Plasmodium falciparum infection is observed in Africa following implementation of new control strategies, malaria is still considered as the major cause of febrile illness in hospitalized African children. The present study was designed to assess the management of febrile illness and to determine the proportion of children with febrile illness hospitalized for primary diagnosis of malaria who had confirmed complicated malaria after implementation of new malaria control strategies in Libreville, Gabon.Although a substantial decline of Demographic, clinical and biological data from hospitalized children with fever or a history of fever, with a primary diagnosis of clinical malaria, aged less than 18 years old, who benefited from hematological measurements and microscopic malaria diagnosis, were recorded and analyzed during a prospective and observational study conducted in 2008 in the Centre Hospitalier de Libreville.Plasmodium falciparum positive blood smears. Age above five years, pallor, Blantyre Coma Score ≤2 and thrombocytopenia were predictive of malaria infection. Respiratory tract infections were the first leading cause of hospitalization (41.1%), followed by malaria (22.7%); co-morbidities were frequent (22%). Less than 5% of suspected bacterial infections were confirmed by culture. Global case fatality rate was 2.1% and 1% for malaria. Almost half (46%) of the children who received antimalarial therapy had negative blood smears. Likewise, antibiotics were frequently prescribed without bacteriological confirmation.A total of 418 febrile children were admitted at hospital as malaria cases. Majority of them (79.4%) were aged below five years. After medical examination, 168 were diagnosed and treated as clinical malaria and, among them, only 56.7% (n = 95) had The use of clinical symptoms for the management of children febrile illness is frequent in Gabon. Information, training of health workers and strengthening of diagnosis tools are necessary to improve febrile children care. In Africa, one in six children dies before its fifth birthday from treatable conditions such as pneumonia, gastroenteritis and malaria. In 2009Presumptive treatment of fever was recommended by WHO for a long time. However, such a strategy is no longer satisfactory because of its poor specificity, leaving non-malarial febrile patients without an appropriate treatment. In fact, evidence suggests a higher mortality in children diagnosed and treated for malaria without microscopic confirmation compared to those who benefited from microscopically confirmed malaria. The bioThe aim of the present study was to assess the management of febrile illness and to determine the proportion of children with febrile illness hospitalized for primary diagnosis of malaria who had confirmed complicated malaria, at the Centre Hospitalier de Libreville, the main public health centre of Gabon, after implementation of new malaria control strategies.P.falciparum. Annual entomological inoculation rate is 33.9 and Anopheles gambiae s.s. is the main vector months; the youngest was one month and the oldest 192 months. Age was distributed as follows: <6 months , 6 to 18 months , 19 to 36 months , 37–59 months , 60–120 months  and above 120 months . More than half of the children  were aged 6 to 36 months. The sex ratio was 1.2.All patients were referred with a primary diagnosis of malaria; 38 9.1%) were sickle cell trait carriers and 32 (7.6%) had a history of asthma. One third  of patients were not well vaccinated according to the Enlarged Program of Immunization and 66.5% (n = 278) used bednets. Main reasons, for consultation aside from fever or history of fever, were asthenia, abdominal symptoms, cough and seizures  , whereas CSF and urine analysis, as well as stools and blood cultures, were realized for 4.5% (n = 19), 2.6% (n = 11), 1.7% (n = 7) and 3.1% (n = 13) of patients respectively. Tuberculin intra-dermal reaction and HIV testing were requested for 54 (12.9%) and 29 (6.9%) patients respectively.Escherichia coli (n = 4) and Shigella (n = 1). Among the 168 patients with a final diagnosis of clinical malaria, 56.6% (n = 95) had confirmed P.falciparum infection with almost in a quarter (24.2%) another infection, mainly suspected lower respiratory tract infection , malaria , gastro enteritis  and meningitis  were the main recorded final diagnosis . However, all patients with sickle cell disease had Hb level below 11 g/dL and were at higher risk of severe anaemia ).Anaemia was found in 355 (84.9%) children: 44 (10.5%) with severe anaemia and 114 (27.3%) with moderate anaemia. Malnutrition and respiratory distress were not associated with severe anaemia  than those without malaria (18 months) (Tablep < 0.01). Bednet use was associated with a reduced risk of malaria (17.0% versus 34.1%) (TableApproximately 23% (n = 95/418) of the participants had confirmed versus 10.2% in uninfected ones; OR: 3.3[1.8-5.9], p < 0.01).At admission, infected patients presented more frequently with pallor , convulsions , vomiting  and prostration . Overall, 20 (21.1%) patients had severe malaria (SM) according to the WHO classification: two had CM, four presented a syndrom of respiratory distress and 14  with SMA. More than three quarters of SM patients (n = 11) were aged between 6 to 36 months old and the two children with CM were 48 and 60 months old respectively. Pallor and presence of neurological symptoms were associated with a higher risk of being infected (Tablep < 0.01) (Tablep < 0.01). None of the HIV positive patients had malarial infection and only two children with malnutrition were infected.The parasite density ranged from 700 to 622,000 p/μL with a median of 22,500p/μL. Hyperparasitaemia was found in five children aged between 16 and 48 months. Most of the parasitemic patients were anaemic (90.5%) and the median Hb level was the lowest in the malaria group compared to the uninfected one. The same trend was observed with platelet count (Table1) Table. The freApproximately two thirds of patients (n = 261) were treated with antibiotics, only 115 having leucocytosis. Quinine was administered to 42.1% (n = 176) of patients, 81 (46.0%) of them were malaria blood slide negative. One patient without clinical and or biological malaria diagnosis was also treated with an antimalarial drug (TableA blood transfusion was given to 96 (27.0%) anaemic children: 41 had severe anemia and 55 had moderate anemia, their Hb level varied from 5.0 g/dL to 7.9 g/dL. Three patients with severe anemia were not transfused.Of the 418 patients, nine died, giving an overall case fatality rate of 2.1%. They were hospitalized for respiratory tract infection (n = 5), gastro-enteritis (n = 2), meningitis (n = 1) and malaria (n = 1). The case fatality rate for malaria was therefore of 1.0%; this patient presented respiratory distress and CM.versus 3 [2.0-5.0] days in the group of survivors (p < 0.01); and with a lower median age: 12 [3.0-14.5] months versus 24[12.0-48.0] months (p < 0.01). Six children were aged between 12 and 36 months and three were less than six months old. Case fatality rate among the less than five years old children was 2.7%.Death was associated with a higher median duration of illness before consultation, 7 [4.8-7.3] days Presumptive treatment of fever with antimalarial drugs is still frequent in public health settings of sub-Saharan Africa, even in countries where ACTs, bed nets, malaria diagnosis and IPT are freely provided to vulnerable population. During In Libreville, malaria declined from being the primary cause of hospitalization among febrile children admitted in the same pediatric ward in 2002, to representing the second cause six years later.P.falciparum infection, either through infection or co-incidental parasitaemia[Hospital case management of seriously ill children is a challenge in middle and low income countries, mostly due to few resources for diagnosis and treatment, staff availability, and laboratory services,15. Respsitaemia-22. Besisitaemia, this issitaemia. ApproxiPlasmodium infections with age observed since 2006 in outpatients was confirmed among the inpatients: children older than five years are 2.6 times more at risk for having complicated malaria. This suggests a change in the transmission profile of the disease as the prevalence of infection increases with age with decreasing transmission[P.falciparum infection often occurs in patients with chronic anaemia and increases its gravity[Globally, prostration, neurological symptoms like seizures and pallor are the main clinical forms of malaria among children with acute febrile illness as observed in other endemic countries,26. Throsmission,30. The smission. The higsmission,30,31. Ismission. This ob gravity.Improvement of medical care is ongoing in Gabon, but most of the population cannot benefit from complementary examinations because of poor financial resources and lack of equipment at health centers. As an example, beside chest X-ray that is systematically prescribed in case of suspected respiratory tract infection, lactate levels cannot be measured at the CHL and material for blood and CSF cultures is always lacking. Therefore, it remains crucial to use clinical and biological signs to target malaria infected children for a rational use of antimalarial drugs in poor resource areas. Pallor, presence of neurological symptoms, BCS ≤ 2 and age >5 years, were predictors of malaria infection among children hospitalized for fever. These risk factors have also been found in children from other countries,34,35. AEven if malaria diagnosis and hematological count were freely provided to patients, overdiagnosis (43%) and overtreatment with antimalarial drugs (46%) were common.P.falciparum-infected patients, lethality was lower (1%) compared to that observed between 2001–2002 in the same paediatric ward (9%) and in 11 African countries (10.5%); probably due to i) the reduced cases of malaria, ii) a better case management because all malaria cases were microscopically confirmed and suspected co-morbidities treated with both antimalarial and antibiotics[Although the low number of ibiotics,38.P.falciparum infection[After the deployment of new preventive measures, decreased frequency of malaria hospital admissions and its various clinical presentations were observed. Howevernfection-41. The This pilot study allowed the inclusion of an enough number of patients to provide reliable data on hospitalized febrile children. A higher number of inclusions would have probably not given more indications on febrile illness management in the paediatric ward, neither more clinical data. Indeed, the main limit of this study concerns the lack of additional investigations necessary to finalize the diagnosis. They are most of the time not performed because of poor financial resources of the parents. Furthermore, there are some limitations on using health facility–based data including the lack of a knowledge on variations in access to, or use of the facilities in the community, extend of real coverage by various interventions.Nevertheless, hospital based studies provide accurate data on children febrile illness etiologies, though they cannot replace epidemiological surveys in the community. The preP.falciparum infection in Gabon in 2008. These results can help for the design of an effective development of new guidelines for fever case management. Priority areas to achieve proper patient care are quality of diagnosis, treatment practices, availability of biological and radiographic diagnosis tools, introduction of malaria diagnosis RDTs to discriminate malarial to non malarial fevers and bring out rational use of drugs, health worker training and adherence to guidelines.This study, and the one performed between 2001 and 2002, highlights the most likely causes of fever as well as a reduction in the frequency of The authors declare that they have no competing interests.MKBA, EK, DPMM and MK were involved in design, implementation, planning of analysis. MKBA and EK were responsible for data analysis, writing up of the results. AEA, ARO and EEA were involved in patient care at hospital; and MKBA, DPMM and MK in the writing up and making critical revisions to the manuscript. MKBA is guarantor. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/216/prepub


