably due to prior infection and subsequent effective treatment. The exclusion criteria included malaria treatment within the previous 2 weeks; some participants may have been treated for malaria; however the HRP-2 detected by these RDTs may have persisted in the blood stream before being completely cleared from the blood. A persistent parasite antigenaemia of HRP-2 has been demonstrated; a study on 240 patients with ollow-up . Others ollow-up , 27. Theollow-up .The disease prevalence in this endemic Lake Victoria region is known to be approximately 37 % . EvaluatThe performance of RDTs compared to PCR was quite different than when compared to microscopy. The sensitivity of RDTs was lower at 71.1–75.4 % when compared to PCR but >90 % when compared to microscopy. The specificity of RDTs in our results is almost similar other studies , 29–31. The opposite was observed for specificity whereby the levels were higher when compared to PCR (80.8–84.8 %) than microscopy (73.3–79.3 %). The specificity of RDTs in our results is almost similar other studies , 29–31. P. falciparum. More evaluations should be carried out around the country, particularly in non-endemic areas with returning migrant workers from endemic areas as well as in epidemic highland areas. After implementation and deployment of RDTs in health facilities, subsequent research should be done on their impact and cost effectiveness in providing reliable diagnosis at the point of care.Rapid diagnostic tests can be applied in a number of settings with a great potential for impact on public health where they can significantly improve the quality of malaria case management in areas where expert microscopy or PCR methods are not available. This study demonstrated that CareStart, SD Bioline, BinaxNow, and First Response RDTs performed reasonably well in this endemic region for the detection of


Circulating endothelial microparticles (EMPs) and progenitor cells (PCs) are biological markers of endothelial function and endogenous repair capacity. The study was aimed to investigate whether COPD patients have an imbalance between EMPs to PCs compared to controls and to evaluate the effect of cigarette smoke on these circulating markers.+CD42b-), apoptotic if they co-expressed Annexin-V+ or activated if they co-expressed CD62E+, circulating PCs (CD34+CD133+CD45+) and the EMPs/PCs ratio between groups.Circulating EMPs and PCs were determined by flow cytometry in 27 nonsmokers, 20 smokers and 61 COPD patients with moderate to severe airflow obstruction. We compared total EMPs (CD31COPD patients presented increased levels of total and apoptotic circulating EMPs, and an increased EMPs/PCs ratio, compared with nonsmokers. Women had less circulating PCs than men through all groups and those with COPD showed lower levels of PCs than both control groups. In smokers, circulating EMPs and PCs did not differ from nonsmokers, being the EMPs/PCs ratio in an intermediate position between COPD and nonsmokers.We conclude that COPD patients present an imbalance between endothelial damage and repair capacity that might explain the frequent concurrence of cardiovascular disorders. Factors related to the disease itself and gender, rather than cigarette smoking, may account for this imbalance. Chronic obstructive pulmonary disease (COPD) is a life-threatening lung disease with systemic impact . The pri1 decline . To. To12]. ytokines  [8,36,37ytokines . Events controls . Samples[+CD42b- , CD31+CDnnexinV+  or CD31+b-CD62E+  micropar6 cells for FMO tubes and at 4x106 cells for sample tubes. Circulating PCs were stained and analyzed by flow cytometry for phenotypic expression of surface markers using pre-conjugated anti-human monoclonal antibodies and isotype controls anti-CD45-FITC , anti-CD34-PECy7 , anti-CD133-PE , anti-IgG1k-PECy7 isotype control , anti-IgG1k-FITC isotype control  and anti-IgG1k-PE isotype control . The fluorescence minus one technique was employed to provide negative controls [The number of circulating progenitor cells was evaluated by flow cytometry using antibodies against CD45 (pan-leukocyte marker), CD133 (sub-population of hematopoietic stem cells) and CD34  as previously described [controls . After 4controls .In the non-adjusted analysis, data are expressed as mean±SD for normally distributed data or as median with interquartile range for skewed distributions. Group comparisons were performed using one or two way ANOVA and post hoc pairwise comparisons using the Student Newman-Keuls test for normally distributed variables or the Dunn’s test for non-normally distributed variables. Correlations between variables were analyzed with Pearson’s or Spearman’s coefficient depending on data distribution.In the statistical adjusted model, as EMP and PC counts were skewed in distribution, values were ln-transformed to improve normality. Linear regression models were used to adjust for potential confounders, which were selected based on biologic plausibility such as age, gender, body mass index (BMI) and Framingham risk score .A p-value <0.05 was considered statistically significant.Anthropometric, clinical and functional characteristics of subjects are shown in In the non-adjusted model, the number of total circulating EMPs was significantly increased in COPD patients and current smokers compared with nonsmokers . In the +CD133+CD45+ PCs showed no significant differences between the different groups  below the median  , and thoe median .In the present study we assessed comprehensively the balance between endothelium injury and repair capacity, which is critical for the maintenance of vascular homeostasis, in patients with COPD. Our results show elevated levels of circulating EMPs, an indicator of endothelial cell damage, and reduced numbers of bone marrow-derived PCs able to maintain and repair the endothelium. The ratio between these two factors was significantly disturbed in COPD suggesting a phenotype of vascular incompetence in those patients ,33. InteIn our study the levels of total and apoptotic circulating EMPs were elevated in COPD patients, in agreement with previous observations –18. SincIn our study, the number of circulating EMPs was not related to indices of COPD severity, suggesting that endothelial damage might be associated with the presence of COPD rather than to its progression. Our results concur with those of Thomashow et al  who showBone marrow-derived circulating PCs are key to endothelial repair . In a reIn our study, we did not find any relationship between the levels of circulating EMPs and PCs, suggesting that both markers may reflect different but complementary physiologic cellular mechanisms of action. However, both markers measured together may characterize a phenotype of vascular competence ,33. Our Some studies have reported that cigarette smoke increases the levels of circulating MPs –15, suggIn our study we did not find any relationship between circulating EMPs and PCs and any of the plasmatic markers assessed. This is consistent with recent studies  and denoThe study has some limitations. Firstly, as we sampled EMPs in peripheral venous blood we cannot be certain that the elevation of EMPs is from pulmonary origin. Secondly, we did not perform CT scans to assess pulmonary emphysema, therefore we were unable to relate the presence of circulating apoptotic EMPs with emphysematous destruction of lung parenchyma. And thirdly, there is no worldwide exclusive procedure to isolate and analyse circulating MPs and PCs from plasma.COPD patients present disturbed vascular competence, as reflected by an imbalance between increased endothelial damage and reduced repair capacity, which might explain the frequent concurrence of cardiovascular disorders. Factors related to the disease itself and gender, rather than the smoking habit, may account for this imbalance.S1 FigA) MPs analysis based on size and fluorescence; B) Sample analyzed by two-color fluorescence histograms as CD31+CD42b- ; C) Sample analyzed by three-color fluorescence histograms as CD31+CD42b-Annexin V+ (apoptotic EMPs) and D) Sample analyzed by three-color fluorescence histograms as CD31+CD42b-CD62E+ (activated EMPs).(EPS)Click here for additional data file.S2 FigA) Peripheral blood mononuclear cells (PBMC) selection based on forward and side; B) Singlet selection with no aggregates; C) Sample analyzed by two-color fluorescence histograms as CD34+CD45+ cells and D) Sample analyzed by three-color fluorescence histograms as CD34+CD45+CD133+ cells.(EPS)Click here for additional data file.S3 Fig+CD42b-Annexin V+ and (B) CD31+CD42b-CD62E+ EMPs expressed per μL of poor platelet plasma (PPP). Number of CD34+CD133+CD45+ labelled cells expressed as percent of lymphomonocytes (C) in women and (D) in men. Number of CD31+CD42b- EMPs/ CD34+CD133+CD45+ PCs expressed as the ln (E) in women and (F) in men. The whiskers extend from the box to the 90th and 10th percentiles. Kruskal-Wallis One Way Analysis of Variance on Ranks.Number of (A) CD31(EPS)Click here for additional data file.S4 Fig+CD42b-Annexin V+ and (B) CD31+CD42b-CD62E+ EMPs expressed per μL of poor platelet plasma (PPP). Number of CD34+CD133+CD45+ labelled cells expressed as percent of lymphomonocytes (C) in women and (D) in men. Number of CD31+CD42b- EMPs/ CD34+CD133+CD45+ PCs expressed as the ln (E) in women and (F) in men. The whiskers extend from the box to the 90th and 10th percentiles. Kruskal-Wallis One Way Analysis of Variance on Ranks.Number of (A) CD31(EPS)Click here for additional data file.S5 FigCorrelation between EMPs and PCs (A) in the whole population, (B) in women and (C) in men. Spearman Rank Order Correlation test. White squares represent nonsmokers, grey triangles represent smokers and black dots represent COPD patients.(EPS)Click here for additional data file.S1 Tableglobal initiative for chronic obstructive lung disease, FEV1: forced expiratory volume in the first second; FVC: forced vital capacity; TLC: total lung capacity; RV: residual volume; DLco: diffusing capacity of the lung for carbon monoxide; PaO2: arterial partial oxygen pressure; PaCO2: arterial partial carbon dioxide pressure; HDL: high-density lipoprotein; LDL: low-density lipoprotein and NA: not applicable. ‡The Framingham risk score can range from -6 to 19, with higher scores indicating greater cardiovascular risk. * p<0.05 compared with men nonsmokers. § p<0.05 compared with men smokers. # p<0.05 compared with women nonsmokers. ¥ p<0.05 compared with women smokers. ⁰ p<0.05 compared with men COPD.Data are shown as mean± SD. COPD: chronic obstructive pulmonary disease; BMI: body mass index, GOLD: (DOC)Click here for additional data file.


