P. falciparum is frequent in areas endemic for malaria and individual diagnosis of clinical malaria attacks is still difficult. We investigated the impact of changes in malaria endemicity on the diagnostic criteria for malaria attacks in an area of seasonal malaria transmission.Asymptomatic carriage of Plasmodium falciparum parasitemia and the risk of fever, with the aim of determining the best parasitemia thresholds for attributing to malaria a fever episode.We analyzed the longitudinal data collected over 20 years from a daily survey of all inhabitants of Ndiop, a rural community in central Senegal, in a logistic regression model to investigate the relationship between the level of P. falciparum asymptomatic carriage declined from 36% to 1% between 1993 and 2013. A total of 9,819 fever episodes were associated with a positive blood film for P. falciparum. Using age-dependent parasitemia thresholds for attributing to malaria a fever episode, we recorded 6,006 malaria attacks during the study period. Parasitemia thresholds seemed to be lower during the low-to-zero transmission season and tended to decrease with changes in control policies. The number of clinical malaria attacks was overestimated for all age groups throughout the study when all fever episodes associated with P. falciparum parasitemia were defined as malaria attacks.A total of 34,136 observations recorded from July 1993 to December 2013 from 850 individuals aged from 1 day to 87 years were included. Pyrogenic thresholds are particularly sensitive to changes in malaria epidemiology and are therefore an interesting tool to accurately assess the burden of malaria in the context of declining transmission. In recent years, the scale-up of new treatments and effective prevention tools has led to major advances in the fight against malaria ,2. HowevThe measurement of parasite density has long been the cornerstone of the approaches to assess malaria morbidity in research and clinical trials in endemic areas ,5. MethoP. falciparum malaria attacks in all age-groups [P. falciparum malaria attack on the road towards malaria elimination.The level of endemicity has also been shown to critically influence the pyrogenic parasitemia thresholds ,13. In tThe study was performed in Ndiop, a village located in central Senegal approximately 290 km southeast of Dakar and 10 km from the Gambian border . The area is characterized by an average annual rainfall of 750 millimeters, concentrated during the rainy season between June and October, followed by a dry season of seven to eight months.A prospective longitudinal study was carried out from July 1993 to December 2013 to investigate the relationship between malaria host, vector, and parasite. Both active and passive detection of fever cases was performed and regular cross-sectional surveys of malaria prevalence conducted.The procedures of medical, parasitological, entomological, and epidemiological surveillance were the same as those used in the village of Dielmo and have already been described ,14.Briefly, inclusion in the cohort was offered to all villagers at the beginning of the study, and to any newcomer thereafter. During twenty years from July 1993 to December 2013, all households included in the study were visited daily except Sundays and nominative information on the inhabitants, including the presence of fever or other symptoms  were recorded. Blood tests were performed for all suspected or confirmed cases of fever. Asymptomatic malaria carriage was investigated by performing cross-sectional surveys for all included individuals at regular intervals: every week during the first six months of the study, then every month unless rare exceptions, and finally three times a year from 2004 onwards: at the beginning and end of the dry season , and at the end of the rainy season (October or November). Blood was taken by finger stick and 200 oil-immersion fields were examined. The parasite/leukocyte ratio for each plasmodial species was measured. For readability and clinical relevance reasons, parasitemia was expressed in trophozoites/μl: to overcome the lack of simultaneous measurement of leukocytemia, we adopted a mean standard leukocyte count of 8,000 per μl of blood for all age groups, following WHO recommendations and in accordance with data from a preliminary investigation in the study area ,16.Four first-line drug regimens were successively used for malaria attack treatment during the study period: oral quinine (July 1993 to December 1994), chloroquine (January 1995 to October 2003), sulfadoxine-pyrimethamine plus amodiaquine (SP+AQ: November 2003 to May 2006), and artesunate plus amodiaquine (Artemisinin-based combination therapy or ACT: from June 2006). Until 2011, antimalarial drugs were systematically given to young children (less than 5 years old) with fever associated with a parasitemia ≥ 2400 trophozoites/μl, whereas the choice to treat or not with antimalarial drugs was left to the appreciation of the physician or nurse when parasitemia was lower. Only symptomatic treatment was generally given to adults and older children permanently living in the village (except for pregnant women). This attitude was justified by the willing to limit parasitic resistance to antimalarials at that time, and by the fact that a number of fever cases with positive parasitemia did not necessarily correspond to malaria, in a context where the close surveillance of the villagers allowed immediate reactivity in case of clinical worsening. The treatment policy was modified in 2011 to limit malaria transmission and since then, ACT has been systematically given to all febrile patients showing the presence of trophozoites in blood films, independent of age and parasite density.LLINs were deployed in Ndiop in July 2008, at the time of their distribution throughout the country. Malaria transmission averaged 43 infected bites per person per year before the introduction of combination therapy, with high annual variations (maximum 169 in 2001), and steeply decreased to very low levels after the deployment of LLINs (average of five for the period from 2008 to 2013) .Parasite prevalence was measured during cross-sectional studies in all villagers enrolled in the project. Comparisons of the prevalence rate between seasons were analyzed using the Fisher exact test to investigate seasonal variations.Febrile cases were identified by active and passive surveillance and compared with asymptomatic controls, identified during cross-sectional investigations. We included only individuals who had participated in at least four consecutive cross-sectional surveys in the period from 1993 to 1997 in the analysis, when these studies were performed monthly, or at least three surveys in one year during the subsequent years. We excluded observations performed during pregnancy and those showing the presence of two or more parasitic species. We established a baseline during the first six months of follow-up to have a reference period, keeping only observations performed before the first antimalarial intake for each individual. Definitions of case and control were the same as those used for the previous work about the Dielmo cohort . BrieflyP. falciparum parasitemia. Odds ratio estimated by the model could be used to measure the association between risk of fever and parasitemia variation. Parasitemia could affect the risk of fever as a continuous variable and also as a binary one: the existence of a threshold effect had been previously demonstrated [ whose values could be estimated by successive fits, as previously described [The same previous method for pyrogenic threshold calculations applied to the Dielmo cohort