Objectives. We investigated the relationship between the stage of diabetic retinopathy and pulse wave velocity (PWV). Methods. This was a cross-sectional study of 689 patients (406 men and 283 women) with type 2 diabetes who were admitted to our hospital from 2004 to 2007. Brachial-ankle pulse wave velocity (baPWV) was measured by an arterial pressure measurement device as PWV/ABI. Diagnosis of diabetic retinopathy was made by ophthalmologists based on the Davis classification: no diabetic retinopathy (NDR), simple retinopathy (SDR), pre-proliferative retinopathy (pre-PDR), and proliferative retinopathy (PDR). Results. There was a significant difference in PWV between patients without diabetic retinopathy (1657.0 ± 417.9 m/s (mean ± SD)) and with diabetic retinopathy (1847.1 ± 423.9 m/s) (P < 0.001). In addition, the stage of diabetic retinopathy was associated with aortic PWV (1657.0 ± 417.9 m/s in NDR (n = 420), 1819.4 ± 430.3 m/s in SDR (n = 152), 1862.1 ± 394.0 m/s in pre-PDR (n = 54), and 1901.1 ± 433.5 m/s in PDR (n = 63) (P < 0.001)). Conclusions. In patients with diabetic retinopathy, even in those with SDR, PWV was higher than that in patients without diabetic retinopathy. Physicians should therefore pay attention to the value of PWV and macroangiopathy regardless of the stage of diabetic retinopathy. Pulse wave velocity (PWV) has been used as a noninvasive clinical index of aortic stiffness. It is reported that PWV of patients with diabetes is higher than that of healthy subjects . In a JaRetinal capillary microaneurysms are the hallmark of diabetic retinopathy and its earliest reliable sign, and individual acellular capillaries are usually visible histologically in the earliest stages of diabetic retinopathy. As retinopathy becomes more severe, larger patches of acellular capillaries are seen. When lesions like cotton-wool spots, intraretinal microvascular abnormalities, venous beading, and retinal hemorrhages are prominent, diabetic retinopathy is considered pre-proliferative, and new vessels are likely to appear soon on the surface of the retina or optic disc. When new vessels appear on the surface of the retina or optic disc, diabetic retinopathy is said to have entered the proliferative stage .To our knowledge, no study has compared PWV with the stage of diabetic retinopathy. Therefore, we investigated the relationship between increased PWV and the stage of diabetic retinopathy. From January 2004 to December 2007, 732 Japanese patients with type 2 diabetes who were admitted to Keio University Hospital  were consecutively observed. Among them, 43 patients with acute illness  were excluded from the evaluation. Consequently, a total of 689 patients with type 2 diabetes who were admitted due to having poor glycemic control were enrolled in this study. All of their purposes of admission were to control glucose metabolism and education for diabetes. The study protocol was approved by the ethical committee of the hospital. Informed consent was obtained from all patients. The diagnosis of diabetic retinopathy was made by ophthalmologists based on the Davis classification: no diabetic retinopathy (NDR); simple retinopathy (SDR); pre-proliferative retinopathy (pre-PDR); and proliferative retinopathy (PDR). During hospitalization, fasting plasma glucose (FPG), 2-hour plasma glucose (PG), C-peptide (CPR), hemoglobin A1c (HbA1c), glycated albumin (GA), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (UN), creatinine (Cr), uric acid (UA) in blood, and 24-hour urine microalbumin were measured. HbA1c was determined by high-performance liquid chromatography  and presented as the equivalent National Glycohemoglobin Standardization Program (NGSP) value . FurtherD1 − D2)/T1, where D1 is the distance from the heart to the left ankle, and D2 is the distance from the heart to the right upper arm. These distances were calculated automatically on the basis of the patient's height. The pressure waveforms obtained at two different sites were simultaneously recorded, and the time interval between the initial rise in the brachial and tibial pressure waveforms was determined as T1. ABI was calculated using the formula ABI = ankle systolic BP/brachial systolic BP.Measurements of brachial-ankle PWV (baPWV) were carried out using an automatic waveform analyzer . Patients lay in the supine position during the test, and occlusion and monitoring cuffs were placed around both the upper and lower extremities. PWV was calculated using the formula: baPWV =  to investigate the relationship between each stage and the value of PWV by the Kruskal-Wallis test. The relationship between the PWV and each factor was evaluated with Spearman's correlation coefficient. The selected variables, which were statistically significant and clinically important, were included in nonparametric multiple regression models to evaluate the association between PWV and each stage of diabetic retinopathy adjusted for some covariates. The purpose of these multivariate analyses was to show the robustness of the results from the univariate analysis.Demographic factors and baseline characteristics were summarized by diabetic retinopathy (DR) and NDR groups. They were compared between the DR and NDR groups using Mann-Whitney Data are presented as mean ± standard deviation (SD) in the text and tables. The significance level for all tests was two-sided, at 5%. All analyses were performed using SPSS 17.0  and SAS 9.2 .Demographic factors and clinical baseline characteristics of patients are shown in P < 0.001). Furthermore, there was a significant positive association between the stage of diabetic retinopathy and PWV. PWV was 1657.0 ± 417.9 m/s in NDR (n = 420), 1819.4 ± 430.3 m/s in SDR (n = 152), 1862.1 ± 394.0 m/s in pre-PDR (n = 54), and 1901.1 ± 433.5 m/s in PDR (n = 63) (P < 0.001) (PWV in patients with diabetic retinopathy (1847.1 ± 423.9 m/s) was significantly higher than that in patients without diabetic retinopathy (1657.0 ± 417.9 m/s) (< 0.001) .P < 0.001).Some sensitivity analyses were performed to evaluate robustness of the results from the univariate analysis. Factors significantly correlated with the PWV by means of Spearman's correlation coefficient were BMI, age, SBP, FPG, and HbA1c . HoweverMeasurement of aortic PWV is considered the gold-standard evaluation of arterial stiffness . Values In the present study, PWV was significantly higher in patients with diabetic retinopathy than in those without. This finding supports the report that diabetic retinopathy is the microvascular complication with the strongest association with increased aortic stiffness . In addiIncreased arterial stiffness is thought to be related to not only hyperglycemia but also to carbonyl and oxidative stress, chronic inflammation, endothelial dysfunction, and formation of advanced glycation end products (AGEs) . It is rSeveral limitations should be taken into account when considering the results of this study. First, the cross-sectional study design and small sample size for each stage of diabetic retinopathy in our study make it difficult to infer the association between PWV and retinopathy. Second, we could not consider the effects of prescribed medication, for instance, anti-platelet agents, which could influence the state of both retinopathy and arterial stiffness. Third, the raw data might have deviated slightly because there was more than one PWV technician and ophthalmologist. However, it was thought that the influence of bias was small because the technicians and ophthalmologists were experts and were not aware of this study when they carried out the examinations. Lastly, the patients with type 1 diabetes were not included in this study. In a recent meta-analysis of observational studies, diabetic retinopathy predicted all-cause mortality and cardiovascular events in patients with type 2 diabetes and also type 1 diabetes . Based oIn conclusion, this study suggested that PWV is significantly higher in patients with diabetic retinopathy than in those without, and that there is a relationship between the stage of diabetic retinopathy and PWV in Japanese patients with type 2 diabetes. Physicians should pay attention to latent macroangiopathy in patients with type 2 diabetes who have diabetic retinopathy, even SDR.


We conducted a prospective study in a cohort of short-term travelers assessing the incidence rate of anti-circumsporozoite seroconversion, adherence to chemoprophylaxis, symptoms of malaria during travel, and malaria treatment abroad.Plasmodium falciparum anti-circumsporozoite antibodies.Adults were recruited from the travel clinic of the Public Health Service Amsterdam. They kept a structured daily travel diary and donated blood samples before and after travel. Blood samples were serologically tested for the presence of Overall, the incidence rate (IR) of anti-circumsporozoite seroconversion was 0.8 per 100 person-months. Of 945 travelers, 620 (66%) visited high-endemic areas and were advised about both chemoprophylaxis and preventive measures against mosquito bites. Most subjects (520/620 = 84%) took at least 75% of recommended prophylaxis during travel. Travel to Africa, use of mefloquine, travel duration of 14–29 days in endemic areas, and concurrent use of DEET  were associated with good adherence practices. Four travelers without fever seroconverted, becoming anti-circumsporozoite antibody-positive. All four had been adherent to chemoprophylaxis; two visited Africa, one Suriname, one India. Ten subjects with fever were tested for malaria while abroad and of these, three received treatment. All three were adherent to chemoprophylaxis and tested negative for anti-circumsporozoite antibodies.Travel to Africa, using mefloquine, travel duration of 14–29 days in endemic areas, and use of DEET were associated with good adherence to chemoprophylaxis. The combination of chemoprophylaxis and other preventive measures were sufficient to protect seroconverting travelers from clinical malaria. Travelers who were treated for malaria abroad did not seroconvert. Half the world’s population is at risk of malaria Plasmodium falciparumTravelers can protect themselves against malaria by using antimalarial chemoprophylaxis and preventive measures against mosquito bites. Recommendations for these preventive measures are based on the anticipated infection rate and drug resistance in Risk estimates for malaria infection among travelers from nonendemic countries are usually based on transmission rates in endemic populations and reports of infections in returned tourists. Neither estimate correlates well with the risk for travelers falciparum malaria among travelers