Plasmodium known to naturally infect humans occur here, along with 20 species of Anopheles mosquitoes confirmed as carriers of malaria. Two species of plasmodia cause the overwhelming majority and virtually equal shares of malaria infections in Indonesia: Plasmodium falciparum and Plasmodium vivax. The challenge posed by P. vivax is especially steep in Indonesia because chloroquine-resistant strains predominate, along with Chesson-like strains that relapse quickly and multiple times at short intervals in almost all patients. Indonesia's hugely diverse human population carries many variants of glucose-6-phosphate dehydrogenase (G6PD) deficiency, most of them exhibiting severely impaired enzyme activity. Therefore, the patients most likely to benefit from primaquine therapy by preventing aggressive relapse, may also be most likely to suffer harm without G6PD deficiency screening. Indonesia faces the challenge of controlling and eventually eliminating malaria across > 13,500 islands stretching > 5,000 km and an enormous diversity of ecological, ethnographic, and socioeconomic settings, and extensive human migrations. This article describes the occurrence of P. vivax in Indonesia and the obstacles faced in eliminating its transmission.Endemic malaria occurs across much of the vast Indonesian archipelago. All five species of  Plasmodium vivax occurs across most of the Indonesian archipelago.1Endemic transmission of the human parasite P. vivax in Indonesia imposes relatively difficult obstacles to diagnosis, treatment, and control. There are 20 confirmed anopheline mosquito vivax malaria vector species in Indonesia scattered across a wide variety of habitats and conditions optimal for malaria transmission.P. vivax places dormant stages in the liver called hypnozoites—a single infectious anopheline bite may result in five to 15 clinical attacks over 2 years.The biology and ecology of P. vivax. But there is, nonetheless, a serious problem with these options. Only one has been very recently confirmed as permitting safe and efficacious administration of primaquine with it (dihydroartemisinin–piperaquine [DHA-PP]).P. vivax is not simply treating the acute attack, but also simultaneously preventing multiple relapses with primaquine. Demonstrating the safety and efficacy of primaquine with each new potential blood schizonticidal partner in radical cure of vivax malaria is a laborious and expensive undertaking that impedes the availability of validated therapeutic options for radical cure of P. vivax.In most of the world, chloroquine is the front-line therapy against acute vivax malaria; but in Indonesia, resistance to this drug arose, spread, and became dominant against sensitive strains.Perhaps the greatest impediment to the control and elimination of vivax malaria in Indonesia is the problem of primaquine toxicity in glucose-6-phosphate dehydrogenase–deficient (G6PDd) patients. This hugely diverse X-linked disorder is highly prevalent among many of Indonesia's hundreds of ethnic groups. The relative sensitivity to primaquine among any of these is not known, but threatening hemolytic reactions to primaquine therapy are widely known by anecdote in Indonesia.In summary, vivax malaria is a very serious and challenging health problem in Indonesia, and the National Malaria Control Program (NMCP) faces formidable technical, logistical, and financial obstacles in dealing with it. Access to safe primaquine therapy may be the most difficult and important among these. Absent such, the elimination of endemic transmission of this parasite would probably be greatly protracted and costly.Plasmodium falciparum but not P. vivax, and vice versa. If the anopheline carries one species, it is very likely to carry the other as well (data on the other more rare human plasmodia are lacking on this point). Two comprehensive reviews of the anophelines of Indonesia, one by Takken and othersP. vivax prevalence (Anopheles stephensi of India). Highest risk occurs in eastern Indonesia, especially East Nusatenggara (Lesser Sundas archipelago), Maluku, and Papua. Most of Kalimantan and Sumatra also have large areas of transmission, but to a much lower level of endemicity than in most of eastern Indonesia.Elyazar and othersThe dotted line drawn through the middle of map of P. vivax  is uniformly low (< 5%) among age groups and varying little with seasonal rains. Sumba does have rather sharp differences in seasonal rainfall, with a distinct monsoon and dry season. The absence of sharp differences between these seasons is probably a product of hypnozoite activation (in the absence of abundant mosquitoes), as well as sufficient diversity of anopheline species to permit transmission during any season . Transmission of malaria on Sumba, as in much of Indonesia, thus occurs year round with little variance according to wet versus dry season.The distribution of vivax malaria in the human population, where it is endemic, tends to be overwhelmingly relatively low-level transmission. Syafruddin and othersPlasmodium. This is clearly seen in P. falciparum in such settings also carries a very high probability of carriage of P. vivax hypnozoites. Douglas and othersP. falciparum experienced a relapse of P. vivax within just 2 months. A diagnosis of any species of plasmodia anywhere in endemic Indonesia should rationally and reasonably prompt therapy against P. vivax hypnozoites.P. falciparum malaria, principally due to the risk of harm caused by primaquine.This prevalence survey, along with many others from all across Indonesia, indicates an important epidemiological principle with malaria—risk across demographic groups is practically invariable between the two dominant species of Anopheles sundaicus. The young and old are attacked with equal frequency, but the higher burden of parasites in younger age groups  is reflected in the trends in hemoglobin levels reported by Syafruddin and othersPrevalence on Sumba rarely exceeded 10% in even the highest risk groups. Typically, it was well below 5%. Children  tended to have the higher prevalence, quite a bit so compared with adults, that is, 4% versus < 1%. These differences are very probably driven by naturally acquired immunity, where older residents are better able to suppress parasitemia to subpatency .P. falciparum and P. vivax across age groups detected by PCR at the study site in Flores of Kaisar and others15A very important consideration in malaria epidemiology is subpatent parasitemia, that is, those beyond diagnostic reach by standard microscopy. Kaisar and othersThus, endemic malaria in Flores (and nearby Sumba and much of Indonesia) may be appreciated as largely cryptic, that is, not causing illness and staying below the level of detection by standard microscopy. Comparing the microscopic and PCR surveys exemplified here, the true prevalence of malaria, even where historically considered hypoendemic , may well exceed 40% in many settings. Further work and evidence on this key question is urgently needed against a backdrop of strategic planning for elimination of such possibly vast reservoirs of infection, that is, the asymptomatic and subpatent and infectious carriers of malaria who apparently dominate many hypoendemic communities.Excepting a few isolated pockets of stable malaria transmission, the island of Java is largely free of malaria transmission.P. vivax because while those soldiers receive prompt and efficient diagnosis and treatment of acute malaria, they do not typically receive primaquine against hypnozoites for want of G6PDd screening and fear of causing harm. The screening and surveillance of one battalion of 650 men followed for 1 year after such a deployment netted only nine cases of falciparum malaria and 143 cases of vivax malaria.The threat of imported malaria is especially acute on Java, where 150 million of Indonesia's 250 million people reside. Javanese people migrate to the outer islands of Indonesia and frequently return home. Military populations represent one key piece of this much larger issue, as they routinely deploy to some of the most malarious areas of Indonesia and engage in relatively high-risk occupational exposure . This problem is especially acute with P. falciparum, whereas 214 were diagnosed with acute P. vivax; again, most attacks occurred in the 4 months after deployment rather than immediately upon return.In another battalion of 532 men similarly deployed later and followed for clinical trial purposes, 18 soldiers were found to have Importation of malaria by travelers between the Outer Islands of Indonesia, especially those to the east, will continue to threaten success of the elimination of malaria from Java. In contrast, Bali also has very few isolated pockets of hypoendemic malaria (in the sparsely populated northwest of the island), but almost no problem with outbreaks. This may be linked to the relative prosperity of the Balinese and the relative infrequency of travel to other islands in search of economic opportunity compared with their Javanese neighbors.P. vivax consistently dominates as the cause of these outbreaks .Elyazar and othersMalaria importation and outbreaks in receptive areas where malaria has been brought under control or eliminated is not only a serious public health problem, but also a direct threat to sustaining elimination where it has been achieved in Indonesia.P. vivax and P. falciparum. All five of the known plasmodia naturally infecting humans occur in Indonesia, but the other species are not reliably diagnosed and reported through routine surveillance. Nonetheless, reliable surveys or other research efforts provide assurance that these other species indeed represent a small minority of malaria cases in Indonesia (probably < 1%).The NMCP provided data on trends in annual parasite incidence (API) and annual proportions of reported cases represented by the two dominant species, P. vivax and P. falciparum. P. vivax as has been witnessed in many other settings in the wake of successful control programs.P. vivax in Indonesia may be representative of what is generally believed to be the southeast Asian/west Pacific pattern represented by the single strain called Chesson.P. vivax and permitted to relapse also showed Chesson-like relapse behaviors. P. vivax from Papua (and Thailand).Relapse behaviors of P. vivax. Studies in neighboring Papua New Guinea lend substantial support for this hypothesis.22One study of American soldiers infected in the Pacific theater of World War II provides a glimpse at multiplicity of relapses with endemic exposure in the region.The very