was used . Risk ofnstrated ,13. Our escribed ,13. The k = 5 dummy variables for the following age groups: 0–23 months, 24–59 months, 5–9 years, 10–14 years, and ≥15 years. Previous studies had shown that parasitemia thresholds for attributing fever episodes to malaria decreased in relation to control policies and decrease in transmission [At first, bivariate analysis was performed to explore the association between risk of fever, parasitemia and age. Comparing several age groups with each other, the best fit based on deviance criterion was obtained using a series of smission ,13. The smission . Analysesmission ,19.zik (with k representing the five age-groups) and parasitemia xij  as a continuous function of parasitemia, different functions were tested: linear, log and power of x (parasitemia). The best criterion of model selection (deviance) was obtained for the rth power function of parasitemia. The parsimonious exponent r was then tested and obtained based on the model deviance for each treatment period and within each treatment period for both transmission seasons for different values with a precision of 0.01  was used in regression analysis to describe the relationship between age and parasitemia  as previously described [a as age of maximum parasitemia, b as the highest parasitemia, c as the parasitemia at age 0, d as the level of parasitemia in the oldest adults, and e as the shape of the decrease. The first equation (h1) concerned the youngest children before age a , whereas the second (h2) was applied to older children and adults after age a:asitemia  Figs. Thescribed ,13. We i on a defined set, we obtained many combinations enabling us to define a binary threshold variable. This variable was coded in 1 or 0 according to whether the parasite density was below or above the curve of h1 and h2, and introduced into the By varying these five parameters P. falciparum clinical malaria attack was defined as any case with fever or fever-related symptoms for which parasitemia was higher than the threshold derived from the above model. Cases were counted separately if they occurred 15 days or more apart.A We then tested four parasitemia definitions and compared the number of malaria attacks determined by each: (A) episode of illness (fever or fever-related symptoms) with parasitemia higher than the age-dependent threshold measured for the corresponding season and treatment period; (B) episode of illness with parasitemia higher than the age-dependent threshold measured for the corresponding treatment period, regardless of the season; (C) episode of illness with parasitemia higher than the constant threshold of 5,000 trophozoites/μl, frequently used in malaria endemic areas; and (D) episode of illness associated with the presence of malaria parasites, regardless of the level of parasitemia.ad-hoc committees of the Ministry of Health, The Pasteur Institutes (Dakar and Paris), and the Institut de Recherche pour le Développement (IRD) regularly performed audits.The project was initially approved by the Ministry of Health of Senegal and the assembled village population. Approval was then renewed on a yearly basis with written informed consent from individuals enrolled in the project and the parents or guardians of the children enrolled. The National Ethics Committee of Senegal and We measured parasite prevalence in 41,334 blood films collected during 159 cross-sectional surveys from all present villagers, irrespective of clinical symptoms. Before the beginning of the project, a preliminary survey carried out in June 1993 (end of the dry season) showed a parasite prevalence of 17%. We observed that prevalence reached 39% on average during the quinine period (which includes two rainy seasons and one dry season) and remained high during the chloroquine period. It started to decrease after the beginning of the combination therapy period , and fell further after the deployment of LLINs (1%). There were consistently wide variations between seasons: parasite prevalence was significantly lower during the low-to-zero malaria transmission season. This difference was found for each treatment period, except at the last years of the study upon the distribution of LLINs .P. falciparum attack. A total of 34,136 observations recorded from July 1993 to December 2013 from 850 individuals aged from 1 day to 87 years were included: 22,827 observations matched the definition for a case of fever and 11,309 for that of a control observation.We excluded 176 individuals (453 observations) because of insufficient follow up, 1,246 observations performed during pregnancy, and 3,201 observations with a thick blood film positive for two or more parasitic species when determining the parasitemia thresholds used to define a P. falciparum trophozoites and the mean level of parasitemia during fever episodes decreased markedly during the 20 years of the study , then decreased during the bitherapy period (62%) and more so after the introduction of LLINs (26%). During the low-to-zero malaria transmission season, the prevalence of fever associated with positive parasitemia was lower: 32%, 44%, and 28% during the quinine, chloroquine, and bitherapy periods respectively, and 4% in years following introduction of LLINs.The proportion of fever cases harboring he study . Parasitvs 32% in the high transmission season during the quinine period, 16% vs 19% during the chloroquine period, and 8% vs 20% during the bitherapy period. Asymptomatic carriage further declined after the introduction of LLINs and was low in both seasons: 1% during the low-to-zero transmission season vs 1% during the high transmission season.Parasite prevalence and the mean level of parasitemia during asymptomatic infections are shown for each period, season, and age group in sij in the model: we found a significant association for each study period. Parameters based on the lowest deviance that define the shape and level of pyrogenic thresholds during the baseline and each season for the four treatment periods by age are given in Our data showed the existence of two distinct groups based on parasite density, which suggested a possible existence of a parasitemia threshold between fever and no fever cases . For eacModeling failed to identify a specific threshold for the low-to-zero transmission season of the ACT+LLINs period, due to the small number of observations with positive parasitemia (38 observations) such that the relationship between parasitemia and risk of fever was no longer significant. There was thus a single threshold for the ACT+LLINs period, independent of the season.The P. falciparum, within which 6,006 could be attributed to malaria using the age-dependent parasitemia thresholds. Overestimation was greatest during the quinine and chloroquine periods during the low-to-zero transmission season , and lowest during the ACT+LLINs period, regardless of the season (25% and 21%). When a constant threshold of 5,000 trophozoites/μl was used (definition C), the number of clinical malaria attacks for the quinine and chloroquine periods was underestimated during the low-to-zero transmission season , but overestimated during the high transmission season , with disparities depending on age. This threshold led to an underestimation of the number of attacks for almost all age groups for the bitherapy period, regardless of the season. Finally, the use of a season-independent threshold led to a substantial overestimation of malaria attacks for the quinine and chloroquine periods .We compared the four definitions of clinical malaria attack. Considering definition A as the reference (episode of illness with parasitemia higher than the age-dependent