declined from 10.0/10,000 in the year 2000 to 3.4/10,000 travelers in 2007, whereas the proportion of travelers who did not use chemoprophylaxis rose from 47% to 52% P. falciparum infection Studies that estimate incidence based on these reports lack information on how many travelers were protected by chemoprophylaxis and how many were treated for malaria abroad. They are, however, valid to estimate trends. In the Netherlands, the incidence of imported Plasmodium falciparum anti-circumsporozoite seroconversion, adherence to chemoprophylaxis, symptoms of malaria during travel, and malaria treatment abroad.In order to contribute to a more definitive assessment of risks for travelers, we conducted a comprehensive prospective study as to the incidence rate of Study protocol was approved by the Medical Ethics Committee of the Academic Medical Center Amsterdam (MEC 06/016). Participants were included only with informed and written consent.P. falciparum malaria A prospective study was performed among persons attending the travel clinic of the Public Health Service Amsterdam from October 2006 to October 2007. All persons 18 years and older were eligible if they were planning to travel for 1 to 13 weeks to one or more malaria-endemic countries. Countries were grouped in continents according to the composition of macro geographical  regions described by the United Nations Statistics Division Plasmodium falciparum malaria and to register adherence to chemoprophylaxis after return. After travel, a nurse checked the diary in the presence of the participant. Participants were then asked if they had taken the advised chemoprophylaxis prior to travel – if applicable.A standard questionnaire in Dutch or English was used before departure to collect data on sociodemographics, travel history, and purpose of travel . Participants were given a thermometer and were asked to take their temperature if they felt feverish. They were also asked to keep a structured travel diary, recording itinerary, use of antimalarial chemoprophylaxis, use of preventive measures against mosquito bites such as a mosquito net and/or an insect repellent containing N,N-diethyl-meta-toluamide (DEET) and/or sleeping in air conditioned rooms, signs of disease (fever), doctor visits, and (self)treatment. Participants made daily diary entries from the day they arrived at their destination to 2 weeks after their return, to encompass the incubation period of The number of days spent in malaria-endemic areas was defined as ‘exposure time’. Participants were considered ‘adherent’ to chemoprophylaxis if they took at least 75% of their recommended tablets as prescribed during their stay in high-endemic areas. Adherence before and after travel were separately described. The use of DEET, use of a mosquito net, and sleeping in an air-conditioned room were quantified in percentages by dividing the number of days the preventive measure was used by the number of days spent in a malaria-endemic area; and we dichotomized these variables by using the mean of this proportion in the total study population as the cut-off. Fever was self-reported (feeling feverish or thermometer confirmed fever with a temperature of 38°C or higher) starting 1 week after arriving in a malaria-endemic area.P. falciparum anti-circumsporozoite antibodies according to standard protocols P. falciparum during present travel.Before departure and 2 to 6 weeks after return, participants donated venous blood samples for serology. All blood samples were immediately stored at 6°C. Blood samples for serologic testing were centrifuged ) and frozen at −80°C within 24 hours until use. The CSP-ELISA (circumsporozoite protein enzyme-linked immunosorbent assay) was used to test for the presence of P. falciparum. ARs were calculated by dividing the number of study subjects displaying seroconversion in the CSP-ELISA by the total number of participants at risk. IRs per 100 person-months were calculated by dividing the number of travelers with exposure to P. falciparum by our calculation of their exposure time. If a traveler was exposed to P. falciparum, we used half of their travel duration in endemic area as their exposure time; for travelers without exposure to P. falciparum, we used their total travel duration. Pearson chi-square tests of association were used to compare categorical variables between any two groups. Independent determinants for adherence to chemoprophylaxis and for use of DEET were identified by multiple logistic regression analysis and expressed as odds ratios with 95% confidence intervals. A p-value <0.05 was considered statistically significant.Data analysis was performed with SPSS version 19.0.0.1 . Attack rates (ARs) and incidence rates (IRs) were based on positive anti-circumsporozoite antibody seroconversion indicative for exposure to A total of 945 travelers to malaria-endemic countries were recruited. Of these, 400 (42%) were male . The majOf the 945 travelers, 620 (66%) were advised of both chemoprophylaxis and preventive measures against mosquito bites. The other 325 participants traveled to low-endemic areas.Of the 620 travelers to high-endemic areas who were prescribed chemoprophylaxis, 449 were prescribed atovaquone-proguanil (48%), 91 proguanil (10%), 70 mefloquine (8%), and 10 (2%) other types of chemoprophylaxis, such as minocyclin, doxycycline, or a combination of chemoprophylaxis.Of 620 travelers, 520 (84%) took at least 75% of the recommended tablets during their stay in high-endemic areas . In multOf 945 travelers, 791 (84%) used DEET, 465 (49%) used a mosquito net, and 544 (58%) slept in an air-conditioned room at least once.Multivariable analysis showed that travelers born in a non-Western country used significantly less DEET and also slept significantly less often under a mosquito net than travelers born in a Western country. Female travelers used significantly more DEET than male travelers. Older travelers used significantly less DEET or mosquito nets than younger travelers. Spending more days in endemic areas was an independent determinant for using less DEET and sleeping less often in an air-conditioned room. Travelers to high-endemic areas in general used a mosquito net significantly more often and slept less often in an air-conditioned room compared to travelers to low-endemic areas. Travelers to Africa or Latin America used a mosquito net significantly more often and slept less often in an air-conditioned room compared to travelers to Asia.Of 945 travelers, only 938 serum samples were tested using the CSP-ELISA method because the sample was insufficient to test in 7 cases. Of the 938 samples, 4 (AR 0.4% (95% CI 0.1–1.0)) showed an anti-circumsporozoite antibody seroconversion. All 4 travelers were born in the Netherlands, had been 100% adherent to their chemoprophylaxis during travel, and none reported fever or had visited a doctor . Of the Of the 945 travelers, 74 (8%) reported fever after a median period of 17 days (IQR 12–26) in endemic areas. Of these 74, 67 (91%) used the provided thermometer and recorded fever with a median temperature of 38.7°C (IQR 38.2°C–39.6°C). Of the 74, 24 (32%) had visited low-endemic areas and 50 (68%) high-endemic areas. Of 24 travelers who reported fever in low-endemic areas, 13 (54%) sought medical attention abroad, 2 of whom were tested negative for malaria. Of 50 travelers to high-endemic areas, 17 (34%) consulted a doctor abroad, of whom 8 were tested for malaria and 3 were actually treated for malaria. These last 3 travelers had been fully adherent to chemoprophylaxis during travel and tested negative for anti-circumsporozoite antibodies.P. falciparum of 0.4% and an overall incidence rate (IR) of 0.8 per 100 person-months.In this prospective study with short-term travelers to malaria-endemic countries, self reported adherence to chemoprophylaxis was good. Best adherence was found among travelers to Africa, travelers using mefloquine as chemoprophylaxis, travelers who spent 14–29 days in endemic areas and travelers who were more adherent to use of DEET. Based on seroconversions for anti-circumsporozoite antibodies, we found an overall attack rate (AR) for Adherence to chemoprophylaxis found in our study is in agreement with self reported treatment adherence found in other studies, which ranged from 70%–89% for atovaquone-proguanil and 72%–95% for mefloquine As in our study, previous studies found longer travel duration to be a determinant of nonadherence Determinants of nonadherence to chemoprophylaxis found in previous studies were visiting friends and/or relatives (VFR), younger age, extensive travel experience, adventurous travel, and (assumed) adverse reactions Furthermore, we did not find a relation between travel experience and adherence, but we did find less adherence to chemoprophylaxis in younger travelers, although not significantly.P. falciparum infection P. falciparum sporozoites. All 4 travelers were infected in high-endemic areas, none reported fever or malaria treatment, and all had been adherent to chemoprophylaxis. This suggests that the use of chemoprophylaxis protected these 4 travelers from clinical malaria. There are a few other prospective studies on malaria infection using anti-circumsporozoite antibody tests in nonimmune travelers using chemoprophylaxis Studies have shown that detecting anti-circumsporozoite antibodies can be used in nonimmune travelers using chemoprophylaxis as a measure of P. falciparum anti-circumsporozoite seroconversion, adherence to chemoprophylaxis, and collection of clinical malaria data among travelers. The prospective nature of this study with blood samples pre- and post-travel allowed estimating the AR and IR of exposure to P. falciparum malaria. The daily diary entries, which minimized recall bias, provided a good record of the use of chemoprophylaxis and antimosquito preventive measures, fever, and treatment for malaria during travel.Our study is one of the few comprehensive studies that combined P. falciparum. Finally, measurement of malaria exposure by anti-CSP seroconversion has limitations due to the methodology. The sensitivity is limited due to small numbers of inoculated sporozoites by infected mosquitoes and their short life span Interpretation of our study results may be influenced by a number of shortcomings. First, the daily diary entries could have served as a reminder for travelers to take their chemoprophylaxis and therefore lead to better adherence to treatment. Second, because of the study design, the end date for travelers to fill in their diaries was 2 weeks after return, so we do not know if travelers using mefloquine or proguanil continued chemoprophylaxis after the 2 weeks. Treatment adherence during stay in endemic areas, however, remains most important in protection against infection with In conclusion, travel to Africa, using mefloquine, travel duration of 14–29 days in endemic areas, and good adherence to DEET were associated with good adherence to chemoprophylaxis. Adherence to chemoprophylaxis in combination with other preventive measures was good enough to protect the travelers who seroconverted from clinical malaria. None of the travelers to low-endemic areas where chemoprophylaxis is not recommended contracted malaria, so there is no reason to adapt the Dutch national guidelines. Similar prospective studies with larger numbers to specific destinations are needed to make more specific destination- dependent advice about the use of chemoprophylaxis.