significant problem of relapse in Indonesia gives substantial weight to the directly linked problem of G6PD deficiency, that is, the phenomenon largely driving poor access to safe primaquine therapy. The great heterogeneity of G6PD deficiency in AsiaSatyagraha and othersP. vivax of that region.A number of clinical trials conducted at Timika, Papua, over the past 10 years provided a wealth of evidence of good efficacy of various ACTs against the chloroquine-resistant blood stages of There is much less assurance regarding the same for primaquine therapy. Only two trials of primaquine safety and efficacy have been reported from Indonesia.P. vivax in Indonesia (see section Drug-resistant P. vivax in Indonesia), prompted the Ministry of Health to recommend an ACT, DHA-PP, as first-line therapy against the acute attack. It also recommends concurrent primaquine therapy (0.25 mg/kg/day × 14 days).A high prevalence of chloroquine-resistant The most recent treatment policy guidelines (2014) recommend antirelapse therapy without mention of G6PD deficiency screening. The guidelines instead offer a warning regarding signs and symptoms of acute hemolytic anemia in patients receiving primaquine (and other drugs), and recommend switching to a weekly dose of 0.75 mg/kg for 8–12 weeks should such signs appear with daily dosing. These policies largely align with those expressed in the new (2015) guidelines for treatment of malaria issued by the World Health Organization (WHO).30P. vivax dominates most of the Indonesian archipelago. The problem seems to have originated in Papua sometime during the 1980s or perhaps earlier. Elyazar and othersP. vivax in Australian travelers to Papua New Guinea in 1989P. vivax.35Resistance to chloroquine by P. vivax since 1952, primaquine is only rarely evaluated for efficacy for its primary therapeutic indication. There is no systematic or standardized means of ascertaining a diagnosis of hypnozoites resistant to standardized doses of primaquine. A number of important confounding factors must be addressed, namely, adherence to the prolonged dosing, quality of drug, reinfection during the long period of risk of relapse, recrudescence by drug-resistant asexual blood stages, pharmacokinetic or pharmacodynamic interference with primaquine efficacy by untried partner blood schizontocides, and possibly polymorphisms in cytochrome-P450 2D6 isozyme.P. vivax acquired in eastern Indonesia.37Resistance to primaquine is a highly complex and difficult question. In Indonesia, as elsewhere, it has yet to be demonstrated. This is not to say sensitivity to primaquine has been examined and proven as the rule. It is a remarkable fact that being the only therapeutic option against relapse of P. vivax–endemic nations, Indonesia faces a therapeutic dilemma with primaquine due to its toxicity in patients with G6PD deficiency. Most providers face patients of unknown G6PD status and, absent such, must choose between potential harm caused by repeated relapses that follow the withholding of primaquine therapy, and potential harm in offering the treatment. The long misunderstanding of P. vivax as benign, and of treating relapse as futile where reinfection occurs, denied this dilemma the attention it required. The NMCP of Indonesia now actively explores options for coping with this dilemma.As in other Historically, the strategy has been to offer the less threatening daily regimen of 0.25 mg/kg with monitoring to mitigate the risks to G6PD-deficient patients. That approach suffers two key pitfalls: 1) probable poor efficacy and 2) poor ability to closely monitor patients where most acquire infection and seek treatment. Acknowledging that many or most variants of G6PD deficiency exhibit severely limited enzyme activity, suggests that strategy as both high risk and low benefit to the patients.As WHO has very recently recommended,P. falciparum or P. vivax malaria during the first trimester of pregnancy elevated risk of spontaneous abortion by a factor of 4, regardless of the species. Plasmodium vivax threatens several million pregnant women, their fetuses, and their newborns every year in Indonesia. This fact, taken together with the lack of access to primaquine therapy against hypnozoites, should raise very serious concerns in the global health community. Indonesia, as many other nations, requires optimized and validated means of preventing relapse without primaquine in these highly vulnerable populations.Pregnant women and infants cannot be offered primaquine, and some experts extend that prohibition to lactating women and children under 4 years of age. WHO recently clarified its recommendation concerning infants less than 6 months of age not to receive primaquine.There are other vulnerable populations, as well, mostly those also lacking access to prompt diagnosis and safe and effective therapy for malaria for reasons of social, economic, or geographic isolation. Primary among these will be patients with G6PDd. The socially isolated may be communities of illegal miners or loggers. The economically isolated may be so impoverished as to be unable to afford even very inexpensive diagnosis and treatment services, or the transport necessary to access them. Likewise, many communities in Indonesia reside at isolated locations where access to care delivery is difficult, time consuming, and relatively expensive. These vulnerable and hard-to-reach populations may suffer chronic vivax malaria and the serious morbidity and mortality risks that come with such isolation. Recently, a young Ministry of Health physician died of vivax malaria at his isolated post in the highlands of Papua because weather prevented his evacuation to hospital by airplane .P. vivax in Indonesia. Reporting systems do not include degrees of illness, but presumably case reporting comes almost entirely from people seeking treatment of febrile illness at government-sponsored care delivery centers. As such, the API  derived from these reporting systems provide some insight on disease burdens. The reported API for 2012 was 1.75/1,000; 256,592 reported cases of malaria. The WHO translated that number  to 5,453,703 cases for Indonesia in 2012.P. falciparum and 150,985 cases confirmed as P. vivax, reflecting the consistency of parity between the two dominant species with respect to both prevalence surveys over the decades .P. vivax attacks, that is, about 18 million cases estimated by WHOP. vivax globally using the Bayesian statistical modeling of prevalent parasitemia.45There are no reliable estimates for morbidity or mortality burdens imposed by  decades , and API decades . The burEven greater uncertainty haunts estimates of mortality due to malaria in Indonesia and elsewhere. In 2011, the NMCP recorded 388 deaths due to malaria and the WHO put the estimated mortality at 8,631 for that year.46P. vivax may evade credible estimation for now, several hospital-based studies carried out in Indonesia since 2005 leave no doubt that death often occurs in association with a diagnosis of P. vivax malaria.P. falciparum were 2.9 times more likely to be classified as seriously ill compared with P. vivax. However, once classified as seriously ill, the odds ratio (OR) of not surviving was equal between the two species . In the very large prospective study at a hospital at Timika, Papua, Tjitra and othersP. vivax and P. falciparum), and also found the odds of death with a diagnosis of P. vivax indistinguishable from a diagnosis of P. falciparum . These trends raise the possibility of P. vivax contributing a very substantial share of the mortality imposed by malaria in Indonesia.Although absolute numbers of fatalities due to Note that 64 deaths attributed to malaria occurred at the hospital at Sumba during 2008 and 2009.Plasmodium vivax seriously challenges health in Indonesia. The long misunderstanding of this species as benign and not threatening drove its neglect by the communities of science, medicine, and public health. The tools needed to combat this threat were not developed, especially a means to safely and effectively attack the tenacious and dangerous hypnozoite reservoir residing in endemic communities. Overcoming the serious problem of primaquine toxicity in G6PD-deficient patients, and thus greatly improving access to this crucial therapy is a top priority in Indonesia.P. vivax circulates its infectious gametocytes even before patients become ill. Health services against malaria must be maintained even after transmission ceases.The NMCP faces challenges in meeting Indonesia's aspirations to eliminate malaria transmission by 2030. A highly mobile population and natural receptivity to malaria transmission across all of Indonesia's islands will demand decades of diligent surveillance and provision of expert diagnosis and treatment services. The dormant and silent hypnozoite is especially threatening in this respect, striking suddenly and unexpectedly months or years after exposure. Further, P. vivax, must be provided alternatives to primaquine to prevent repeated clinical attacks. The same will be true of the many patients diagnosed as G6PD deficient. Chemoprophylactic or presumptive therapeutic regimens will need to be developed, optimized, validated, and implemented for these patients.Patients unable to receive primaquine pose another important challenge. Pregnant women or lactating women and their infants, who are especially vulnerable to The many people living in geographic isolation across Indonesia also pose a special challenge to the NMCP. They need knowledge, tools of personal protection, diagnostics, and safe and effective therapies put into their hands. Eliminating malaria will require reaching well beyond where health-care providers live and work. Delivery of innovative implements against malaria to those isolated from health services would greatly advance the elimination agenda in Indonesia.Plasmodium vivax occurs all across Indonesia as a codominant species with P. falciparum. Both impose substantial burdens of morbidity and mortality across an enormously diverse landscape of anopheline vectors, habitats, and human genetic diversity. Indonesian P. vivax is widely resistant to chloroquine, tolerates lower doses of primaquine therapy, exhibits the aggressive relapse behavior of Chesson-like strains, and may have selected for some of the most severely deficient G6PD variants known. Indonesia's malaria research community already works hand in hand with the NMCP to address these challenges, and progress has and will continue to be made against this very stubborn and difficult parasite.