threshold measured for the corresponding season and treatment period), definition D  considerably overestimated the number of clinical malaria attacks for all age groups and all periods . During In this cohort of villagers living in an initially moderate and seasonal malaria transmission area, we observed that malaria epidemiology and the pyrogenic parasitemia thresholds guiding the definition of malaria attacks tend to change over time. These results corroborate those observed in Dielmo, a neighboring locality with initial intense and perennial malaria transmission and where the same treatment policies and control measures were implemented in parallel ,19.P. falciparum prevalence was 69% during the first year of the study in 1990 (vs 26% during the first full year of study in Ndiop), and fell to 0.3% in 2012 [Parasite prevalence progressively decreased with the implementation of new malaria control strategies. It decreased in the early years of the study, possibly due to the project itself, which resulted in more administered treatments. Thereafter, it decreased when combination therapy replaced chloroquine as first-line treatment. Finally, the most important decline followed the deployment of LLINs: parasite prevalence fell to 1% by the end of the study, and gametocyte carriage to 0.2%. These changes mimic those observed in Dielmo, where mean Asymptomatic carriage was more frequent during the first two periods than during subsequent periods, but parasite densities were lower: high prevalence of low density carriers in all age groups could reflect robust immunity acquired before the beginning of the survey. During the study, the peak prevalence of fever with presence of trophozoites on blood film (regardless of the level of parasitemia) shifted gradually towards older children and young adults. In correlation with the dramatic decline observed in asymptomatic carriage, this result highlights the fact that reduced exposure consecutive to malaria control intensification is reflected in waning immunity in adults. It illustrates that was observed in serological studies .The pyrogenic parasitemia thresholds seemed to vary substantially during the study period and to be highest during the high transmission season in all periods except the last years, although the method used here do not allow us to demonstrate a significant difference between periods and between seasons. Pyrogenic threshold is an old concept in malariology . It invoMalaria control strategies seem to modify pyrogenic parasitemia threshold levels, as previously observed in Dielmo . The onlP. falciparum during the dry season in areas of seasonal malaria transmission [Pyrogenic threshold levels varied depending on the season: they tended to be lower during the low-to-zero transmission season. Similar results have been reported in areas of similar endemicity in several studies with various approaches to determine the parasitemia thresholds used for case definition of malaria attack ,11,22,23smission –26.Our study shows the persistence of a pyrogenic threshold during the ACT+LLINs period, although malaria became hypoendemic and asymptomatic carriage quite rare. This may reflect, in part, the long-term persistance of some immunity as observed in immigrants exposed to malaria after years or even decades without contact with the parasite . The rolOur results confirm that parasite density is a key determinant of malaria morbidity in endemic areas with seasonal transmission. Defining malaria attacks as all fever cases associated with any level of parasitemia overestimated the malaria burden for all age groups in an endemic area. This was observed during all study periods and remained true during the last years of the study, although to a lesser degree, when malaria became hypoendemic.a, b, c, d, and e estimated by the model remained unchanged whether the random-effect was included or excluded.It must also be acknowledged that the choice to group data by periods did not take into account dynamic changes within treatment periods, such as variations observed in annual parasite prevalence, wear and tear of the nets, or changing in clinicians appreciation to treat or not with antimalarial drugs a fever case with low parasitemia. However, implementation of new strategies at the village level seemed to be the most relevant breaking point for study epidemiological changes. The thresholds determined for Ndiop may not be extrapolated to other settings with similar endemicity: malaria transmission is spatially heterogeneous and a recent study showed the complexity of such spatial distribution in eight neighboring villages of Ndiop [This study has several limitations. First, it is well known that there are rapid changes in parasite density during the same fever episode . Followiof Ndiop . FinallyThe pyrogenic parasitemia threshold model is applicable to cohort studies in the context of seasonal malaria transmission, as previously documented for intense and perennial transmission settings. Pyrogenic thresholds are not fixed and particularly sensitive to the evolution of the epidemiological profile, and are therefore an interesting tool to accurately assess the burden of malaria in the context of declining transmission.S1 FigA: Parasite density by age (loess curves in solid red line), B: zoom of A on low values.(TIF)Click here for additional data file.S2 Figr is the exponent of the power function of parasite density used for modeling the relationship between parasitemia and fever risk as a continuous function.The parameter (TIF)Click here for additional data file.S3 FigThe thresholds presented for Ndiop are those of the high transmission season.(TIF)Click here for additional data file.


In sub-Saharan Africa, where there is the scarcity of proper diagnostic tools, febrile illness related symptoms are often misdiagnosed as malaria. Information on causative agents of febrile illness related symptoms among pastoral communities in Ethiopia have rarely been described.Salmonella enterica serotype Typhi (S. Typhi) and Rickettsia infections. Rose Bengal Plate Test (RBPT) and Complement Fixation Test (CFT) were used to screen Brucella infection. Thin and thick blood smears were used to diagnosis malaria.In this a community based cross-sectional survey, we assessed the prevalence of typhoid fever, typhus, brucellosis and malaria among individuals with a set of given symptoms in Amibara district, Afar Region, Ethiopia. Blood samples were collected from 650 study participants, and examined by Widal and Weilfelix direct card agglutination test (DCAT) as well as test tube based titration test for S. Typhi infection. Among these, 46 (55.4%) were reactive by the titration test at the cut off value ≥ 1:80. The combined sero-prevalence for S. Typhi by the two tests was 7.3% (46/630). The seroprevalence for Rickettsia infection was 26.2% (165/630) by DCAT and 53.3% (88/165) by the titration test at the cut off value ≥ 1:80. The combined sero-prevalence for Rickettsia infection by the two tests was 14.0% (88/630). The sero-prevalence for Brucella infection was 12.7% (80/630) by RBPT, of which 28/80 (35%) were positive by CFT. The combined sero-prevalence for Brucella infection by the two tests was 4.4% (28/630). Out 650 suspected individuals for malaria, 16 (2.5%) were found positive for P. falciparum infection.Out of 630 sera screened by DCAT, 83 (13.2%) were reactive to H and/or O antigens for In this study, typhoid fever, typhus, brucellosis and malaria were observed among symptomatic individuals. The study also highlighted that brucellosis cases can be misdiagnosed as malaria or other disease based solely on clinical diagnosis. Therefore, efforts