This parasite may be transmitted from macaques to humans. Plasmodium knowlesi are endemic to Southeast Asia. To determine the prevalence of P. knowlesi malaria in malaria-endemic areas of Thailand, we analyzed genetic characteristics of P. knowlesi circulating among naturally infected macaques and humans. This study in 2008–2009 and retrospective analysis of malaria species in human blood samples obtained in 1996 from 1 of these areas showed that P. knowlesi accounted for 0.67% and 0.48% of human malaria cases, respectively, indicating that this simian parasite is not a newly emergent human pathogen in Thailand. Sequence analysis of the complete merozoite surface protein 1 gene of P. knowlesi from 10 human and 5 macaque blood samples showed considerable genetic diversity among isolates. The sequence from 1 patient was identical with that from a pig-tailed macaque living in the same locality, suggesting cross-transmission of P. knowlesi from naturally infected macaques to humans.Naturally acquired human infections with  Plasmodium knowlesi circulates mainly among long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) that inhabit a wide area of Southeast Asia , but individual cases have been increasingly identified in countries in Southeast Asia  in several malaria-endemic areas bordering Myanmar, Cambodia, and Malaysia  living in Tak Province were included for comparative analysis. Of these samples, 143 were obtained during May–July; the remaining 67 samples were obtained during October–December. Blood samples were preserved in EDTA and stored at −40°C until use. Results of malaria species distribution during 2006–2007 from our analysis  derived from small subunit rRNA for a primary PCR , respectively. PCR products were analyzed by 2% agarose gel electrophoresis.DNA was extracted from 200 μL of blood by using a DNA Minikit  according to the manufacturer’s instructions. Malaria species was identified by using a nested PCR with M. nemestrina monkeys, 1 M. fascicularis monkey, and 1 Semnopithecus obscurus monkey in Narathiwat Province had P. knowlesi infections (P. knowlesi (Pkmsp-1) from these pig-tailed macaques and humans in the current survey were obtained by direct sequencing of PCR-amplified products as described  and Narathiwat Province (n = 566) showed that 5 2 test, or Fisher exact test. A 2-tailed p value <0.05 indicated statistical significance.Sequences were aligned by using ClustalX with minor manual adjustments made by visual inspection  or P. vivax (48.21%); the prevalence of co-infections with both species was 0.18%. Of 432 samples containing co-infections with P. falciparum and P. vivax detected by PCR, only 3 (0.69%) samples were correctly diagnosed by microscopy. Microscopy showed negative results for 37 (8.56%) samples and failed to detect cryptic P. falciparum for 181 (41.90%) samples and cryptic P. vivax for 211 (48.84%) samples ; PCR failed to detect malaria in 46 microscopy-positive samples.During October 2008–September 2009, microscopic examinations of blood samples from 3,770 febrile patients showed that 3,300 had malaria parasites and 470 did not. Most malaria cases diagnosed by microscopy were either  samples . ConversP. knowlesi in these samples. In contrast, PCR identified 23 (0.67%) patients with P. knowlesi infections (8 monoinfections and 15 co-infections with other Plasmodium species)  .P. falciparum (26.19%) and P. vivax (45.71%), respectively. The remaining 59 patients (28.10%) had mixed species infections with P. falciparum and P. vivax (n = 50), P. vivax and P. malariae (n = 7), P. vivax and P. knowlesi (n = 1), and P. falciparum, P. vivax, and P. malariae (n = 1). These findings indicate that P. knowlesi has circulated among humans in Thailand for at least 12–13 years.PCR analysis of 210 blood samples from microscopy-positive patients in Tak Province obtained during 1996 identified 55 and 96 patients as having monoinfections with P. falciparum and P. vivax displayed spatial variation in Thailand. PCR assays showed that P. falciparum and P. vivax contributed almost equally to malaria cases in Tak Province during 2008–2009 , P. vivax was more prevalent than P. falciparum in Chantaburi Province, and P. falciparum was more prevalent than P. vivax in Yala and Narathiwat Provinces. Despite a low overall prevalence of the remaining malaria species (<2%), P. knowlesi could be detected more often than P. malariae and P. ovale in these malaria-endemic areas . Conversely, distribution of P. knowlesi remained stable over these periods . If one considers that samples from 3 periods  were available for comparison only for Tak Province, the contribution of P. knowlesi to malaria cases was not different in all pairwise comparisons, which suggested stable prevalence of this simian malaria for more than a decade in this malaria-endemic area. In contrast, a significant difference was found in the distribution of P. falciparum, P. vivax, and P. malariae obtained during the 3 periods in each malaria-endemic area , and P. ovale was not detected in samples obtained during 1996 but comprised 1.41% and 0.21% of samples obtained during 2006–2007 and 2008–2009, respectively . The opposite result was observed for isolates from Yala Province . No significant seasonal difference in prevalence of mixed species infections occurred in isolates from Tak Province  and Narathiwat Province .Prevalence of mixed species infections showed regional and seasonal variations characterized by a significantly higher prevalence during the dry season than in the rainy season in isolates from Chantaburi Province (χP. knowlesi was detected in 34 patients (0.65%) , had uncomplicated malaria symptoms , and most (73.50%) cases occurred in persons 16–45 years of age. P. knowlesi malaria was diagnosed in male patients ≈2× more often than in female patients (M:F ratio 2.18:1), which was similar to the sex distribution of total malaria cases (M:F ratio 2.16:1). Thus, no sex preference was observed for P. knowlesi (p = 0.97).Of 5,254 PCR-positive samples obtained during 1996, 2006–2007, and 2008–2009,  (0.65%) , Table 3P. knowlesi–infected malaria patients acquired infections in southern Thailand near Malaysia. Monoinfections with P. knowlesi were observed in 10 patients whose parasite densities ranged from 0 to 145,000 parasites/μL. The remaining 24 patients had co-infections with P. falciparum (n = 16) or P. vivax (n = 9) or triple infections with P. falciparum and P. vivax (n = 5). Although the geometric mean parasite density for P. knowlesi moninfections was higher than that for mixed species infections, no significant difference was observed  of P. knowlesi malaria occurred in the rainy season. However, the ratio of P. knowlesi–infected patients to total malaria patients for the rainy and dry seasons was not significantly different . Most (73.50%) human infections with P. knowlesi occurred in areas with macaques living nearby.Initial microscopic diagnosis identified Pkmsp-1 gene sequences were analyzed for 10 isolates from humans and 5 isolates from pig-tailed macaques. These human isolates were obtained from 8 patients in the current survey  and from 2 patients from Prachuab Khirikhan Province obtained during 2006–2007  was observed among Pkmsp-1 genes of these isolates.Fifteen complete Pkmsp-1 genes, similar to those found in the msp-1 gene of P. vivax  and a monkey isolate from Narathiwat Province (isolate HB3), and the remaining isolates belonged to the other cluster (Pkmsp-1 gene sequence from 1 patient (NR280) in Narathiwat Province was identical with that isolated from a pig-tailed macaque (HB149) living in the same locality. Furthermore, isolates from 2 patients (YL975 and YL978) who lived in Yala Province had identical Pkmsp-1 gene sequences. The patients were concurrently infected with P. knowlesi in the same malaria-endemic areas. The other identical sequences were found in isolates from 2 patients (CT157 and CT190) in Chantaburi Province who had onset of febrile illness a few days apart  along the Thailand–Myanmar border that contrasts with a low prevalence of 3% and 5% in malaria-endemic areas bordering Cambodia and Malaysia, respectively . When 1 of these 2 species coexisted with other malaria species (including P. knowlesi), only P. falciparum or P. vivax infection was diagnosed by microscopy. Failure to diagnose mixed species infections could result in repeated diagnosis and treatment, economic losses, misinterpretation of drug or vaccine efficacies, and inadequate control policies  malaria patients had P. knowlesi monoinfections  showed that Pkmsp-1 gene sequences found in 2 patients in Chantaburi Province who lived in the same area and had symptomatic malaria during the same week suggests that both patients acquired the infections from a common source. Alternatively, parasites harboring this identical Pkmsp-1 allele could predominate among infected macaques in the region. Unfortunately, no extensive survey of malaria in wild macaques in Chantaburi Province was performed during the study. Despite limited number of samples in our study, Pkmsp-1 gene sequences from humans in eastern and southern Thailand were found in both clusters of the phylogenetic tree, which suggested that both dimorphic types are widely distributed in Thailand. Whether both dimorphic types of Pkmsp-1 genes from infected persons co-exist in other malaria-endemic regions of Southeast Asia remains to be investigated.Identical P. knowlesi (,P. knowlesi malaria had febrile illness and associated symptoms that were indistinguishable from those caused by other malaria species (,P. knowlesi requires 24 hours to complete its asexual erythrocytic cycle, which results in a unique quotidian type of fever pattern different from that for the other 4 human malaria species (P. knowlesi malaria is responsive to chloroquine treatment and would likely be responsive to other antimalarial drugs (P. knowlesi among patients with mixed infections after initial treatment for infection with another malaria species in Thailand seems less likely. P. knowlesi infections in humans caused severe symptoms and deaths in 6.5% and 1.8%, respectively, in the Kapit population in Sarawak where it comprised 70% of all malaria cases (P. knowlesi malaria had a high prevalence of complicated symptoms, the probability of observing such severe and fatal consequences from P. knowlesi infections among patients in Thailand would be low.A spectrum of clinical manifestations has been observed in persons infected with P. knowlesi has occurred in Thailand for more than a decade. Despite variations in the prevalence of all 4 human malaria species, P. knowlesi has shown stable prevalence rates, which suggests different transmission cycles. Human infections with P. knowlesi in Thailand could result from macaques, as shown by identical Pkmsp-1 gene sequences of human and macaque origins.In conclusion, human malaria caused by