Plasmodium spp. antibodies and its meaning in blood donation. Therefore, this study aims to assess the longevity of different subclasses of anti-Plasmodium spp. antibodies in individuals with previous stays in endemic areas, as well as to assess how those antibodies are related to personal features and travel characteristics. Based on those results, the suitability of the Portuguese blood donors screening method was addressed, i.e. the method to search for an eventual risk of transfusion–transmitted malaria among the population studied.Cases of transfusion-transmitted malaria have been described around the world and highlighted in some studies. Semi-immune individuals are more likely to transmit malaria as they may be asymptomatic. Some countries allow blood donations only based on epidemiological criteria while others reinforce their criteria with serological tests. However, little is known about the longevity of anti-Plasmodium spp. antibodies and some travel characteristics, namely to be born in endemic area versus non endemic and previous episodes of malaria. The intersection between seropositive results and the last year of stay in endemic areas showed a longer longevity of anti-Plasmodium spp. antibodies than previously reported. Those results represented a considerable portion of the individuals having returned from their last stay in endemic areas more than 10 years before enrolment in this study. Considering the study population as potential blood donors, serological results also indicated that if epidemiological criteria alone were applied to screen blood donors, an important percentage of seropositive individuals would be approved for blood donation. Because the nature and meaning of those antibodies in the blood donation context is still not understood, those approved individuals could represent a risk for blood transfusion safety.Statistical associations were found between the presence of total anti-The place of birth and past episodes of malaria seem to be related to the serological outcome. Epidemiological criteria to screen potential blood donors are insufficient to guarantee the safety of the blood, if applied alone.The online version of this article (doi:10.1186/s12936-016-1586-x) contains supplementary material, which is available to authorized users. Plasmodium infective to humans. Malaria is endemic in tropical and sub-tropical regions, being the sub-Saharan region the most affected, where Plasmodium falciparum is the most prevalent species . The distribution of the levels of total anti-Plasmodium spp. antibodies among the population studied (n = 505) are presented in Fig. The prevalence and level of total anti-Plasmodium spp. antibodies are present in the population studied, we analysed the distribution of the levels of those antibodies according to age group and the length of time since the last stay in endemic areas of malaria   , such as a previous history of malaria   comparatively to ‘Not born in endemic areas’, which means that having been born in an endemic area increases in 132.4% (2.324 − 1×100) the possibility of having antibodies. Notwithstanding, the odds ratio of having a previous history of malaria was 8.872  comparatively to not have a previous history of malaria. In this case, a previous history of malaria increases in 878.2% the probability of an individual be seropositive . Results are summarized in Tables A group of 505 people, ranging from 18 to 65 years old, with previous stays in endemic areas of malaria were included. The majority of the individuals were born in Portugal (a nonendemic area), and thus its immune system was not stimulated against Plasmodium spp. antibodies is not related to the age of subjects, being a confounding variable of the studied population. However, analysing the percentiles of the variable “age” it is possible to notice that there are more seropositive subjects in the two older percentiles . Although it has been described that children under 5 years old are one of the most vulnerable groups to malaria infections [Plasmodium spp. antibodies until adulthood. Mackinstosh and colleagues analysed the blood of 272 children under 10 years old and indicated the presence of anti-Plasmodium falciparum antibodies only in parasitized children, suggesting that those antibodies may be short-lived [To be born in endemic areas seems to be an important feature that may justify the presence of fections , the prert-lived . To be t2 = 68.50, p < 0.001), , although it does not take into account the moment when it occurred. Those results are consonant with a study of Nguyen including blood donors in the USA, in which the authors analysed the risk of transfusion-transmitted malaria between a group of deferred donors and a group of accepted blood donors [Plasmodium spp. antibodies seemed to be also dependent of the length of stay in endemic areas  according to bivariate analysis, the logistic regression model indicated that this is another confounding variable. Regarding other variables studied, as the number of travels to endemic areas and the time passed since the last stay in endemic areas , statistical analysis indicate that serological results are independent of those variables, showing no differences statistically significant.According to the statistical results obtained in this study, the presence of total antibodies is related to a previous history of malaria  should also be assessed in broader and representative populations.New studies should be performed to deeply investigate molecular characteristics and immunological effects of those antibodies detected decades after exposure. Other subjects or travel characteristics should be considered to contribute to the longevity of anti-Plasmodium spp. antibodies circulating in their bloodstream.In the present study, an important percentage of individuals returned from endemic areas more than 10 years before their enrolment was detected with total anti-Plasmodium spp. antibodies over time were identified: (a) had been born in endemic areas and (b) previous history of malaria. On the other hand, living in endemic areas during childhood does not seem to be related to the longevity of total anti-Plasmodium spp. antibodies, as well as the number of travels to endemic areas or the length of time spent in endemic areas, for the population studied. Although the length of time since the last stay in endemic areas was not statistically significant, the presence of total anti-Plasmodium spp. antibodies in the bloodstream of individuals many years after exposure, with no history of malaria in the meantime, is important to highlight.Some factors that may influence the longevity of total anti-In the blood donation context, epidemiological questionnaires should be reinforced with serological tests to guarantee the safety and quality of the blood donated. New studies should be performed to understand the biological nature of those long-lived immunoglobulins, their specific role in the immunity of malaria and their implication in blood transfusion.


Anti-malarial herbal preparations (HPs) continue to enjoy high patronage in Ghana despite reports that the artemisinin-based combination therapy (ACT), the recommended first choice for treatment of uncomplicated malaria in the country, remains efficacious. A major issue with the use of these preparations is inadequate or unreliable data on their efficacy and quality. An assessment of the potency and quality of the most popular commercial anti-malarial HPs in Ghana was, therefore, carried out. The outcome of this investigation is herein discussed preceded by a short literature review of herbal medicines in Ghana.Plasmodium berghei in vivo was assessed using standard methods.Using a questionnaire survey of 344 individuals in parts of Ghana, five of the most frequently used HPs were identified and selected for test of their efficacy and quality. The effect of the selected compounds on P. berghei in vivo. However the degree of parasites inhibition is significantly lower compared to the WHO-recommended artemether–lumefantrine combination . Using the Solomon Saker’s Test, two of the preparations were found to contain chloroquine or compounds with chemical properties like that of chloroquine.All five tested HPs  showed chemo-suppressive activity against Popular anti-malarial HPs used in southern Ghana were found to have chemo-suppressive properties. Intentional addition of chloroquine or SCs to these preparations in order to enhance their effectiveness has serious public health concerns as it may induce cross resistance to amodiaquine, one of the partner drugs in the recommended ACT for use in Ghana. The global fight against malaria is multi-faceted, employing a number of approaches including vector control and effective case management. The use of the ACT as first-line treatment against uncomplicated malaria was recommended by the World Health Organization (WHO) in the mid-2000s and up to date, ACT has been used extensively in most disease endemic countries to manage the disease. In Ghana, artesunate–amodiaquine (AA) is the first choice, with artemether–lumefantrine (AL) and dihydroartemisinin-piperaquine (DHAP) as alternatives. The use of the ACT coupled with the implementation of other interventional measures has resulted in significant reduction in the incidence of malaria in Ghana lately. Despite this gain and reports that the ACT still remains efficacious in the country, commercial anti-malarial herbal preparations continue to enjoy high patronage in Ghana. This situation may be due to the fact that herbal medicine offers a cheaper alternative to allopathic medicines. Worldwide, it is estimated that over 1200 plant species are used for the treatment of malaria and fevers, and herbal preparations are potentially important sources of new anti-malarial treatments [Cryptolepis sanguinolenta, also known as nibima, which has been demonstrated to be clinically efficacious against malaria [Tridax procumbens [Phyllanthus amarus [Theobroma cacao [Haematostaphis barteri [Plumeria alba [The practice of using herbal medicine to treat malaria in Ghana is indigenous and widespread. An ethno-botanical survey of plants used by indigenous households in the Dangbe West District of Ghana revealed that a total of about 30 different species of plants are used by the people to treat malaria . This tr malaria . Herbal ocumbens , Phyllans amarus , Theobroma cacao , Haemato barteri  and Plumria alba .Plants showing anti-malarial activity need to be examined further in order to identify and unearth the potential phytochemicals present that may be responsible for the activity. Pursuing this will make herbal medicine a route for anti-malarial drugs discovery. In spite of the health benefit of herbal products, these preparations could also be a potential death trap. For instance, if the claimed cure by the peddler is not true, the life of the consumer may be at risk as high number of parasites may build up and may results in severe disease condition. Another health risk is when the preparation contains a toxic component. Again, if the HPs have active ingredients whose chemical properties are similar to certain allopathic drugs, then there could be cross resistance to such drugs as a consequence of selection pressure. Adulteration of HPs with allopathic drugs in order to increase their potency has been reported . Such prWhilst the Food and Drugs Authority (FDA) monitors the toxicity of the commercially available HPs, the efficacy of most of these preparations remains unverified. With a public health approach, this study, therefore, explored the potency of some selected HPs in Ghana.The survey was conducted in three Coastal regional capital cities shown in Fig. This is an open gender-unbiased study. Individuals below the age of 18 years at the time of the questionnaire administration