are needed to improve disease awareness and laboratory services for the diagnosis of brucellosis and other zoonotic diseases to identify other causes of febrile illness in this pastoral setting. Many diseases such as typhoid fever, typhus, brucellosis and malaria show common symptoms such as fever, headache, joint pain and back pain. Hence, in countries where there is a problem of appropriate laboratory based diagnostic tools, health workers cannot properly diagnose these diseases and provide appropriate treatment. A community- based studies of the causative agents of the above mentioned illness would provide important information for health workers about some of the common causative agents in that particular area. In this study, we assessed the prevalence of typhoid fever, typhus, brucellosis and malaria among individuals who were complaining illnesses such fever, headache, joint pain and back pain in the pastoral of the Amibara district, Afar Region, Ethiopia. Among 650 individuals who were complaining symptoms, 46 (7.3%), 88 (14.0%), 28 (4.4%) and 16 (2.5%) were diagnosed for typhoid fever, typhus, brucellosis and malaria in that order. However, for the majority of the participants (75.4%), the cause of their illness remained unknown, and further investigations on the causative agents of febrile illness related symptoms is important in the present study area. Sub-Saharan Africa is plagued by a myriad of infectious diseases posing significant public health and economic challenges. In addition, the often non-specific clinical signs of these diseases and the scarcity of proper diagnostic tools are the major challenges for health professionals in properly diagnosing and treating adequately patients , 2. StudS. Typhi has been reported as the leading cause of over 21 million febrile cases and over 200,000 deaths each year in many low- and middle-income countries [Many studies have shown that diseases such as typhoid fever, rickettsioses, brucellosis, Q fever, and leptospirosis are the leading causes for febrile illness with symptoms such as fever, headache, joint pain and back pain –9. For iountries –9.Brucellosis has been considered as an important zoonotic disease worldwide and is responsible for big economic losses as it causes abortion in livestock , 11. It Although many malarious countries including Ethiopia are scaling up malaria intervention programs towards elimination, the disease remains one of the worst health problems with an estimated 216 million cases and 445 000 deaths globally in 2016, while most of the cases and deaths occurred in African [In Ethiopia, there have been few health facilities based studies to determine the prevalence of typhoid fever, typhus, malaria and brucellosis among individuals presented with febrile illness related symptoms –19. CommThe study was conducted in the pastoral community of Amibara district in the Afar Region of Ethiopia, around 260 km from Addis Ababa. The majority of the study population are pastoralists, depending on livestock for their livelihoods, while some started to practice agro-pastoralism and growing crops along river Awash. The study area and the population has been previously described in detail , 21.A community based cross-sectional survey was carried out between September and December 2016 to determine the prevalence of typhoid fever, typhus, brucellosis and malaria among individuals who were complaining of a range of symptoms. The result of previous health facilities based sero-prevalence of brucellosis (34%) in other pastoral community areas of Ethiopia was used to estimate the sample size  for the In this study, six accessible pastoral kebeles of the district were conveniently selected, and a house-to-house survey of all households in the selected kebeles was conducted by community health workers under the supervision of the research team. The heads of the households (husband or wife) or individuals over 18 years were asked if there was any family member (age ≥ 2 years) who manifested symptoms such as fever, fatigue, headache, joint pain, and back pain for short or long periods of time. The individuals were asked to come to the nearest health post, and they were interviewed in their local language (Afar language) using a structured questionnaire that captured common signs/symptoms they felt, onset of illness, treatment sought, and information on socio-demographic characteristics of the individuals. Body temperature was also recorded using a digital thermometer. All individuals equal or older than 2 years, who reported the above symptoms, came for examination and willing to provide blood, and gave informed consent and/or assents were included in the study.Three ml venous blood sample was collected into plain vacutainer test tube and transported to Melka Werer Health Center. Serum was separated and tested for typhoid fever, typhus and malaria on the same days. The remaining serum was stored at -20°C until transported to the laboratory of Aklilu Lemma Institute of Pathobiology, Addis Ababa University and tested for brucellosis and also tested for typhoid and typhus by test tube based titration method. Anemic individuals and pregnant women were included in the malaria study and provided only finger prick blood sample.S. Typhi and Rickettsia infections, respectively following the manufacturer’s instructions , and as previously described [Brucella infection as previously described [Widal and Weilfelix direct card agglutination tests (DCAT) were used for the serological screening of escribed , 23. A tescribed . Rose Beescribed . All serescribed . The guiescribed .Data was entered into EpiData 3.1 and analyzed with Stata/SE 11.0. Descriptive analysis was used to summarize the data in the form of frequencies and percentages of variables. Pearson chi-square test was used to evaluate the statistically significant difference in the level of prevalence of typhoid fever, typhus, brucellosis and malaria between male and female study participants and according to the reported clinical features. Bivariable and multivariable logistic regression analyses were performed to explore associations of socio-demographic characteristics of the study participants with increased odds of having higher prevalence of typhoid fever, typhus, brucellosis and malaria. P-value below 5% was considered as indicator of statistical significance.This study received ethical clearance from the Institutional Review Board of Aklilu Lemma Institute of Pathobiology, Addis Ababa University (ALIPB/IRB/005/2015/16)). Permission was obtained from Amibara Health Office. Participants’ information sheet which contains the objective of the study, inclusion/exclusion criteria, the required data and methods of data collection as well as informed consent document were prepared in Amharic the national language of the country. Then, the elements of participants’ information sheet initially were orally translated to the local language and described to community leaders and to each of the study participant or parent in case of children under 18 years by trained local health personnel. Informed written consent was obtained from illiterate participants and/or assent in children aged between 12 and 18 years by signing with their finger. Blood sample was collected under aseptic condition by experienced laboratory technicians. Study participants who were found positive for the investigated diseases were treated accordingly as per physician recommendation.A total of 657 individuals who were complaining various symptoms such as headache, joint pain fever and back pain appeared for clinical examination. However, seven individuals were not volunteers to consent