Plasmodium falciparum and the relationship between household exposure and parasite density.At the local level, malaria transmission clusters in hotspots, which may be a group of households that experience higher than average exposure to infectious mosquitoes. Active case detection often relying on rapid diagnostic tests for mass screen and treat campaigns has been proposed as a method to detect and treat individuals in hotspots. Data from a cross-sectional survey conducted in north-western Tanzania were used to examine the spatial distribution of P. falciparum, with the parasite density of positive samples being estimated by quantitative PCR. Household exposure was estimated using the distance-weighted PCR prevalence of infection. Parasite density simulations were used to estimate the proportion of infections that would be treated using a screen and treat approach with rapid diagnostic tests (RDT) compared to targeted mass drug administration (tMDA) and Mass Drug Administration (MDA).Dried blood spots were collected from consenting individuals from four villages during a survey conducted in 2010. These were analysed by PCR for the presence of Polymerase chain reaction PCR analysis revealed that of the 3,057 blood samples analysed, 1,078 were positive. Mean distance-weighted PCR prevalence per household was 34.5%. Parasite density was negatively associated with transmission intensity with the odds of an infection being subpatent increasing with household exposure (OR 1.09 per 1% increase in exposure). Parasite density was also related to age, being highest in children five to ten years old and lowest in those > 40 years. Simulations of different tMDA strategies showed that treating all individuals in households where RDT prevalence was above 20% increased the number of infections that would have been treated from 43 to 55%. However, even with this strategy, 45% of infections remained untreated.The negative relationship between household exposure and parasite density suggests that DNA-based detection of parasites is needed to provide adequate sensitivity in hotspots. Targeting MDA only to households with RDT-positive individuals may allow a larger fraction of infections to be treated. These results suggest that community-wide MDA, instead of screen and treat strategies, may be needed to successfully treat the asymptomatic, subpatent parasite reservoir and reduce transmission in similar settings. Malaria transmission is spatially heterogeneous over all geographical scales. At a global level, countries or regions experience varying levels of transmission . Within ACD currently relies on rapid diagnostic tests (RDT) or microscopy to identify infected individuals. There is, however, a growing body of evidence that shows that these diagnostic tests miss a substantial proportion of malaria infections in endemic areas compared to PCR,22, primet al. found a positive relationship between transmission and parasite density, with the proportion of infections that are subpatent being highest in low transmission settings [Using aggregated survey prevalence estimates, Okell settings . This suThis study has used intensive cross-sectional sampling in north-western Tanzania to examine household-level heterogeneity in parasite exposure and density. The data obtained were used for simulations of different screen and treat strategies to maximize impact on subpatent malaria infections.Misungwi district  is located in the north-west of Tanzania at an altitude of 1,178 m above sea level. The district has a moderate level of malaria transmission (meso-endemic). The district has two annual rainy seasons, the long rains between February and May and the short rains between November and December. The dry and relatively hot season is June to September. Transmission intensity has a seasonal cycle, with peaks in malaria incidence one to two months after the rains start. The district is situated 60 km from Mwanza town; the prevalence of malaria infection in the region is estimated to be 31.4% by microscopy during a Demographic and Health Survey (DHS).The population of the district is 308,134, living in 37,468 households, 18.1% of whom are aged five years or under. The district is rural with an economy based around cotton production, rice plantation and fishing from Lake Victoria. The district has a total of 19 wards.Paracheck-Pf®, Orchid Biomedical Systems, Goa, India) and referred to a study clinician for management of their febrile illness. Filter papers were dried overnight and stored individually with desiccant at −20°C for later molecular analysis.A census of four villages in a single ward was carried out between August and November 2010  targets were run in duplicate for each sample in a 354-well format ABI qPCR machine, model 7500. DCT values between the two targets were estimated for each sample and the mean DCT of duplicate wells normalized to the within-run quantitative standard, comprising the WHO International Standard for Plasmodium falciparum DNA (IS) [-1. The ratio of parasite density in the sample relative to the IS was then multiplied by 500 parasites mL-1 to obtain the estimate of parasite density. Any samples that were negative by qPCR but positive by nPCR were assigned an arbitrary parasite density value of half the minimum density detected.DNA was extracted from filter papers using the Chelex® extraction method described previously  in 96-deet al.  with theDNA (IS) ,31, and et al. showed that this method provides a suitable index for exposure, as it was predictive of individual infection in the index household [Household exposure to malaria infection was estimated using distance weighted local prevalence of malaria infection (detected by PCR) . This meousehold .versus continuous values of household exposure and age was compared using Akaike Information Criterion (AIC) values and likelihood ratio tests.Due to the distribution of the parasite density data, parasite density was modelled as a binary outcome; subpatent (> 0 and <100 parasites/μl) 1; patent (>100 parasites/μl) 0. This classification was based on the density typically quoted as the detection limit for RDTs . UninfecIn order to estimate the number of infections that would be detected by RDT and treated accordingly, and to explore whether it was possible to increase the proportion of PCR positives that would be correctly treated using RDTs, simulations of targeted mass drug administration (tMDA) were conducted. This system works by first defining a threshold household prevalence. In households where the prevalence of infection by RDT exceeds this threshold, all individuals in that household are treated irrespective of their RDT result. For example, if the threshold was set at 50%, all individuals in households where at least 50% tested positive by RDT would be treated. Simulations using thresholds of 10%-100%, in increments of 10%, were conducted. Using the parasite density estimates obtained from the qPCR, individuals were assumed to test positive to an RDT if they had a parasite density of >100 parasites/μl. Simulations using detection limits of 50 parasites/μl and 200 parasites/μl were also run for comparison. These represent a more ideal detection limit and the lowest density used by WHO in quality control tests respectively . This siThe census revealed that approximately 3,800 individuals lived in the 4 study villages. Dried blood spots were collected from 3,057 individuals (80.4%) and 52.7% of participants were male. Overall prevalence of infection by nPCR (two rounds of amplification) was 35.2%. The single round qPCR was less sensitive, as expected, such that 601 of the 1,078 nPCR positives were negative by qPCR. For the purpose of further analysis and simulations, these were assigned a density value of half the minimum density (eight parasites/μl) of the 477 samples that tested positive by qPCR. Having corrected for qPCR negative/nPCR positive samples, geometric mean density of infection was 153 parasites/μl ; 56.2% of infections (n = 606) were of a density <100 parasites/μl.Mean household exposure as estimated by distance-weighted nPCR prevalence was 34.5% (range 0–94.7%). Figure vice versa  , using a novel application of qPCR on DNA from filter-paper blood-spots. In contrast to studies conducted over larger geographical areas, this study found that in a moderate transmission setting, at the household level, the proportion of infections defined as subpatent is positively associated with exposure. If similar findings are found in other settings, this suggests that microscopy and RDTs are unlikely to have adequate sensitivity to identify transmission hotspots where acquired immunity allows people to control infections to very low densities; yet these are the exact places one needs to detect cases during a mass screen and treat campaign. Simulations showed that the proportion of infections that are correctly identified and treated can be increased using tMDA. However, this approach still misses a large proportion of infections, suggesting that MDA of entire foci may be required for interruption of transmission.This paper investigates the micro-epidemiology of et al. who estimated that where prevalence of infection is 35% by PCR, around 60% of infections are missed by microscopy [et al., who showed that with increasing proximity to the River Gambia, the prevalence of infection increased but the density of infection decreased [Results of qPCR analysis showed that 56.2% of infections were of a density <100 parasites/μl, the density typically quoted as the limit of detection for routine microscopy and RDTs. This finding fits with the analysis of Okell croscopy . This stecreased .et al. [Multiple factors can influence the relationship between parasite exposure and the density of blood stage infection, and these factors may be more or less apparent at different spatial scales. Potential explanations for a negative association between exposure and parasite density within a focus, seen in this study at a microscale, is that highly exposed individuals acquire greater blood stage immunity, controlling parasite densities more effectively . On the et al.  is that et al. ,37.If, over small geographical regions, density of infection is indeed lowest among those at highest exposure, there are substantial implications for mass screen and treat campaigns that plan to rely on RDTs for diagnosis. As this data suggest, due to a positive association between RDT sensitivity and parasite density, RDTs are likely to display lowest sensitivity in transmission hotspots. Failure to properly target individuals in hotspots may allow transmission to persist. Use of more sensitive diagnostics might be able to circumvent this problem. However, those currently available, such as PCR and LAMP, are not yet practical for widespread routine field use , and point of care serological tests, that may be able to differentiate hotspot households, remain in development. An alternative approach, extrapolating from qPCR data of this study, is to target MDA to those households with highest prevalence of infection by RDT. The simulations show that such an approach increases the proportions of true positives who would be treated. However, even with a threshold of 10% prevalence of RDT positives in a household, where nearly half the population would be treated, an estimated 36.5% of infections would still go untreated.The importance of the subpatent asymptomatic parasite pool rests on the understanding that subpatent infection can transmit malaria. While subpatent infections are known to transmit infection ,39, the Plasmodium malariae and Plasmodium ovale spp. to the density estimates cannot be ruled out [-1) may overestimate the numbers of infections that would be missed, but would not change the overall finding that RDT sensitivity is likely to be least adequate in hotspot households. Thirdly, it is possible that PCR produced some false negatives and false positives which could affect conclusions. That said, false negatives would presumably be due to very low density infections which likely play a minor role in transmission. False positives are unlikely, but may have arisen due to contamination, although all steps were taken to minimize this possibility. Lastly, assumptions were made that, distance-weighted PCR prevalence provides a suitable estimate of exposure and location of hotspots. This assumption is based on the study by Olotu et al. [This study has several limitations. Firstly, the qPCR method applied here is novel, as this is the first study to use qPCR on filter-paper blood-spot DNA to provide an estimate of parasite density in a cross-sectional survey. This was achieved without having to measure the volume of blood used in the assay. This method is yet to be fully validated on filter-paper samples from the field; further, the parasite target used is pangenus, and some contribution from uled out ,30. Secou et al. , which sP. falciparum in Tanzania. The study found a negative relationship between density of infection and exposure, with the proportion of subpatent infections increasing with increasing exposure. Simulations of different tMDA strategies suggest that treating all individuals in households where RDT prevalence was above 20% would increase the number of infections treated from 43 to 55%. However, 45% of infections would remain untreated, suggesting that community-wide MDA may be needed to successfully treat the asymptomatic parasite reservoir in communities such as the one studied.This study has examined the micro-epidemiology of malaria, exploring the spatial relationship between parasite prevalence and density using molecular methods of detection, in a site with moderate transmission of The authors declare that they have no competing interests.JFM was involved in the study design, supervised the implementation of the study and data collection, analysed data, drafted and revised the manuscript. HJWS and BG were involved in data analysis, interpretation of the data, drafted and revised the manuscript. DC and RG were involved in overall study design and supervision, interpretation of the data and revisions of the manuscript. TB, CS and CD were involved in supervision of laboratory work, interpretation of the data and revision of the manuscript. BG and GK were involved in interpretation of the data and revisions of the manuscript. NG and SA performed the real-time PCR testing and revised the manuscript. All authors have read and approved the final version of the manuscript.