were excluded in the survey. Sampling at the street level was done randomly by interviewing every third individual or individuals from every second open shop. At the community level, every second house was chosen and the individuals in it were selected and interviewed. Based on the 2010 population census in Ghana, the sample size was estimated using the formula proposed by Cochran [The herbal preparations mentioned by respondents in the questionnaire survey as their choice for treatment of malaria are shown in Fig. Male ICR mice (25–30 g) were housed in the animal facility of the Department of Biomedical and Forensic Sciences, University of Cape Coast (UCC). The animals were housed in groups of five in stainless steel cages (34 × 47 × 18 cm) with soft wood shavings as bedding. They were fed with normal commercial pellet diet . Water was given ad libitum, under laboratory conditions. The cages were kept in an environment of constant temperature (25–27 °C). All procedures and techniques used in these studies were in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals . All proPlasmodium berghei NK65) used in this study was provided by the Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana. The parasites were initially stored at −70 °C in the laboratories of the Department of Biomedical and Forensic Sciences, University of Cape Coast until used.The rodent parasite , were treated with one of three different doses, 10, 30 and 300 mg/kg p.o. of a chosen HP once per day. A group of five mice also received 4 mg/kg p.o. of artemether/lumefantrine (A–L). The negative control group, made up of also of five mice, was given 10 ml/kg p.o. normal saline. To determine the daily parasitaemia level, a drop of blood was collected from the tail of each mouse for preparation of thick and thin blood films on glass slide. The thin film was fixed with methanol and both films were stained with 10% Giemsa. The stained blood smears were then examined under the light microscope at 100× magnification. Parasitaemia was estimated as usual. The mean survival time of the mice in each treatment group was determined over a period of 28 days.The liquid HPs  were lyophilized with a freeze dryer. The anti-malarial effect of the selected HPs on y et al. . BrieflyThe Solomon Saker’s test  was perfp < 0.05 was considered statistically significant.GraphPad Prism for Windows version 4.03  was used for all statistical analyses. All data were expressed as mean ± SEM (duplicate measurement). The time-course curves were subjected to two-way (treatment × time) repeated measures analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. From the questionnaire survey, a total of 31 different herbal medicines were mentioned to have been used. The names of the herbal preparations mentioned as used by the respondents to treat malaria are shown in Fig. P. berghei. However, almost all the test compounds were observed to be able to reduce parasitaemia significantly (p < 0.05) and consistently throughout the entire test period . This is indicative of the presence of high level of chloroquine or compounds with chemical properties similar to chloroquine in the two products.P. falciparum infections in Ghana. Whether these herbal medicines are able to provide the claimed cure or protection against the disease remains largely unknown. This study therefore ascertained the efficacies of five of the most popular herbal preparations in Ghana and the implication of the findings on malaria control in Ghana is herein discussed.For various reasons, a number of Ghanaians prefer herbal medicine over the ACT for the treatment of malaria, although available evidence indicates that the latter are still potent against P. berghei in vivo. Notably, HP-E was able to protect the mice against the parasites for up to 28 day post-infection making it worthy to receive attention for further future development. None of the test HPs could completely bring down parasitaemia in the first 2 days post-treatment. This could probably be due to low bioavailability of the HP in the mice or insufficient dosage among others.Medicinal plants have been used in the treatment and prevention of malaria in various parts of the world and if found to have excellent anti-malarial properties, a herbal preparation stands a chance of becoming a candidate for further development on a similar scale as that of the quinine or artemisinin. The entire HPs investigated in this study exhibited chemo-suppression against Since the HPs are chemo-suppressive, their inability to completely clear parasites should be of concern to the malaria control program as it poses a potential danger to the health of the users due to anticipated persistency or delayed parasite clearance. Another danger is the likelihood of cross resistance if the HP shares similar chemical component with any of the recommended first line anti-malarial drugs, especially if the herbal medicine is not administered in the correct dosage. These observations made in this study are important as herbal medicine plays a significant role in the management of malaria in Ghana. Reports indicate that about 9% of the Ghanaian population use HPs for malaria treatment . With thP. berghei developed resistance to quinine administered in doses near the maximum amounts tolerated by mice [Solomon-Saker’s test indicated what could be the presence of chloroquine in the herbal preparation. This could be a case of intentional adulterations of the preparations in order to boost its efficacy. It is possible then, that chloroquine or compounds with chemical properties similar to chloroquine was the active ingredient responsible for the chemo-suppression exhibited by HP-B and HP-C. However the Solomon-Saker’s test has been shown to give false positive results for the anti-malarial drugs, quinine and proguanil . It is,  by mice . This in by mice . Elsewhe by mice .Aside the possibility of adulterations with allopathic drugs or their analogs, the fact that these preparations were unable to completely clear parasites should be of public health concern as this could lead to the development of severe form of the disease. It is however assuring that the HPs showed some degree of anti-malarial activity. There is the need therefore to identify the active components in these HPs for further investigation and possible development to regulated drugs.P. berghei in vivo, its comparison or extrapolation to field situation in Ghana, which is dominated by P. falciparum infections, is limited by differences in both the rodent host and the strain of malaria parasite. Nonetheless, results of this study should stimulate further investigations of Ghanaian HPs using other methods, such as the in vitro susceptibility test of Plasmodium falciparum to these preparations.It must however be emphasized that since this study was performed using It is however interesting to note that in the course of preparation of this manuscript, Amoah et al. published the outcome of a similar study conducted in Ghana which may likely include some of the HPs assessed in this study . Though Put together, both studies have demonstrated that most of the HPs in Ghana possess a significant anti-malarial activity which justifies their use for the treatment of ‘fever’. This should open the way for a systematic interrogation of the chemical components of HPs present in Ghana in order to identify their active anti-malarial ingredients for isolation and further development.P. berghei in vivo. Two of the tested HPs have possibly been adulterated with chloroquine or compounds with chemical properties similar to chloroquine. Intentional adulteration of HPs with standard anti-malarial drugs should be of concern to stake holders in the health sector.All five tested HPs showed various degree of chemo-suppressive activity against


The malaria burden in sub-Saharan Africa (SSA) has fallen substantially. Nevertheless, malaria remains a serious health concern, and Uganda ranks third in SSA in total malaria burden. Epidemiological studies of adult malaria in Uganda are scarce and little is known about rates of malaria in non-pregnant adult women. This pilot study assessed malaria prevalence among adult women from Wakiso district, historically a highly malaria endemic region.Adult women using public health services were screened for malaria, HIV and pregnancy. A physician-selected subset of women presenting to the Outpatient Department of Entebbe General Hospital (EGH) with current fever (axillary temperature ≥37.5 °C) or self-reporting fever during the previous 24 h, and a positive thick smear for malaria in the EGH laboratory were enrolled (n = 86). Women who self-identified as pregnant or HIV-positive were excluded from screening. Malaria infection was then assessed using HRP2/pLDH rapid diagnostic tests (RDTs) in all participants. Repeat microscopy and PCR were performed at a research laboratory for a subset of participants. In addition, 104 women without a history of fever were assessed for asymptomatic parasitaemia using RDT, and a subset of these women screened for parasitaemia using microscopy (40 women) and PCR (40 women).Plasmodium falciparum infection by research microscopy and PCR. Subset analysis of hospital diagnosed RDT-negative participants detected one sub-microscopic infection with Plasmodium ovale. Compared to RDT, sensitivity, specificity and PPV of hospital microscopy were 100% (CI 19.8–100), 0% (CI 0–5.32) and 2.33% (CI 0.403–8.94) respectively. Compared to PCR, sensitivity, specificity and PPV of hospital microscopy were 100% (CI 31.0–100), 0% (CI 0–34.5) and 23.1% (CI 6.16–54.0), respectively. No malaria was detected among asymptomatic women using RDT, research microscopy or PCR.Of 86 women diagnosed with malaria by EGH, only two (2.3%) had malaria confirmed using RDT, subsequently identified as a Malaria prevalence among adult women appears to be low in Wakiso, but is masked by high rates of malaria overdiagnosis. More accurate malaria testing is urgently needed in public hospitals in this region to identify true causes of febrile illness and reduce unnecessary provision of anti-malarial therapy. Although great progress has been made over the past one and a half decades in reducing the burden of malaria , the disSub-Saharan Africa health facilities tend to over-diagnose malaria in patients presenting with symptoms such as fever, due to traditional perceptions  and issues related to laboratory testing –14. The The objective of this pilot study was to assess malaria prevalence among adult women with and without a history of fever, who were accessing public health services in Entebbe, Wakiso district, Uganda. Historically, Entebbe has been classified as a malaria hyper-endemic area , 21 withPlasmodium falciparum, but infection by Plasmodium malariae and Plasmodium ovale has also been observed [Entebbe is located in Central Uganda Fig. . This reobserved . EntebbeAs a precursor to studies on the immune impact of malaria infection, women aged 18-45 years were screened for malaria, HIV and pregnancy between July-December 2015  and/or quinine according to the Uganda clinical guidelines. HIV rapid testing was conducted using the Uganda Ministry of Health testing algorithm . PregnanP. falciparum or mixed Plasmodium species infection. The First Response Malaria Ag pLDH/HRP2 , validated by previous studies from Uganda, India and Yemen to have high performance characteristics [Malaria infection was then assessed using RDT in all participants. At all sites, two RDTs meeting the WHO performance criteria  were emperistics , 27, 28,Confirmatory thick and thin smear LM analysis was performed for a subset of participants (n = 16) at the UVRI-IAVI HIV Vaccine Programme’s research laboratory as follows. Blood was transported to the research laboratory, where two blinded microscopists prepared thick and thin blood films using Field stain and separately performed LM. A blood film was considered negative if no parasites were detected after 100 high power fields had been examined. If parasites were observed, counting was performed against 200 white blood cells on the thick film. The thin film was used for malaria species identification and known positive/negative samples were used as test controls. Discrepant LM results were resolved by the UVRI Medical Research Council Clinical Diagnostics Lab Services (MRC/UVRI CDLS). The UVRI-IAVI research microscopists receive extended practical training in malaria parasitology and LM at MRC/UVRI CDLS and participate in quarterly inter-laboratory comparisons with the MRC/UVRI CDLS experts. In the last three years, the UVRI-IAVI research laboratory has also participated with satisfactory performance in the Royal College of Pathologists of Australasia (RCPA)-Malaria External Quality Assurance Programme.Plasmodium spp.