to provide blood sample and they were excluded. Out of the 650 study participants, 630 provided venous blood and 20 provided finger prick blood sample due to anemia and/or pregnancy. The participants’ age ranged from 2 to 80 years with mean age 34.25 ±17.38 years. The majority were illiterate (75.2%) and pastoralists (78.8%) .S.Typhi infection either against flagella (H) antigen  or against somatic (O) antigen  or against both H and O antigens .Out of 630 sera screened by the DCAT, 83 (13.2%) were reactive for S. Typhi as indicated either by H and/or O antigen was considered as 7.3% (46/630) at the cut off value ≥ 1:80. The cases were more common among females than among males  as detected by DCAT and by the titration test . All the randomly selected 25 samples which were non-reactive by DCAT were also found non- reactive by the titration test.Among the reactive sera to O antigen, 17(19.54%), 8(9.20%), 1(1.2%) and 1(1.2%) were reactive at the titration of 1:80, 1:160, 1:320 and 1:640, respectively. Among the reactive sera to H antigen, 15 (22.73%), 9 (13.64%) and 1(1.52%) were reactive at the titration of 1:80, 1:160 and 1:320, respectively. Thus, the overall sero-prevalence of current infection with S. Typhi infection  and duration of illness above a month  were found to be associated with a high sero-positivity for nfection .Rickettsia infection by DCAT, 165 (26.2%) were reactive. Out of these sera, 41(21.8%), 33(17.6%), 9(4.8%) and 5(2.7%) were reactive at the titration of 1:80, 1:160, 1:320 and 1:640, respectively. Hence, 88 (53.3%) samples were reactive by the titration test at the cut off value ≥ 1:80. The combined sero-prevalence for Rickettsia infection by the two tests was 14.0% (88/630). The sero- prevalence was frequent among females compared to males  by DCAT, as well as by titration test . It was also higher among those individuals who reported headache compared to who did not  by DCAT and  by titration test. All the 25 samples which were non-reactive by DCAT were also non-reactive by titration test.Of the 630 sera screened for Rickettsia infection  and reporting headache  were significantly associated with sero-positivity for nfection .Brucella infection among the study participants was 12.7% (80/630) by RBPT and 35% (28/80) by CFT. The combined sero-prevalence for Brucella infection by the two tests was 4.4% (28/630). The sero-prevalence for Brucella infection was relatively high in the age group between 2–14 and 15–24 . In multivariable logistic regression analysis, agropastoralism by occupation was associated with a high risk  for Brucella infection. None of the 68 samples which were negative by RBPT was found positive by CFT.In the univariable logistic regression analysis; being children  was found to be associated with high seropositivity for Brucella infection was not significantly associated with clinical symptoms reported by the study participants  were found positive for P. falciparum malaria cases were more common among males than among females . The case was also high in the age group between 2–14 years  and among individuals with body temperature ≥ 37.5°C . It was also high among individuals felt the illness for a week or less . Multivariable regression analysis showed that being a male  and having fever ≥ 37.5 °C  were independently associated with increased odds of having P. falciparum malaria infection  were found to be positive for one or more of the infectious agents by the screening tests . However, only 24.6% (160/650) were found to be positive for one or more of the infectious agents by the confirmatory tests .Among the total 650 study participants who were tested for S.Typhi, Rickettsia, Brucella infection by confirmatory tests, and/or positive for P. falciparum infection by microscopy. This result is in line with previous health facility based studies in other parts of Ethiopia on causes of febrile illnesses [We investigated the prevalence of typhoid fever, typhus, brucellosis and malaria among individuals reported signs of fever, headache, joint pain and back pain in Amibara district, Afar Region, Ethiopia, through a community-based cross-sectional study. A quarter of the individuals (24.6%) were sero-positive for llnesses , 19, andRickettsia infection in this study was lower than the one reported from other parts of Ethiopia [Rickettsia infection was high among individuals reported headache. Headache has been shown to be one of the main clinical symptoms of endemic typhus [Rickettsia infection was more common among females than among males. This high sero-prevalence for Rickettsia infection among females could be due to the large number of female study participants involved in this study. A previous retrospective sero-prevalence study of typhus among prisoners in Ethiopia also showed a higher seroprevalence among males than among females which could be due to a higher proportion of male study participants (86%) compared to that of female study participants (14%) [However, the sero-prevalence of Ethiopia , 28, butEthiopia  and BirhEthiopia . The varEthiopia . The serc typhus . The stuRickettsia infection like through increasing community’s awareness in the present study area.Nevertheless, our study showed that typhus is one of the major public health concerns in the study area. Hence, emphasis should be given to appropriate diagnosis/treatment and prevention of S.Typhi infection was slightly high among females which is similar to the results of other previous study in Ethiopia [S.Typhi infection among females. However, a previous health facility-based study in Ethiopia revealed a slightly higher seroprevalence for S.Typhi infection among female study participants (22.5%) compared to that of males (16.7%) despite a higher proportion of male participants (60%) compared to that of females (40%) [S.Typhi infection and favors the spread of the disease. Moreover, the high seroprevalence for S.Typhi infection among females could be associated with the daily living habits of females like frequency of exposure to contaminated water during fetching water from river and wells or washing clothes as most of these activities are usually performed by females.The second most common disease found was typhoid fever (7.3%). Our result is comparable with other health facility based studies in different parts of Ethiopia , 24. TheEthiopia . In the es (40%) . In presIn addition, health facilities in the study area had limited laboratory facilities to accurately diagnosis and treat the disease. Thus, increasing access to safe water, strengthening health facility/system for the diagnosis of typhoid fever and treatment as well as increasing community awareness are very important in order to reduce the mortality and morbidity due to this disease.Brucella infection as detected by RBPT (2.1%) and CFT (0.0%) [Several studies have shown the occurrence of brucellosis in livestock in different parts of Ethiopia –35. HoweT (0.0%) .Brucella infection, would not have been diagnosed for brucellosis if the physician would have based the diagnosis solely on clinical signs. Therefore, efforts need to be made to improving laboratory services for the diagnosis of brucellosis in the present study area.Brucellosis has an overlap of clinical symptoms with many other febrile diseases, and can be misdiagnosed with malaria or other diseases due to lack of awareness of medical staff and lack of diagnostic capabilities in the present study area. In this study, 4.4% of symptomatic study participants who were found sero-positive for Brucella infection among males than in females [Brucella infection. Unlike results from Uganda [Brucella infection in the younger age group and