Plasmodium falciparum and P. vivax.Although rapid diagnostic tests (RDTs) have practical advantages over light microscopy (LM) and good sensitivity in severe falciparum malaria in Africa, their utility where severe non-falciparum malaria occurs is unknown. LM, RDTs and polymerase chain reaction (PCR)-based methods have limitations, and thus conventional comparative malaria diagnostic studies employ imperfect gold standards. We assessed whether, using Bayesian latent class models (LCMs) which do not require a reference method, RDTs could safely direct initial anti-infective therapy in severe ill children from an area of hyperendemic transmission of both We studied 797 Papua New Guinean children hospitalized with well-characterized severe illness for whom LM, RDT and nested PCR (nPCR) results were available. For any severe malaria, the estimated prevalence was 47.5% with RDTs exhibiting similar sensitivity and negative predictive value (NPV) to nPCR (≥96.0%). LM was the least sensitive test (87.4%) and had the lowest NPV (89.7%), but had the highest specificity (99.1%) and positive predictive value (98.9%). For severe falciparum malaria , the findings were similar. For non-falciparum severe malaria , no test had the WHO-recommended sensitivity and specificity of >95% and >90%, respectively. RDTs were the least sensitive (69.6%) and had the lowest NPV (96.7%).RDTs appear a valuable point-of-care test that is at least equivalent to LM in diagnosing severe falciparum malaria in this epidemiologic situation. None of the tests had the required sensitivity/specificity for severe non-falciparum malaria but the number of false-negative RDTs in this group was small. Plasmodium species P. falciparum in a variety of settings P. falciparumP. vivaxThe World Health Organization (WHO) advocates treatment of malaria based on universal access to light microscopy of blood smears (LM) and/or antigen-based rapid diagnostic tests (RDTs) The currently recommended application of malaria diagnostic tests depends on the clinical situation. PCR-based methods are not yet in routine field use. Quality-assured LM and RDTs are considered equivalent in uncomplicated malaria P. vivax and mixed-species infections are increasingly recognized as important causes of severe disease Plasmodium species There have been few studies comparing diagnostic modalities in severe malaria and these have been conducted in sub-Saharan Africa where falciparum malaria is predominant The application of Bayesian latent class models (LCM) is one approach to addressing the limitations of LM as a gold standard in studies of RDTs. LCM assume no gold standard and the true disease state (disease or no disease) for each individual is unknown. Bayesian approaches to LCM are increasingly used to validate diagnostic tests for infectious diseases without assuming a gold standard P. falciparum and P. vivax, we performed LM, RDTs and nested PCR (nPCR) to diagnose malarial illness in the setting of severely unwell, hospitalised children. Rather than assume that either LM or PCR was the gold standard, we developed a Bayesian LCM to determine the diagnostic performance of each test in the absence of a gold standard. The primary aim of the study was to determine whether the overall diagnostic performance of RDTs was equivalent to, or better than other diagnostic methods for severely ill children in an epidemiological setting where multiple Plasmodium species are transmitted. We also aimed to assess the diagnostic performance of RDTs according to infecting Plasmodium species and presenting clinical features.As part of a prospective study conducted in an area of PNG with hyperendemic transmission of both P. falciparum and P. vivax malaria The present study was performed at Modilon Hospital in Madang Province on the north coast of mainland PNG, an area hyperendemic for both Between October 2006 and December 2009, all children aged 0.5–10 years admitted to Modilon Hospital, the provincial hospital to which the majority of children with severe illness are referred, were assessed for recruitment to an observational study of severe pediatric illness. Inclusion criteria included i) impaired consciousness (Blantyre Coma Score (BCS) ≤4, or ≤2 at 0.5, 1 or 6 hours after correction of hypoglycemia, a seizure or parenteral anticonvulsant therapy, respectively), ii) prostration (inability to sit/stand unaided), iii) multiple seizures, iv) hyperlactatemia (blood lactate >5.0 mmol/L), v) severe anemia (hemoglobin <50 g/L), vi) dark urine, vii) hypoglycemia (blood glucose ≤2.2 mmol/L), viii) jaundice, ix) respiratory distress , x) persistent vomiting, xi) abnormal bleeding, and/or xii) signs of shock. These criteria reflect the World Health Organization (WHO) definition of severe malarial illness P. falciparum histidine-rich protein-2 (PfHRP-2) and aldolase from P. falciparum and non-falciparum malaria species, respectively. All RDTs were read by trained research nurses according to the manufacturer’s instructions. The presence of a single PfHRP-2 and aldolase line was considered diagnostic of mono-infection with P. falciparum and P. vivax, respectively. A positive test for both antigens indicated either P. falciparum alone or mixed infection. Giemsa-stained thick blood smears were examined independently by two skilled microscopists who were blinded to the RDT and nPCR results, with discrepancies adjudicated by a third microscopist. The peripheral blood parasitemia was quantified by counting the number of malaria parasites per 200 leucocytes assuming a peripheral blood leucocyte count of 8,000/µL. After parasite DNA extraction , we performed nPCR Plasmodium DNA and species.A baseline venous blood sample was taken for LM and an ICT Malaria Combo Cassette Test MR2 . This RDT detects Additional on-site tests comprised whole blood glucose  and lactate , a full blood count  and blood culture. We also subsequently assayed plasma creatinine and bicarbonate .After recruitment, a standardized case report form that recorded demographic and medical data was completed. This included details of immunizations, past medical history and recent treatment with antimalarial drugs and antibiotics, as documented in each child’s hand-held medical record book. Clinical examination, inpatient management and follow-up were as described previously 3) possible response profiles. These are denoted in the text and figures by a positive (+) or negative (–) result for each test. Outcomes of the diagnostic tests were assumed to be independent conditionally on the latent classes (disease or no disease). Formal model fitting was performed using Markov-Chain-Monte-Carlo (MCMC) simulations using a Gibb’s Sampler  P-value comparing observed and calculated frequencies together with graphical representations for each response profile  fulfilled the criteria for severe illness and were recruited. The median [IQR] age of this sub-group was 37 [18.3–45.2] months and 56.6% were males. All were of Melanesian racial background. There were 797 children (94.5%) with nPCR, LM and RDT results available and who were included in the present analyses see .P. falciparum by LM was 45,540 /µL whole blood and that in the 44 children with P. vivax was 1,125 /µL. Fifty-seven (7.2%) children died.The clinical features of deep coma, metabolic acidosis, hyperlactatemia, severe anemia and respiratory distress were present in 104 (13.0%), 90 (11.3%), 109 (13.6%), 120 (15.1%) and 210 (26.3%) of children, respectively. The median [inter-quartile range] parasite density in the 299 children with P-value was 0.46, indicating good overall LCM fit and consistent with the assumption that the three diagnostic tests were independent conditional on the patient’s true disease status. The prevalence of malaria, and sensitivity, specificity, PPV and NPV’s for RDT (either test line positive), quality-assured LM (any species) and nPCR (any species positive) using a two-class LCM in which the true disease state for each individual was unknown are shown in The observed and predicted frequencies for different response profiles along with MCMC estimations of 95% credible intervals (95% CI) for any malarial infection are shown in P. vivax mono-infections. RDTs also had the lowest NPVs in these two situations but the estimates were still >96%.Based on the LCM model, the estimated overall prevalence of any severe malaria was 47.5%. RDTs had a similar sensitivity (96.0%) and NPV (96.1%) to nPCR . LM was the least sensitive test and had the lowest NPV (<90% in each case), but it had a higher specificity and PPV than the other two modalities (≥98.0% in each case). The diagnostic performances of all three tests for severe falciparum malaria were similar to those obtained for any severe malaria, consistent with the former group providing most of the cases see . For non2 (four) response profiles for any severe malaria. The model-derived disease prevalence (56% [51–61%]) was higher than that in the LCM involving all three diagnostic modalities, while the sensitivity and NPV of nPCR increased to 99.8% [99.1–100] and 99.8 [98.8–100]), respectively. For the RDT/LM combination, sensitivity (94.4% [88.0–99.7]) and NPV (93.3% [85.0–99.7]) were between the values obtained for RDT and LM when they were considered as individual diagnostic modalities.In some settings, both RDT and LM may be routinely and promptly available. To explore the diagnostic implications of this scenario, a further LCM was generated using RDT and LM results pooled as a single diagnostic modality with either test positive considered an overall positive result. The RDT/LM combination was compared to nPCR in a multinomial model with 2The main presenting clinical features of the patients were deep coma (BCS≤2), metabolic acidosis (plasma bicarbonate <12.2 mmol/L), hyperlactatemia (blood lactate >5 mmol/L), severe anemia (hemoglobin <50 g/L) and respiratory distress . The sensitivity, specificity, NPV and PPV for each of these clinical features by diagnostic test using LCMs is shown in P. falciparum is the major cause of severe malarial illness but P. vivax is also a significant contributor.The ideal diagnostic test is one that is easy to perform and interpret, can be widely deployed, and has high sensitivity and specificity. RDTs for malaria have the potential to provide valuable diagnostic information promptly and cost-effectively when a detailed initial clinical and laboratory assessment of a severely ill child is problematic, such as in a rural setting in a developing tropical country. There is increasing evidence that PfHRP-2-based RDTs are useful alternatives to LM for diagnosing severe falciparum malaria in African children, with sensitivities and NPVs relative to LM between 91–94% and 85–91%, respectively The sensitivity and specificity of a malaria diagnostic test should be at least 95% and 90%, respectively, compared with expert LM P. vivax parasite densities <200/µL in uncomplicated cases P. vivax under controlled conditions and would be better alternatives in future similar comparative diagnostic studies. However, it is important to note that, although the ICT MR2 RDT had the lowest sensitivity for non-falciparum malaria in our study, neither LM nor PCR had a sensitivity >95% in LCM models for mixed-species/P. vivax mono-infections or P. vivax mono-infections alone. Although these two groups contained a relatively small number of children, they have a mortality rate at least that of severe P. falciparum malaria P. vivax and mixed-species infections than severe falciparum malaria in PNG children In the case of severe non-falciparum malaria, the type of RDT assessed in the present study did not perform as well as either nPCR or LM. The ICT MR2 test was selected prior to the publication of the first WHO testing rounds that are now in their fourth iteration and which demonstrated recently that the ICT MR2 had comparatively low sensitivity at Plasmodium species. In the case of P. falciparum, a false negative RDT could be due to either the prozone effect P. falciparum parasitemias >100,000/µL P. vivax may reflect limited antigen availability and low test sensitivity at parasitemias <200/µL. The generally lower specificities of both RDTs and nPCR relative to LM are consistent with persistence of PfHRP-2 and nucleic acids in the absence of viable forms after successful prior treatment.Extrapolating from the LCM model in the 797 children enrolled in which the malaria prevalence was 47.5% and the overall RDT sensitivity was 96%, there was a relatively small number of false negative RDT results (n = 16) that would include children infected with either or both P. vivax malaria in an RDT-negative child.Although the present data show that none of the three diagnostic modalities assessed have optimal performance characteristics in the setting of hyperendemic mixed-species malaria transmission such as found in PNG and other parts of Oceania, as well as in Asia and South America, there appears a clear role for RDTs as a valuable point-of-care test that is at least equivalent to LM in diagnosing severe falciparum malaria. On-site field LM is often not as reliable as the expert LM used as a comparator in the present study, making a further argument for the use of RDTs as part of the initial evaluation of a severely ill child. However, diagnostic tests should only be part of this evaluation. Two important considerations are the prior probability of severe malaria and the presenting clinical features. In areas such as PNG and sub-Saharan Africa where falciparum malaria is the true diagnosis in up to 60% of severely ill children With declining malaria mortality and hospitalizations in Africa