-specific PCR was then performed at the University of Toronto, as described elsewhere [For PCR investigation, dried blood spots (DBS) were prepared by blotting blood onto Whatman FTA cards  kept at room temperature until DNA extraction. Nested lsewhere  on thirtTo assess malaria prevalence among asymptomatic individuals, women without a history of fever (n = 104) who were attending the family planning or child vaccination clinics at either the Mother and Child Health (MCH) department of EGH or the Uganda Virus Research Institute (UVRI) outpatient clinic were enrolled for malaria screening using RDT. In addition, a randomly chosen set of 40 women underwent thick and thin smear LM at UVRI-IAVI and another set of 40 participants was tested using nested PCR.P. falciparum positive. Out of fourteen participants that had been initially scored positive by the EGH laboratory, none were scored positive by research LM. In a subset of eleven RDT-negative participants that were also scored negative by research LM, one sample was positive for P. ovale by PCR  nor in a smaller sample subset, by research LM (88%) or PCR (77%). Overall, hospital LM had high sensitivity (100%), but very low specificity and positive predictive values compared to RDT, research LM and PCR Table . This reImportantly, a very high false-positive rate of malaria diagnosis would be expected to lead to substantial malaria overtreatment, with the ensuing possibility of adverse drug effects, as well as under-management of other potentially important clinical causes of fever in the OPD. In agreement with the latter, SSA clinical studies found a higher case fatality rate among hospital patients misdiagnosed with malaria, compared to true malaria cases . In addiRecent data regarding adult malaria prevalence rates in SSA and Uganda are limited , 4, as mIn the most recent survey from three sub-counties  LM-based rates among adults peaked at 5.0%. The survey also uncovered six to tenfold higher rates of parasitaemia using a combination of LM and molecular techniques, highlighting high levels of previously unappreciated sub-microscopic parasitaemia . In thisP. ovale DNA in a sample scored negative by both research LM and RDT. This highlights an important limitation of RDTs- their low sensitivity for detection of P. ovale and P. malariae infections [P. ovale or P. malariae infections that were identified by the hospital microscopists but were not flagged by the RDT. Interestingly, the P. ovale PCR-positive individual was scored negative by research LM, implying that the patient had sub-microscopic parasitaemia. Sub-microscopic malaria is often not associated with clinical symptoms [The RDT used in this study had high sensitivity and specificity compared to research LM, but lower sensitivity compared to PCR Table , which dfections , which afections . It is psymptoms , 38, andP. falciparum ensured detection of a majority of symptomatic malaria cases, however a minority of cases consisting of P. ovale and P. malariae would have been missed by the test. RDTs also tend to be less reliable at low parasite density settings, where PCR testing is recommended to confirm RDT performance [The findings presented here have limitations. First, this study was designed as a pilot to explore the prevalence of malaria among febrile outpatients and was intended neither to be a true malaria survey of the population nor a rigorous comparison of different malaria diagnostics. Rather, the study relied primarily on a previously validated RDT for malaria screening. The RDT’s validated high sensitivity for formance . Howeverformance . Data weformance , recruitIn summary, a lower than anticipated prevalence of malaria was detected among adult women in Entebbe, Wakiso district and high rates of malaria overdiagnosis by LM were observed in a local public health facility. This confirms previously described difficulties in the LM-based diagnosis of malaria, and emphasizes the importance of regular quality control and RDT use to avoid misdiagnosis and mistreatment of fever. These findings may not be uncommon in other regions of Uganda and SSA, where successful reductions in malaria transmission may be masked by sub-optimal diagnostic practices leading to over-inflation of perceived malaria burden. In many regions, malaria in pregnant women and children remains a major public health problem and more efficient allocation of resources for treatment and eradication of this infection may be urgently needed. This pilot study provides background for future more detailed investigations of malaria dynamics in Wakiso.


Background. Malaria is known to have devastating effects on mortality in tropical and subtropical regions with the effect being magnified in people with weakened immunity such as those in pregnancy. We assessed the effect of malaria on renal function of pregnant women receiving antenatal care in a mesoendemic area of Ghana. Methodology. A case-control study that enrolled a total of 100 pregnant women . Sociodemographic characteristics, obstetric history (obtained with a questionnaire), urea, creatinine, sodium, and potassium were analyzed using a chemistry automated analyzer. Results. Plasma urea and creatinine were significantly increased  among cases compared to the controls. Also the levels of urea (P = 0.033), creatinine (P = 0.032), and parasitaemia (0.016) were significantly increased with increasing gestational age. Conclusion. Malaria has a significant impact on renal function  and is also significantly associated with increasing gestational age among our study participants. Malaria during pregnancy is a major public health problem in tropical and subtropical regions with 30.3 million African women becoming pregnant in malaria endemic areas, and, in spite of this, only a fraction of these women have access to effective interventions , 2. It iPregnancy and malaria mutually tend to present aggravating conditions. The physiological and the pathological changes due to malaria have a synergistic effect on the patient which makes life difficult for the mother, child, and the treating physician . MalariaOne of the dreaded causes of foetal mortality and morbidity is malaria in pregnancy. Anaemia, hypoglycemia, and other complications associated with malaria can adversely affect the foetus. Interestingly, malaria caused by falciparum has been shown to cause problems leading to foetal mortality up to 15% , 8. SponA contemporary study to understand the relationship between malaria and renal failure found a significant increase in serum creatinine with 67.14% of the patients recording serum creatinine level above normal. The researchers also reported that 52.86% of their participants had their serum urea level above the normal range which the World Health Organization describes as an indication of renal failure .Even where antenatal screening for malaria is a national policy, the WHO criteria acknowledge that some biochemical and haematological features should raise suspicion of severe malaria and renal screening for malaria in pregnant women should sporadically be implemented at best .Few estimates exist as to how well this problem is being addressed in areas of the world where screening and treatment could have an enormous impact . MeanwhiThis hospital-based case-control study was carried out between October, 2015, and April, 2016, at the Koforidua Polyclinic. A total of 100 patients  were recruited for this study. The flow chart for sample selection and inclusiveness is shown by Pregnant women with singleton pregnancies receiving antenatal care at the study center were eligible to participate in this study.Exclusion criteria were participants with preexisting renal diseases, chronic kidney disease, hypertension and diabetes mellitus, human immunodeficiency virus, and acquired immune deficiency.Ethical clearance for the study was obtained from the University of Cape Coast Institutional Review Board (UCC/IRB) and from the authorities of Koforidua Polyclinic. Consent was sought from participants having explained to them the purpose of the research and its relevance. Participants were made to willingly opt out anytime they felt uncomfortable or had a change of mind.With the aid of a questionnaire a resident or an intern nurse obtained sociodemographic characteristics and obstetric history  of consented participants.μL of blood), moderate (1000–9999 parasites/μL of blood), and severe  [Five (5) milliliters of venous blood was taken from each patient for the blood film and the biochemistry assay. Blood smears were prepared and stained for the examination of malarial parasites using appropriate techniques . The thif blood) . Biochemf blood)  and Jafff blood) , respectP value, and one-way ANOVA were used to assess the statistical significance. Statistical significance was decided when P < 0.05.Data collected were coded, entered into a computer, and cleaned. The statistical package GraphPad Prism-6 was used to analyze the data. Results were presented in tables using means and percentages. Chi-square, P = 0.002), urea (P = 0.0003), and creatinine (P < 0.0001) were associated with malaria infection. However, age, gravidity, parity, sodium, and potassium showed no significant association with malaria infection.P < 0.05 and P < 0.0001, resp.). However, the electrolytes showed no significant differences.There was a significant difference in levels of urea and creatinine and the degree of parasitaemia when stratified according to the gestational age . HoweverThis study investigated the effect of malaria infection on renal function in pregnant women in New Juaben Municipality, a mesoendemic area of seasonal malaria transmission . Our finIn contrast to other studies , 19, 20, Plasmodium falciparum infection with clinically significant renal and renal related biochemical disorders has been previously established [Reports on the levels of urea and creatinine among malaria patients are varied. Whereas others  reveal dablished . These dablished , 27.Usually in normal pregnancy, there is a fall in plasma urea and creatinine concentration due to several factors, including the dilutional effect of an expanding plasma volume, decreased production , and increased renal excretion as a consequence of a pregnancy-induced increase in the glomerular filtration rate . DespiteP > 0.05) in the levels of serum electrolytes of the malarious subjects compared with the control subjects. The study also showed no significant correlation between the intensity of infection and the electrolyte levels. This finding contradicts the findings of [There was no significant difference , with cases of increasing gestational age having a higher risk of the consequences of malaria. We recommend that pregnant women who report to hospitals with malaria should also be examined for kidney function. We further recommend that a larger sample size should be considered in future studies.