children. Children can be exposed to Brucella infection by regularly drinking raw milk, contaminated soil with the bacteria and having regular close contact with livestock, particularly goat and sheep [In the present study, significant difference was not found in the prevalence of brucellosis between males and females, which is similar with other studies done in Ethiopia , Tanzani females . This cam Uganda  and Bangm Uganda , where end sheep .P. falciparum which causes the most severe form of malaria is the widely distributed species as previously reported [This study, was undertaken during a high malaria season though it showed a low prevalence of undiagnosed and untreated malaria (2.5%) among the study participants compared to results of previous community-based prevalence study of malaria among non-febrile individuals in Gondar town, North Ethiopia , and in reported . Hence, falciparum malaria. Previous studies also suggested that increased body temperature could be helpful in diagnosing and treating children with febrile illness [In this study, among other clinical features, body temperature ≥ 37.5 °C was found to be strong indicator for infection with  illness , 47.In Ethiopia, serological tests  using the DCAT are commonly used to diagnose typhoid fever and typhus. A number of subsequent studies indicated that these tests are extremely valuable in the absence of adequate laboratory facilities and culture methods like in resources limited countries –51.S.Typhi infection using serological based screened tests, but revealed absence or very low prevalence using blood culture or fecal samples which are considered as gold standard tests for the diagnosis of a current infection with S.Typhi [Brucella. RBPT was found to be simple and useful for the screening for Brucella infection in health institutions where bacterial culturing facilities are not available [Brucella infection [On the other hand, several previous studies have shown a high seroprevalence of  S.Typhi , 52, 53.vailable , though nfection . WhereasS.Typhi, Rickettsia and Brucella infections, respectively despite the fact that these serological based tests are not convincing tests for the diagnosis of current infection because of short comings such as false positivity due to previous exposure or false negativity in an endemic setting [In this study, we have used Widal, Weil-Felix, and RBPT/CFT tests to report the seroprevalence for  setting , and thiThe study participants were recruited based on the clinical signs/symptoms reported by the study participants that may not necessarily have been caused by an infectious agents that cause acute or chronic illness and this might result in a selection bias. The primary objective of this study was to identify the prevalence of brucellosis, malaria, typhoid and typhus among symptomatic individuals with febrile illness related symptoms. However, in an endemic area, these diseases could be prevalent among asymptomatic individuals. Hence, the findings of this study cannot be generalized to the entire population in the study area.In pastoralists like in the present study area who are living in close contact with their animals on a daily basis, many neglected zoonotic diseases such as campylobacteriosis, Q fever, and leptospirosis can cause a significant health problems both in humans and animals . In the In this study, typhoid fever, typhus, brucellosis and malaria were observed among symptomatic individuals. The study also highlighted that brucellosis cases can be misdiagnosed as malaria or other disease based solely on clinical diagnosis. Therefore, efforts are needed to improve disease awareness and laboratory services for the diagnosis of brucellosis, which should be considered in the routine differential clinical diagnosis of febrile illness in the study area. Only a quarter of the study participants (24.6%) were diagnosed for one or more of the above mentioned diseases. In the majority (75.4%) of the symptomatic individuals, the cause of their illness remained unknown. In addition, a high prevalence of unexplained abortions in women (35.7%) was observed. Hence, further community based studies on other zoonotic diseases like leptospirosis and Q fever are warranted to identify other causes of febrile illness in this pastoral setting.S1 Checklist(DOC)Click here for additional data file.


Background: RTS,S/AS01E, the most advanced malaria vaccine confers partial immunity. The vaccine-induced pre-erythrocytic immunity reduces exposure to blood-stage parasites, delaying acquisition of antibodies to blood-stage antigens.  However, the duration of this effect is unknown.Methods: We measured, by enzyme-linked immunosorbent assay, IgG-antibodies to 4Plasmodium falciparum blood-stage antigens  on 314 children randomized to receive RTS,S/AS01E or Rabies vaccine at 5 – 17 months of age in a phase 2b trial in Kenya, and thereafter participated in a 7-year study of the duration of vaccine immunity.Results: Antibody levels to MSP142, AMA1 and EBA175 were slightly lower among the RTS,S/AS01E recipients, relative to the Rabies-control vaccinees, during the first 48 months of surveillance. Irrespective of vaccine arm, antibody levels to merozoite antigens were positively associated with the risk for malaria. However, this was only apparent at high levels for EBA175 and AMA1 and was not evident after adjusting for heterogeneity in malaria-exposure. Among children with asymptomatic parasitaemia, antibody levels were associated with reduced clinical malaria.Conclusions: The reduction in levels of antibodies to blood-stage antigens induced by vaccination with RTS,S/AS01E can last for several years. In absence of asymptomatic infection, anti-merozoite antibody levels were unreliable correlates of clinical immunity. Progress in malaria control has stalled and may have reversed in some areas2.Despite the recent gains in malaria control, the disease remains a major public health risk, with 216 million cases and 445,000 deaths associated with malaria in 2016E is the most advanced candidate malaria vaccine and is based on the circumsporozoite protein (CSP) that targets the pre- erythrocytic cycle ofPlasmodium falciparum in humans. Vaccination with RTS,S/AS01E has been partially efficacious against malaria in phases II and III trials in Africa4. RTS,S/AS01E induces pre-erythrocytic immunity. In contrast, naturally acquired immunity to malaria is largely dependent on antibodies to blood-stage parasites including the merozoite stage. Although there are no unambiguous correlates of natural immunity5, antibodies to merozoite antigens have been associated with protection through multiple mechanisms including the inhibition of erythrocyte invasion and replication6, complement-dependent mechanisms7, and enhancement of uptake and clearance by circulating phagocytes9. Antibodies to antigens expressed on the surface of infected red blood cells (iRBCs) have also been associated with immunity, which could inhibit or reverse sequestration of iRBCs, inhibit formation of rosettes, and promote opsonization of iRBCs for uptake by phagocytes12.RTS,S/AS01E -vaccination that reduce exposure to blood-stage antigen will affect the rate at which antibodies to merozoite and other blood-stage antigens are acquired. Previously, we and others demonstrated that RTS,S/AS01E and RTS,S/AS02 vaccinations reduced blood stage antibody levels, likely as a result of reducing the exposure to blood stage parasites due to induction of partial pre-erythrocytic immunity14. However, the duration of this effect remains unknown. It is important to determine the duration of this effect as RTS,S/AS01E vaccination could delay the