Nutritional iron deficiency may limit iron availability to the malaria parasite reducing infection risk, and/or impair host immunity thereby increasing this risk. In pregnant women, there is evidence of an adverse effect with iron supplementation, but the few reported studies are strongly confounded.A case control study in pregnant Malawian women was undertaken in Chikhwawa southern Malawi in order to describe iron status in relation to placental malaria controlling for several confounding factors. Pregnancy characteristics were obtained and a blood sample at delivery. A full blood count was performed and serum ferritin and transferrin receptor quantified by enzyme-linked immunoassay. DNA analysis was used to identify genetic polymorphisms for ABO phenotype, hemoglobin HbS, and glucose -6 phosphate dehydrogenase deficiency. Placental tissue was obtained and malaria histology classified as active, past or no malaria infection.Plasmodium falciparum infection. In those with acute, chronic or past placental infections the odds ratio for iron deficiency was 0.4, 95% CI 0.2-0.8, p = 0.01; for acute and chronic infections 0.4, 0.2-0.8, p = 0.006; for acute infection 0.3, 0.1-0.7, p = 0.001. The association was greater in multigravidae.112 cases with placental malaria were identified and 110 women with no evidence of placental infection. Iron deficiency was less frequent in women with placental Women with either acute, or acute and chronic placental malaria were less likely to have iron deficiency than women without placental malaria infection There is a priority to establish if reversing iron deficiency through iron supplementation programs either prior to or during pregnancy enhances malaria risk. Plasmodium falciparum transmission, 54.7 million of which occur in areas with stable transmission and 30.6 million in areas with unstable transmission [The influence of iron deficiency on infection risk has been a long-standing clinical concern , relatedIron-malaria interactions in young non-pregnant women also have been little studied and it is unknown whether improving iron status of young women prior to pregnancy living in malaria endemic areas would increase their risk for pregnancy related malaria. Weekly iron and folic acid supplementation for all women of child-bearing age also has been recommended by the World Health Organization , althougPlasmodium vivax infection which was not related to parity [P. falciparum infection in multigravidae with HbAS genotype [P. falciparum malaria at delivery [Only five published studies on the effects of iron treatments or status during pregnancy on the prevalence of maternal malaria have been identified-10. One o parity , anothergenotype . Two uncdelivery ,9, one oP. falciparum infection, which is attributed to parity specific immunity to malaria [Multigravidae have reduced prevalence of  malaria , althoug malaria  and adol malaria . The ratThis was a case control study conducted between January and July 2005 at Montfort Hospital, in Southern Malawi amongst all pregnant women who attended the hospital for delivery and who provided informed consent. Malaria was highly endemic in this area and transmission occurred year round but intensifies during the rainy season from April to December. A total of 647 women were enrolled who attended the hospital for delivery. Women were approached during the first stage of labour and informed consent was requested for participation. A questionnaire was completed by a trained midwife requesting information on age, parity, literacy, obstetric history, bed net and anti-malarial use. Women with emergency obstetric problems , or with recent blood transfusion were excluded. Maternal mid-upper-arm-circumference (MUAC) was measured at the mid-point of the left arm.Following delivery of placenta a tissue sample was obtained by cutting a one cm cube of tissue from both the central and peripheral cotyledons. These were fixed in 10% neutral buffered formalin. Biopsies were embedded in paraffin wax, sliced into thin sections and stained with Giemsa and/or haematoxylin and eosin. Slides were examined at the Histopathology Department, College of Medicine, Blantyre, by two observers, with discrepant findings assessed by a pathologist. Cases were women with placental malaria, and controls those with no evidence of infection. Placental malaria was histologically classified as acute, based on the presence of parasites, chronic, based on the presence of parasites and haemozoin, and past infection if haemozoin alone was present. Active infection included both acute and chronic categories .http://www.coulter.com). Ferritin and sTfR were quantified by enzyme-linked immunoassays . Iron deficiency was defined as a sTfR:log ferritin ratio > 1.6, as this cut-off has been shown to best predict iron deficiency based on bone marrow iron stores in Malawi, an area with high infection pressure [A maternal peripheral blood sample was obtained after delivery and placed in a EDTA tube. Thick and thin blood smears were stained with Giemsa and screened for malaria parasites. A full blood count was performed by an automated Beckman Coulter AcT 8 S/N. AA322060 . There were no significant differences between cases and controls for the following parameters: mean age and parity, proportion with low mid-upper arm circumference (< 23 cm), marital or literacy status, adolescents, frequency of use of insecticide treated bed nets or uptake of IPTp-SP during pregnancy . 44.1% of cases and 42.5% of controls were anaemic (Hb < 11g/dl), There were no differences between cases and controls in prevalence of severe anaemia , G6PD deficiency (mean 18.7%), or sickle cell trait (mean 44.5%), .Blood group O phenotype was less frequent in multigravidae with active , or acute infection  than controls (54.8%), while the converse was true for primigravidae . Sera was available for iron biomarkers for 92 cases and 68 controls. Of cases 57.6% had acute, 9.8% chronic, and 32.6% past infection. Women with either acute (p = 0.001), or acute and chronic (p = 0.006) placental malaria were less likely to have iron deficiency than women without placental malaria infection . Factors in the model were those with P < 0.1 in univariate analysis. These were iron deficiency, parity, ABO phenotype and adolescence. Risk of iron deficiency was reduced for combined infection categories (p = 0.008), active infection (p = 0.007), or acute infection (p = 0.001)Table . In multIron deficiency was less frequent in women with evidence of acute or chronic placental malarial infection indicating that women with placental infection have better iron status than those without. There was no difference in use of IPTp-SP during pregnancy between cases and controls, and the association remained highly significant controlling for maternal ABO phenotype.The importance of these results relates to the influence which maternal iron status has on parity specific malaria immunity, especially as the results were most significant for multigravidae. The cumulative iron requirements of successive pregnancies would increase prevalence of iron deficiency leading to reduced risk for placental malaria in multigravidae. As there is a need to supplement these women with iron this is likely to increase malaria risk despite acquisition of parity specific malaria immunity. These results support the findings from a study in Tanzania based on intervillous blood smears although that study reported malaria was less prevalent among women with iron deficiency especially during the first pregnancy . It willThe calculation of body iron store based on the sTfR:SF ratio provides the best estimate of iron status in adult men and non-pregnant women in populations where infectious disorders are not prevalent. Its utility where women are living under endemic conditions for malaria is less clear and in severely anaemic HIV infected adults had low sensitivity based on bone marrow iron stores . It has P.falciparum parasites; and altered erythropoiesis with down regulation of parasite invasion of young red cells.Four hypotheses might explain these effects of iron status in the context of malaria. These are: the availability of non-transferrin bound iron which might influence the growth of the malaria parasite which may be an acute effect related to recent iron intake or supplementation; an effect on the oxidant/anti-oxidant balance and oxidative free radical reactions in the placental intervillous blood which may alter cellular immune function and macrophage iron metabolism; altered expression of vascular endothelial molecules which may influence adhesion and sequestration of All these women received haematinics of iron (60 mg) and folic acid (5 mg) in short daily courses mostly in the second half of pregnancy. Women were not routinely screened for anaemia at first antenatal attendance or during pregnancy and there was no preferential prescription of haematinics for selected women with anaemia. This is important because women with malarial anaemia could receive therapeutic iron supplements for anaemia treatment, reducing iron deficiency, and then independent of their iron status, these same women may be more likely to have placental malaria due to a higher malaria exposure risk. Although the possibility that some women received additional iron treatments cannot be excluded, the number was nevertheless likely to be small and the policy was to uniformly prescribe haematinics to all women.This study needs to be replicated in women living under different levels of malaria transmission in order to determine the magnitude of these effects in subjects with different levels of malaria immunity. The small sample size and lack of available sera from all subjects limited detailed sub-analyses, for example, placental malaria iron interactions in younger adolescents less than 16 years. A large multi-centre case control study is required to adequately address this.As many millions of pregnant women are exposed to malaria annually there is a priority to establish if reversing such iron deficiency states through routine supplementation programs either prior to or during pregnancy enhances malaria risk especially in areas where malaria control and surveillance is limited. It is particularly important to assess this in adolescents as their malaria immunity is lower than that in older women and many will experience pregnancy while still adolescent.The authors declare that they have no competing interests.ES carried out the field study, participated in the analysis and preparation of the manuscript; GH co-ordinated and participated in the laboratory analyses; GK supported the data preparation and analysis; PK participated in the study design and co-ordination and helped to draft the manuscript; BB conceived the study and participated in its design, co-ordination, analysis and helped draft the manuscript. All authors have read and approved the final manuscript.