Malaria rapid diagnostic tests (RDTs) enable point-of-care testing to be nearly as sensitive and specific as reference microscopy. The Senegal National Malaria Control Programme introduced RDTs in 2007, along with a case management algorithm for uncomplicated febrile illness, in which the first step stipulates that if a febrile patient of any age has symptoms indicative of febrile illness other than malaria , they would not be tested for malaria, but treated for the apparent illness and receive an RDT for malaria only if they returned in 48 h without improvement.Plasmodium falciparum infection identifiable by RDT. Health post personnel enrolled patients of all ages with fever (≥37.5 °C) or history of fever in the previous 2 days. After clinical assessment, a nurse staffing the health post determined whether a patient should receive an RDT according to the diagnostic algorithm, but performed an RDT for all enrolled patients.A year-long study in 16 health posts was conducted to determine the algorithm’s capacity to identify patients with Over 1 year, 6039 patients were enrolled and 58% (3483) were determined to require an RDT according to the algorithm. Overall, 23% (1373/6039) had a positive RDT, 34% (1130/3376) during rainy season and 9% (243/2661) during dry season. The first step of the algorithm identified only 78% of patients with a positive RDT, varying by transmission season , malaria transmission zone , and age group .In all but the lowest malaria transmission zone, use of the algorithm excludes an unacceptably large proportion of patients with malaria from receiving an RDT at their first visit, denying them timely diagnosis and treatment. While the algorithm was adopted within a context of malaria control and scarce resources, with the goal of treating patients with symptomatic malaria, Senegal has now adopted a policy of universal diagnosis of patients with fever or history of fever. In addition, in the current context of malaria elimination, the paradigm of case management needs to shift towards the identification and treatment of all patients with malaria infection. Senegal is a malaria-endemic country in the Sahel zone on the west coast of Africa. Malaria transmission is highly variable across the country, with very low transmission in the north, increasing towards the south. Dakar, the capital city, experiences heterogeneous transmission. Transmission is highly seasonal, with the majority of cases occurring during and just after the rainy season, which generally lasts from July through October. The Senegal National Malaria Control Programme (NMCP) has rapidly scaled up malaria control interventions during the last decade. Artemisinin-based combination therapy (ACT) was introduced in 2006 , first aAfter lengthy consultations with stakeholders, including infectious disease physicians, regional and district medical officers, other Ministry of Health officials, and financial and technical partners, a symptom-based case management algorithm was introduced, along with RDTs, which changed the definition of a suspected malaria case. In 2007, Senegal had just entered the phase of rapid scale-up of malaria control interventions, and the number of cases of fever exceeded available funding needed to test all fever cases. In view of conserving RDTs and testing those most likely to have malaria, the first step of the case management algorithm directed that not all patients with uncomplicated febrile illness or history of fever should receive an RDT. While those without an obvious fever source other than malaria were recommended to receive an RDT, those that also had symptoms or signs indicative of another source of fever  were recommended to be treated for that indication and return for a follow-up visit 48 h later, at which point if there was no clinical improvement, an RDT was recommended. Patients with positive RDTs were to receive treatment with an ACT Fig. . At commAs the nation gained experience with the algorithm, concerns grew that the algorithm might miss an unacceptable number of patients with parasitaemia accompanying other symptoms, with dangerous consequences, particularly for children under 5 years old. Health workers in higher transmission zones complained that if they followed the algorithm, they missed patients with malaria. The proportion of patients with fever associated with signs suggestive of other diseases, but also suffering from malaria, was unknown.Attempts to quantify the malaria-attributable fraction, the proportion of fevers among people with malaria infection that are actually due to malaria, have been made by a number of authors, using both cross-sectional household surveys and health facility-based patient enrolment, both to determine malaria-attributable fraction for a population through modelling and to determine age and season-based cut-offs, though this was found to vary greatly by age, season and transmission intensity –9. MalarThis study was designed to evaluate the first step of the case management algorithm, which recommends testing for malaria if no signs or symptoms of an alternative fever source are present, as a screening test, considering the RDT as the definitive test, to determine the sensitivity, specificity, positive predictive value, and negative predictive value of limiting malaria RDT testing based on the first step of this algorithm, compared to universal RDT testing for febrile patients throughout the malaria transmission zones of Senegal and throughout the year to guide case management policy. These values were to be determined for patients under 5 years old and 5 years and above, during rainy and dry season, and in each of four malaria transmission zones.A longitudinal health facility-based study was conducted at 16 health posts in eight health districts, with two health districts in each of the four malaria transmission zones considered by the NMCP: north ; centre ; south ; and, the capital city of Dakar (heterogeneous) Fig. . Health In order to have an adequate sample size to analyse results by age group (<5 and ≥5 years), season (rainy and dry), and transmission zone , with a 95% confidence interval, with a margin of 5%, at least 369 patients were required to be enrolled at each site. The sample size was set at 6144, with each of the 16 health posts expected to enrol 32 patients monthly for 12 months (384 per site).Health post staff were trained on study methods and conducted the enrolment. During a full year (April 2013–March 2014), health post staff recruited patients 4 days per week, enrolling the first patient of age at least 2 months  but younger than 5 years and the first patient 5 years or older each day of enrolment who presented with fever (axillary temperature of ≥37.5 °C) or history of fever in the previous 48 h, for their first visit to the health facility for that illness. If a febrile patient of either age group did not present on a given day of enrolment, catch-up was allowed on other days of the week. Pregnant women and patients with signs of severe disease were excluded, as they are excluded from the algorithm.All recruited patients gave informed consent, or consent of caregiver in the case of minors. Health post staff then completed a questionnaire documenting region, district, health facility, age, gender, duration of symptoms, previous care sought, temperature, symptoms, and physical examination. Based on these, they determined whether testing with an RDT would be recommended by the algorithm, and documented their determination. Following this, all enrolled patients were tested with RDT , with results and treatment documented. During the first 9 months of the study, as an RDT quality control measure for patients enrolled on 2 of the 4 days each week, a thick blood film was also performed for quality control. All patients were treated according to the result of the RDT with an ACT for positive tests as directed by national policy, with ancillary therapy as indicated. All patients were given an appointment for follow-up in 48 h.Every month, a team composed of a research assistant and a laboratory technician visited every health post, reviewed study materials, collected questionnaires and blood films, did any needed trouble shooting, and supervised and corrected blood film technique. Blood films were dried and labelled with pre-printed ID stickers on site, and were stained in 10% Giemsa for 15 min and read in Dakar by expert malaria microscopists in the national reference laboratory (University Cheikh Anta Diop Parasitology Department). Two parasitologists read the films in a blinded manner, with a third reading in case of discrepancy between the results of the two readings. In case of discrepancy, the third microscopist’s reading was used. There were discrepant readings on five slides. Asexual malaria parasites were enumerated against 200–500 white blood cells (WBC), transformed to parasites/µL using the assumption of 8000 WBC/µL; 250 fields were read before declaring any film negative.Questionnaires were entered into an Epi-Info  database by double entry. Every quarter, the research team met to review the data and address any data quality issues. Data were analysed using Epi Info.The sample was restricted to patients enrolled from 1 April, 2013 to 31 March, 2014. During some months, health post personnel, trying to make up for months during which they had not enrolled the desired sample size per month, enrolled a large number of patients. Up to double the month’s quota of 32 were accepted. For any month during which a site enrolled more than 64 patients, enrollees were randomly selected from the highest enrolment days for removal. This exercise was performed for three health posts: two during July and one during August.Rainy season was defined as July–December and dry season was defined as January–June. Fever was defined as an axillary temperature of ≥37.5 °C, and history of fever was defined as a complaint of a hot body in the previous 48 h. In order to determine if health providers had implemented the algorithm and correctly assigned patients to the groups to be tested, two senior members of the research team reviewed the symptoms recorded for each patient and determined whether the algorithm recommended testing. Concordance between health post workers and expert review was assessed. Sensitivity, specificity, positive predictive value, and negative predictive value of the algorithm as a screening test were calculated, compared to RDT. This calculation was done across age groups, transmission zones and rainy/dry season to determine for which age groups, in which transmission zones, and for which seasons the algorithm demonstrated sufficient sensitivity to diagnose an adequate proportion of febrile patients with parasitaemia.Comité National d’Ethique pour la Recherche Santé #0109) and by the Centers for Disease Control and Prevention Human Subjects Board, where it received a non-research determination. Participation was strictly voluntary. Patients who refused consent received the same evaluation and treatment as they would have if they had participated, other than an RDT for patients that would not have met criteria for RDT according to the algorithm.The protocol was reviewed and approved by the Senegal National Ethical Committee  were children under five, reflective of the instructions given, and 48.4% were male (95% CI 47.2–49.7). Of the 5988 for whom data