development of naturally acquired immunity, increasing the possibility of continued susceptibility in older children after the waning of the vaccine induced immunity15.Antibodies to malaria parasites are acquired as a result of exposure. As such, interventions like insecticide impregnated bed nets and RTS,S/AS01E vaccination, relative to Rabies control vaccines13. We analysed plasma samples collected from children during a seven-year extended follow up of a phase IIb randomized, controlled trial of RTS,S/AS01E among young children in Kilifi, Kenya, examining antibodies to 4 different merozoite antigens by enzyme-linked immunosorbent assay (ELISA). We then analysed the effect of RTS,S/AS01E vaccination on the acquisition of these antibodies and tested for potential correlations between antibody levels and protection from clinical malaria episodes.In this study, we aimed to determine the durability of the previously reported reduction in antibody levels to merozoite antigens in children receiving RTS,S/AS01E or Rabies vaccine in a phase 2b trial, to evaluate the efficacy and safety of RTS,S/AS01E against clinical malaria episodes byP. falciparum infection. Details have been published elsewhere3.447 healthy Kenyan children aged 5 – 17 months were randomized in a 1:1 ratio to receive 3 doses at monthly intervals of either RTS,S/AS01P. falciparum parasite density of 2500 parasites/microlitre of blood. Active surveillance was implemented with weekly home visits, where children were screened for fevers associated withP. falciparum parasites, both during the trial and the extended follow up period. A parallel passive surveillance was implemented by field workers residing in the study villages and health care staff in local health facilities.The primary end point was a clinical episode of malaria, defined as an axillary temperature of >37.5°C, with aAsymptomatic infections were detected by both microscopy and blood-smears during the cross-sectional data and sample collecton surveys described below.Vaccines doses were given at month 1, 2 and 3. Blood samples were initially taken (1) before vaccination (in March 2007), (2) 1 month after dose 3, (3) in March 2008 , and (4) 12 months after dose 3. Subsequently, the study was extended to test the duration of vaccine induced immunity, and further blood samples were collected in March (5) 2009, (6) 2010, (7) 2011, (8) 2012, (9) 2013, and (10) 2014. Separated plasma was aliquoted and stored at 80°C until assayed.13. In the current study, we extend the analysis to samples collected during the extended study, including 10 timepoints taken over 7 years; i.e. pre-vaccination (i.e. month 0), then at 4, 6.5, 8, 14, 24, 36, 48, 60, 72, and 84 months.Previously, we reported anti-merozoite antibody responses for the samples collected from four time points during the first 14 months of follow upP. falciparum antigens as described elsewhere16: MSP142, 3D7 sequence expressed inEscherichia coli17; MSP3, FVO sequence, expressed inE. coli18; the receptor-binding domain II (PfEBA175RII) of EBA175, 3D7 sequence, expressed inP. pastoris19; and AMA1, 3D7 sequence expressed inE. coli20. In brief, each antigen was coated onto high absorbance plates (Immulon4 HBX) at a con- centration of 0.5 micrograms/mL and stored at 4°C overnight. The plates were washed 3 times in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBS-T) and blocked for 3 h with blocking buffer (1% w/v dried skimmed milk powder in PBS-T). After 3 additional washes, 100 microlitre of each plasma sample were added to duplicate wells at a final dilution of 1/1000 in PBS-T. The next day, after 5 washes, 100 microlitre of horse radish peroxidase–conjugated antihuman IgG (DAKO) at a dilution of 1:5000 in blocking buffer was added to each well, and plates were incubated for 3 h. The plates were then developed using H202 as substrate and OPD (Sigma) as the colorimetric indicator for 20 min in the dark. Plates were read at 492 nm on a Molecular Devices Versa Max ELISA reader. Tests were repeated if duplicate optical density (OD) values for an individual plasma sample varied by more than a factor of 1.5. A pool of serum samples from an area in Africa where malaria is highly endemic was titrated on each plate and acted both as a positive control and provided values for a standard curve for converting optical density (OD) readings into arbitrary units, minimizing inter-plate and inter-day variations. A 3-parameter sigmoid ligand binding model was used to least-squares fit a curve to the values of the hyperendemic serum sample pool, and this was used to calculate sample antibody levelss on each plate.Samples were tested by ELISA for the presence of human IgG against the following21, to estimate exposure to malaria based on geographical location.Antibody scores from ELISAs were expressed relative to the OD readings obtained from the hyperimmune standard, with a score of 1000 scaled to be the maximum reactivity seen at the lowest dilution used in the hyperimmune standard curve, and then log-transformed before analysis. Student’s T-test with comparison of means and non-parametric analyses with comparisons of medians and rank sum tests were used to compare groups. For the prospective association with malaria risk, the antibody levels were split into deciles, and a Poisson regression analysis was conducted with the unit of analysis being the period of time after each antibody level was estimated, hence including up to 10 observations per child, using the clustered sandwich estimate in Stata 15 (StataCorp LLC). We used the exposure index, as previously described1735 plasma samples collected from 10 time points: at 0 (i.e. pre-vaccination), 4, 6.5, 8, 14, 24 , 36 , 48 , 60 , (72) , and 84  months of the third dose of vaccination were tested for antibody levels. The antibody levels varied widely, with the majority of the children being unresponsive , while the rest had values lying within the straight part of the hyperimmune standard curve .13, but it was not possible to assess seasonality once sampling was scaled back to 1 sample per child per year, timed to occur in the dry period just before the main transmission season. Antibody levels for AMA1, EBA175 and MSP142 diverged after vaccination, with levels being higher among the Rabies control vaccinees than the RTS,S/AS01E vaccinees at months 12, 24 and 36, 24 and 36, and 6, 12, 24, 36 and 84 of the third dose of vaccination for AMA1, EBA175 and MSP142, respectively  with statistical significance  for AMA1 and EBA175 (irrespective of vaccine arm). However, the incident rate ratios for the effect of antibodies on clinical malaria reduced after controlling for heterogeneity in malaria exposure using an exposure index, suggesting that these anti-merozoite antibodies are markers of exposure, rather than immunity.Antibody levels were split into deciles, which were then tested for prospective associations with protection from malaria in the transmission period after each, but before, the next sampling time-point. Pre-existing antibody levels for the 4 different merozoite proteins were not associated with clinical immunity . InsteadFurthermore, when all the data were stratified by asymptomatic-parasite positivity at sampling by microscopy, the highest levels for AMA1 and MSP3, and all the of levels for EBA175 above the non-reactive group, were associated with reduced rate ratios for clinical malaria, among the children with asymptomatic parasitaemia at the time of sampling. Associations between higher antibody levels and increased incident rate ratios were maintained among the children without asymptomatic parasitaemia .E vaccination resulted in a reduction in