Plasmodium falciparum histidine rich protein-2 (PfHRP-2), and PCR provide superior analytical sensitivity and specificity for quantifying malaria parasites infections. This study reports on parasite prevalence, sick visits parasite density and species composition by different diagnostic methods during a phase-I malaria vaccine trial.Compared to expert malaria microscopy, malaria biomarkers such as PfHRP-2 and Plasmodium lactate dehydrogenase (pLDH) ELISAs and real time quantitative PCR (qPCR) were collected during scheduled (n = 298) or sick visits (n = 38) from 30 adults participating in a 112-day vaccine trial. The four methods were used to assess parasite prevalence, as well as parasite density over a 42-day period for patients with clinical episodes.Blood samples for microscopy, PfHRP-2 ELISA  detected higher parasite prevalence than pLDH ELISA  and all methods were more sensitive than microscopy . All microscopically detected infections contained P. falciparum, as mono-infections (95%) or with P. malariae (5%). By qPCR, 102/119 infections were speciated. P. falciparum predominated either as monoinfections (71.6%), with P. malariae (8.8%), P. ovale (4.9%) or both (3.9%). P. malariae (6.9%) and P. ovale (1.0%) also occurred as co-infections (2.9%). As expected, higher prevalences were detected during sick visits, with prevalences of 65.8% (qPCR), 60.5% (PfHRP-2 ELISA), 21.1% (pLDH ELISA) and 31.6% (microscopy). PfHRP-2 showed biomass build-up that climaxed (1813±3410 ng/mL SD) at clinical episodes.During scheduled visits, qPCR  and PfHRP-2 ELISA and qPCR may be needed for accurately quantifying the malaria parasite burden. In addition, qPCR improves parasite speciation, whilst PfHRP-2 ELISA is a potential predictor for clinical disease caused by P. falciparum.NCT00666380ClinicalTrials.gov  Current malaria control goals are ambitious and include elimination and ultimately eradication Plasmodium falciparum histidine rich protein-2 (PfHRP-2) and Plasmodium lactate dehydrogenase (pLDH) Microscopy, the gold standard for malaria diagnosis, has detection limit of 10–50 parasite/µL pLDH ELISA and qPCR detect circulating parasites, and can therefore only account for parasitemia at the time of drawing blood and not for the sequestered parasite population. In contrast, PfHRP-2 is released into circulation from all stages of P. falciparum parasites, and therefore is able to account for trophozoites and schizonts that are sequestered away from peripheral circulation Like microscopy, methods such as PfHRP-2 and pLDH ELISAs and qPCR during a malaria vaccine trial to 1) detect Plasmodium events; 2) determine species composition; and 3) show infection dynamics prior to sick visits and following antimalarial treatment.This paper reports on the performance of microscopy, The protocol for this trial and supporting CONSORT checklist are available as supporting information; see P. falciparum are about 95% during long rains and 75% during the short rains PfHRP-2 and pLDH ELISAs. In addition, samples were taken during unscheduled sick visits, and clinical malaria episodes were treated with oral tablets of artemether/lumefantrine (20 mg/120 mg) given as follows: 4 tablets under direct observation at the time of initial diagnosis, 4 tablets after 8 hours and then 4 tablets twice daily (morning and evening) on each of the following two days. The phase I clinical trial is designed to evaluate safety and not designed or powered to evaluate vaccine efficacy. No difference was shown in the ability of serum to inhibit parasite growth in the vaccinated and control groups and therefore no attempt was made to dichotomize the study samples into vaccinated and controls.The FMP-10 phase I malaria vaccine trial was conducted between December 2008 and June 2009 at the KEMRI/Walter Reed Project Clinical Trial Centre in the Kombewa Division of Kisumu West District, western Kenya . This area is holo-endemic for malaria with peak transmission occurring during the long rains (March–June) and short rains (November–December). Cumulative malaria attack rates for Written informed consent was obtained from all participants involved in the study. Scientific and ethical approvals for the study were obtained from the Kenya Medical Research Institute (KEMRI) Ethical Review Committee (KEMRI SSC # 1337) and the Walter Reed Institute of Army Research (WRAIR) Human Subject Protection Committee (WRAIR # 1417b).Microscopy examination involved thick and thin blood smears that were made as described previously PfHRP-2 and pLDH ELISAs were carried out according to methods described previously PfHRP-2 ELISA were generated from the PfHRP-2 recombinant antigen . pLDH standards curves were generated from recombinant antigen supplied in the pLDH kit  as a positive control. The concentration range of the pLDH standard curve was 0.0423 to 132 ng/mL and 1.9 to 500 ng/mL for PfHRP-2.P. falciparum 3D7 ring stage parasites, obtained by D-sorbitol synchronization of a 3% parasitemia laboratory culture For the qPCR analysis, 200 µL of EDTA blood was used to extract nucleic acids (combined DNA and RNA) using QIAamp® MinElute® Virus Spin kit (Qiagen Inc USA). The samples were eluted in 100 µL elution buffer. Plasmodium using primers and probes targeting the 18S ribosomal RNA gene (18S rRNA) as described previously Quantitative PCR was carried out first for the genus pLDH and PfHRP-2 ELISA and qPCR were compared to microscopy and between each of the methods using odds ratios. As performance of the various techniques depends on parasite density , we have adjusted our analysis for visit day (time since start of the study), and for the interaction between visit day and method. General linear models were used to test whether parasite density estimated by the 4 methods differs with time before onset of disease, and if any time point could serve as a predictor of clinical disease. Robust standard error was used to adjust for repeated measurements.All statistical analyses were performed using SAS Version 9.1 . Generalized Estimating Equations for dichotomous outcome was used to test for differences between the four diagnostic methods in detecting parasite prevalence, because multiple samples of each patient were tested. pLDH and PfHRP-2 ELISA) for the detection of malaria parasites during a vaccine trial. Samples were not dichotomized into vaccinated and control groups as the study showed no differences between the groups. Blood samples of 30 individuals were collected during scheduled visits (n = 298) and were evaluated for parasitemia events by the 4 different diagnostic methods. There were significant differences between the 4 methods in detecting malaria parasite prevalence. Using data from adjusted odds ratio, qPCR  and PfHRP-2 ELISA  performed similarly (P = 0.80) and both detected higher prevalences compared to pLDH ELISA  (P<0.0001). Microscopy  9.6–17.3%) detected lower prevalences than the three other methods (P<0.05)  .P. falciparum was the most common malaria parasite species and was present in all samples diagnosed positive by microscopy (N = 40), either as mono-infections  or in combination with P. malariae . Of the 119 samples positive by genus qPCR, 102 were successfully amplified by the species-specific qPCRs. P. falciparum was present in 89.2% of these infections, 71.6% (N = 73) as mono infection, 8.8% (N = 9) with P. malariae, 4.9% (N = 5) with P. ovale and 3.9% (N = 4) with P. malariae and P. ovale. P. malariae and P. ovale mono-infections accounted for 6.9% (N = 7) and 1.0% (N = 1) respectively and 2.9% (N = 3) for co-infections. P. vivax was not detected in any of the samples.PfHRP-2/pLDH ELISAs, and qPCR. Only twelve samples (31.6%) had microscopically confirmed malaria parasites and these patients were treated with artemether/lumefantrine as described in the methods section. Following treatment, patients continued to be evaluated for malaria during the scheduled visits.We have evaluated the utility of the 4 diagnostic methods in quantifying malaria parasite load in patients with clinical signs suggestive of malaria. A total of 38 acute care malaria blood smears were requested by the clinical team over the 112 days follow-up period for study volunteers presenting with any of the following indicators of malaria: fever (ancillary temperature >37.5°C), headache, backache, malaise and generalized body pain. Blood samples from these patients were analyzed by microscopy, PfHRP-2 ELISA), 21.1% (pLDH ELISA) and 31.6% (microscopy). PfHRP-2 ELISA and 7/12 (58.3%) for the pLDH ELISA. Two microscopy confirmed cases were negative for all three other diagnostic methods, suggesting that the parasites identified by microscopy may have been misdiagnosis, which would suggest sensitivities to be 70% (pLDH) and 100% (qPCR and PfHRP-2 ELISA). In addition, PfHRP-2 ELISA and qPCR detected over double the amount of malaria cases detected by microscopy and pLDH ELISA, most likely as these methods detect parasites densities way beyond the detection limit of microscopy and PLDH ELISA; also PfHRP-2 ELISA detects the antigen secreted by parasites in previous multiplication cycles within the infection, allowing an estimate of cumulative parasite biomass.As participants were clinically unwell, the probability of malaria parasites in samples taken during these visits is higher, with prevalences of 65.8% (qPCR), 60.5%  on the day the patients reported being sick. Following anti-malarial treatment (day 0), PfHRP-2 levels declines slowly and remains detectable up to 21 days post treatment, whilst the other methods cannot detect parasites or parasite products on days 7, 14 and 21 post treatment. As shown in PfHRP-2 and/or qPCR. Their mean PfHRP-2 levels at the day of presentation (day 0) was 260 ng/mL and respectively 134 ng/mL, 253 ng/mL and 12 ng/mL on days 7, 14 and 21 before disease onset.PfHRP-2 and pLDH ELISAs and qPCR for detecting Plasmodium events and evaluates parasite density build-up that culminates in malaria attributable sick visits in a cohort participating in a 112-day malaria vaccine trial. Each of these methods has particular attributes that appeal to different end users. Microscopy is the gold standard for malaria diagnosis, it is cost effective and simple to use but has a low sensitivity. When not done well, its main drawback includes poor reproducibility, variable sensitivity, and unacceptably high false-positive rates pLDH is a secreted antigen that is found in all Plasmodium species, and because it is short lived in blood, it is a good indicator of current infection. However, pLDH is produced in small quantities and this limits its sensitivity, especially for the non P. falciparum species whose parasitemias are usually low. In contrast, PfHRP-2  P. falciparum parasites PfHRP-2 is the mainstay for P. falciparum rapid diagnostic tests. PfHRP-2 is water soluble and its blood level originates from parasites in the peripheral circulation as well as from those sequestered in endothelial vessels and in organs such as liver, kidneys, brain and placenta PfHRP-2 has a long half-life and therefore can account for historical parasitemia. Because of these attributes, circulating levels of PfHRP-2 reflect the total cumulative parasite biomass pLDH, but unlike PfHRP-2, qPCR only detect current parasitemias and therefore cannot account for diurnal fluctuation or for mature trophozoite and schizont stages that are unavailable in the peripheral circulation.This study provides a dataset for judging the performance of microscopy, PfHRP-2 ELISA (36.9%) and qPCR (39.9%) . However, some studies have reported lower sensitivity of PfHRP-2 assays compared to PCR in pregnant women at delivery PfHRP-2 assays is regularly observed PfHRP-2 as a diagnostic tool is that reported PfHRP-2 gene deletions in parasites from South America While the number of subjects taking part in the study is small (N = 30), the numbers of samples available for analysis for the 112 days follow-up period is large (N = 298), and by using statistical methods that account for repeated measurements, we were able to fully benefit from this sample size. Our results show that microscopy detects the lowest proportion of individuals with non-clinical malaria (13.4%), followed by pLDH ELISA (16.8%) and  (39.9%) , Table 2pLDH ELISA and microscopy indicated that approximately 20% of the participants had malaria parasites (PfHRP-2 ELISA. This enrollment parasitemia reflects the timing at the end of the short transmission season with 33% of infections being submicroscopic (PfHRP-2 positive and qPCR positive) and a considerable proportion of individuals having signs of recent but not current infection (PfHRP-2 positive but qPCR negative). The parasite prevalence declines over time, with more substantial declines measured by PfHRP-2 ELISA (45%) and qPCR (15%), compared to almost no decline for microscopy and pLDH ELISA . Our unreported data show that, malaria infected red blood cells have up to 10 times more PfHRP-2 than in the plasma. In the future, and as part of standardizing of these assays, sample choice and genetic polymorphisms will need to be evaluated.Clearly, microscopy detects only the proverbial ears of the hippo while the massive body of parasites remains sub-microscopic. These data are corroborated by other studies that show that microscopy considerably underestimates parasite rates by up to 50% PfHRP-2 ELISA demonstrates a cumulative parasitemia that is highest at the time patients reported to investigators with clinical malaria and high parasite densities. For this reason, and as reported recently PfHRP-2 levels may be useful for predicting potential clinical episodes in prospective research studies. In the Hendriksen study PfHRP-2 concentration of >1000 ng/mL was shown to represent “true” severe malaria and had a case fatality rate of >10%. In our study, we are proposing that a blood PfHRP-2 concentration of >1813 ng/mL as an indicator of a true clinical malaria. In comparison, 26 individuals who reported sick but did not have detectable malaria parasites by microscopy showed mean PfHRP-2 levels at the day of presentation (day 0) that was four times lower than those with clinical malaria. Although 16 of these individuals had submicroscopic parasitemia by PfHRP-2 and/or qPCR  will be needed for accurate evaluation of the stated goals.The study described here reports on the combined performance of four diagnostic methods of malaria for monitoring parasite prevalence in a longitudinal study set up. The findings illustrate scores of submicroscopic Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.