were recorded, 32.2% (95% CI 31.0–33.4) sought initial care elsewhere prior to this consultation. Of 6035 for whom temperature was recorded, 83.1% (95% CI 82.1–84.0) were febrile at consultation, with a median duration of symptoms of 2.4 days at the time of consultation. The breakdown by quarter and malaria risk zone is found in Table Overall, 57.7% (95% CI 56.4–58.9) (3483/6039) were determined eligible to receive an RDT according to the algorithm Fig. , which vRegardless of whether they were eligible for an RDT or not according to the algorithm, all enrolled patients were tested for malaria with an RDT; results were available for 6038/6039. Overall, 22.7% (95% CI 21.7–23.8) were positive for malaria. RDT positivity rate was 11.7% (95% CI 10.5–13.0) (298/2554) for those for whom an RDT was not recommended by the first step of the algorithm, compared to 30.9% (95% CI 29.4–32.4) (1075/3483) for those for whom an RDT was recommended by the algorithm. Children under 5 years had an RDT positivity rate of 16.5% (95% CI 15.1–17.9) compared to 28.2% (95% CI 26.7–29.8) for patients 5 years or older. The positivity rate noted by risk zone confirmed the highly stratified epidemiology. In the low risk northern zone, the RDT positivity rate was only 1.3% (95% CI 0.8–2.1), varying from 0.5% (95% CI 0.1–1.5) in the dry season to 2.0% (95% CI 1.2–3.2.) in the rainy season. In the high-risk southern zone, the RDT positivity rate was 56.5% (95% CI 53.9–59.0), varying from 27.1% (95% CI 23.7–30.9) in the dry season to 77.6% (95% CI 74.7–80.3) in the rainy season. The centre (moderate) and Dakar (heterogeneous) were intermediate in RDT positivity, with a five-fold difference from dry to rainy season , were both 99.1%, demonstrating excellent reliability in the hands of well-trained health post staff.During the first 9 months of the study (April–December 2013), blood films were collected for 49.3% of patients (2396/4856), and 23.3% (21.7–25.1) of blood films were positive, compared to 25.5% (95% CI 24.3–26.7) of RDTs from this period. All positive samples were identified as Overall, sensitivity of the first step of the diagnostic algorithm  compared to RDT was 78.3% (95% CI 76.0–80.4), specificity was 48.4% (95% CI 46.9–49.8), negative predictive value was 88.3% (95% CI 87.2–89.4), and positive predictive value was 30.9% (95% CI 30.0–31.7). Calculation of the diagnostic values for both age groups, rainy and dry season, and every transmission risk zone separately demonstrated large differences in diagnostic values depending on these categories  among patients 5 years and older, and 45.0% (95% CI 39.4–50.7) among children under 5 years. Given that malaria incidence is generally low in Senegal, positive predictive value was relatively low, save in the south during rainy season, where it was 77.5% (95% CI 73.5–81.2) among children under 5 years and 87.4% (85.5–89.0) among patients 5 years and older.The sensitivity of the study team’s determination of RDT eligibility according to the algorithm compared to RDT was determined to be 82.8% (95% CI 80.7–84.8), compared to 78.3% (95% CI 76.0–80.4) for the determination by the health post workers. Specificity was 39.4% (95% CI 38.0–40.8), compared to 48.4% (95% CI 46.9–49.8) for the health post workers. Other diagnostic values were within 5% points.Considering only patients for whom a blood film was positive (n = 621), 22% (137) had been judged not to be eligible to receive an RDT. Patients for whom a blood film was positive but who were judged not eligible to receive an RDT had a mean parasite density of 23,075 parasites/µL , while patients with positive smear who had been judged eligible to receive an RDT had a mean parasite density of 22,639 parasites/µL . For all positive smears, mean and median parasite density were similar for children under 5 years , children 5–9 years , and patients 10 years and older .All patients were given a 48-h follow-up appointment, which 70.0% kept. Only eight (0.2%) were judged to have had a non-favourable evolution, two of whom had had a positive RDT and had been treated with ACT. Of the five patients that had a negative RDT but positive blood film on the initial visit, parasite density was available for four and ranged from 667 to 10,160 parasites/µL at the initial visit. Four of the five attended the 48-h follow-up visit, and were all judged to have had a favourable clinical evolution.Senegal was one of the first countries in sub-Saharan Africa to recommend laboratory-confirmed malaria diagnosis at all health posts nationwide and was forced to do so in the context of resource constraints. The case management algorithm, as it was designed, represented the consensus of malaria scientists and programme managers in Senegal as the best way to use limited resources to diagnose the greatest number of malaria sufferers. It was implemented with the intention to evaluate and modify it as the situation evolved.This study was the first to measure year-round malaria test positivity rate among patients with uncomplicated febrile illness throughout Senegal, and confirms what has been seen in the routine health information system and cross-sectional surveys regarding the highly seasonal and graduated nature of malaria transmission in Senegal. The RDT positivity rate was very low during dry season and in the north, and quite high in the south in rainy season. While the proportion of patients eligible to be tested according to the algorithm (patients without another fever source) varied by transmission risk zone, the variation was within 15% points from north to south, and the variation between dry and rainy transmission seasons was only 5% points, with roughly 60% of febrile patients recommended for testing according to the algorithm.Surprisingly, children under 5 years old were substantially less likely than patients 5 years and older to be determined eligible for a test, though young children have frequent fevers due to many upper respiratory and other infections, which diminish with age, while adults have fever more rarely, and those fevers may be more likely to be considered malarial.While this study was not an attempt to estimate malaria-attributable fraction, the diagnostic algorithm may be considered a method to help healthcare providers identify patients among whom fever is likely to be attributable to malaria. The RDT positivity rate was indeed higher, by almost three-fold, among those recommended for RDT testing than among those not recommended for RDT testing by the first step of the algorithm. Studies that examined the effect of seasonality found a higher malaria-attributable fraction during the rainy season , 9 and sWhile sensitivity and specificity for laboratory tests are theoretically intrinsic and unchanging values, for a case management algorithm there is variation; this algorithm is a way of assigning testing to those with higher pre-test probability. The sensitivity of the algorithm for identifying patients with malaria among patients under 5 years was lower than that for patients 5 years or older, despite the lower test positivity rate among children under 5 years, and was lowest in the highest transmission risk zone. While the diagnostic algorithm permits testing and diagnosis of almost 80% of febrile patients with malaria parasites nationwide over the course of the year, this proportion is unacceptably low for use as a screening test. In addition, in high transmission zones during the dry season, just over one half of children under 5 years with parasites would be detected by the diagnostic algorithm, validating the complaints of the health providers that if they follow the algorithm, they miss too many cases of malaria. Low specificity, on the other hand, would not be as problematic for a screening test. Negative predictive value is generally greater than 85%, consistent with relatively low burden, except for in the high transmission zone during the rainy season, where it falls to only 45 and 24% among children under 5 years and among patients 5 years and older, respectively; while otherwise low, positive predictive value is greater than 75% among all ages in the high transmission zone during rainy season.Some of the patients ineligible for a test according to the diagnostic algorithm that actually had malaria parasites may in fact have had symptoms due to another cause, with simultaneous asymptomatic malaria parasitaemia. When the algorithm was adopted, it was assumed that these patients likely had low density parasitaemia not requiring treatment. However, among patients for whom a blood film was obtained, and among whom that film was positive, patients for whom an RDT was recommended and patients for whom an RDT was not recommended had very similar mean parasite densities. As Senegal moves toward the goal of pre-elimination, every opportunity to diagnose and treat patients with parasites becomes increasingly important. Senegal is adopting strategies of reactive active case detection, even testing asymptomatic household contacts in elimination zones . TheoretThis study had several limitations. Due to budgetary constraints, health post nurses were trained to carry out the study procedures instead of hiring dedicated study staff. Hence, a simplified enrolment scheme was used, and enrolment was dependent on the care providers’ comprehension of procedures as well as work schedule, though it enabled understanding of how the care providers were evaluating patients. Providers struggled to enrol the planned numbers of patients during the dry season, and among children under 5 years, and were not required to report total numbers of patients or total numbers of febrile patients seen on days of enrolment. Results were presented by age group, season and transmission stratum separately to minimize bias due to this. The limitation to 16 health posts made it impossible to get a nationally representative sample, however sites from across all four of the malaria transmission risk zones were included. While relying on a care provider to assess the need for an RDT according to the algorithm may have been a weakness, the classification by study personnel, including a member of the NMCP, had good concordance with that of the provider. Concern for another potential weakness, that of using RDT for gold standard diagnosis, was mitigated by the very high sensitivity and specificity of RDT compared to reference laboratory microscopy for the half of the samples for which a blood slide was obtained.Despite the limitations, this study provided unequivocal evidence that the first step of the case management algorithm caused providers who followed it strictly to miss an unacceptable proportion of malaria infections. Based on the poor sensitivity of the diagnostic algorithm, the Senegal NMCP is phasing out the use of the first step of the algorithm in favour of universal testing of febrile patients, while rolling out the policy over several years to assure that adequate RDT supplies could be ordered to support the new policy. Starting in 2015, all febrile children under 5 years nationwide and year-round were to be tested with an RDT, while universal testing for febrile patients 5 years or older was limited to the rainy season. The routine malaria information system in Senegal demonstrated that the number of children under 5 years tested for malaria increased from 136,847 in 2014 to 364,771 in 2015 . Startin