antibody levels to blood stage malaria antigens14. However, the duration of this effect is unknown. Here, we investigated the longevity of the reduction of antibody levels to four blood stage antigens after an extended follow up of the vaccines and controls for up to 7 years post-vaccination. We found that immunization with RTS,S/AS01E and the associated clinical protection resulted in the reduction of antibody response to MSP142, AMA1 and EBA175 antibody levels but not for MSP3. While the antibody levels for AMA1 and EBA175 among RTS,S/AS01E vaccinees were below those measured in the Rabies control vaccinees during the first 48 months of monitoring, antibodies to MSP124 remained lower in the RTS,S/AS01E vaccinees than in the controls throughout the study period. This latter, persistent difference was statistically significant except at the very last timepoint, when statistical significance was only marginal, considering that there are multiple comparisons by timepoint and by adjuvant (p=0.019).We and others reported previously that RTS,S/AS0122). In the presence of asymptomatic infection, anti-EBA175 antibodies at all the deciles were higher than the lowest (i.e. non-reactive) decile, and some of the higher deciles for AMA1 and MSP3 antibodies were associated with protection from clinical malaria (irrespective of the vaccine arm). This finding is consistent with several previous studies where analyses of single antigen-specific antibody responses within whole populations demonstrated no protective effect of antimalarial antibodies, but the same antibodies were associated with clinical immunity when parasite-positive individuals were analysed separately23. Our analysis involves only four antigens and there is evidence that the breadth of antibody positivity is also important for protection25.In this study, antibody levels to four specific merozoite antigens were not associated with clinical protection. Rather anti-merozoite antibody levels were positively associated with the risk of clinical malaria for the group as a whole. The most likely explanation for this is that antibody responses are markers of exposure, and therefore represent ongoing risk of future exposure to malaria, and this interpretation is supported by the fact that the positive association was reduced after controlling for the exposure index. It is possible that higher antibody titers might have been protective .Data are available under the terms of theThe study protocol and its subsequent amendments received ethical and scientific approval from the Kenyan Medical Research Institute National Ethics Committee. The study was overseen by an independent data-monitoring committee and local safety monitors and was conducted in accordance with the Helsinki Declaration of 1964 (revised 1996) and Good Clinical Practice guidelines. Written informed consent in the local languages (Swahili or Giriama) was required from parents/guardians for participation.FMN and PB conceptualized the study, supervised and managed the collection of immunology data, analysed and interpreted the data, and wrote the paper. JM performed the antibody measurements, JW conducted and supervised surveillance for malaria, and sample collection, PN conducted and supervised the the vaccine trial, supervised and sample collection. KM and PB obtained the funding, and supervised the overall conduct of research. CD was involved in antibody measurements and interpretation of the data. PB conceptualized and provided supervision for the study, supervised and managed the clininical trial, analysed and interpreted the data, prepared the metadata and wrote the paper. All the authors reviewed the manuscript. General Comments In this study, the authors take advantage of a rich collection of longitudinally sampled plasma from an RTS,S malaria vaccine trial to address questions of blood-stage antibody responses after vaccination. In their prior work, they showed that RTS,S vaccinees have reduced antibodies against four merozoite antigens during the 12 months after vaccination (Bejon JID 2013). Here, as a follow-up, they compare antibody levels in RTS,S and control vaccinees at multiple intervals up to 84 months and assess the relationship between Pf-specific antibodies and prospective malaria risk during the interval prior to the following antibody time point. Overall, the unadjusted data supports  the main conclusion that humoral immunity to tested blood-stage antigens is hampered or delayed by RTS,S vaccination, and this reduction in immunity is extended for years after vaccination. As the authors mention in the Discussion, they only test four merozoite antigens that may not elicit naturally protective antibody responses, and so the connection to RTS,S-associated long-term reductions in protective blood-stage immunity still remains a question. As shown in their previous work, the differences in antibody responses after vaccination was affected by RTS,S vaccine-induced differences in prior malaria episodes. Thus, it would be important to show if the significant difference between comparisons in Fig 2 and Table 1 still hold after adjusting for prior episodes. Related to this, and to provide more conservative interpretations of their results, if one were to account for multiple comparisons (three comparisons per time point), only EBA175 and MSP1 have significant differences at 24, 36, and 60; and at 8.5, 24, 36, respectively. In the Discussion, the statement “In the presence of asymptomatic infection, anti-EBA175 antibodies at all the deciles were higher than the lowest (i.e. nonreactive) decile” is redundant and unclear as written. Was the intention to state “…[malaria protection] for anti-EBA175 antibodies at all the deciles [was] higher…,” given the lower IRR in the higher deciles for EBA175? The subsequent clause, “and some of the higher deciles for AMA1 andMSP3 antibodies were associated with protection from clinical malaria (irrespective of the vaccine arm)” is not supported by the plots in Fig. 3 given most of the 95%CI bars cross unity (the lone exception being the highest decile for AMA1). It is notable that the lone antigen that clearly did not show a significant group difference in Fig. 2 was MSP3, which, interestingly, was also the only antigen expressed as FVO, with the others being 3D7. As the authors are aware, the RTS,S vaccine was most effective against the vaccine strain (3D7) . Thus, a possible explanation for the lack of difference for MSP3 is similarity in incidence of malaria episodes caused by non-vaccine strains. This possibility should be added to the discussion. It would be interesting to compare antibody data for 3D7 antigens vs. antigens from heterologous strains, especially a genetically divergent one such as FVO. Minor Comments: To harmonize with the main text, it would be more useful for the reader if Fig. 1 showed the best fit curve (fitted to standards) for each antigen with sample values superimposed to provide a sense of how many samples were on the linear versus lower and upper plateaus.  4 or 5 parameter logistic models are more frequently used for ELISA dilutional standard curves as they most often give the best fit. The authors should briefly mention why they opted for a less conventional model. Also, can the authors explain the rationale for using Poisson regression to estimate malaria risk at each interval in this study when Cox regression was used in their previous study? Did some of the underlying assumptions change when the evaluation was extended from 12 m to 84 m? Although the timing of the sampling can be somewhat inferred from Fig. 2, Table 1, and the text, it would be helpful for readers if there was a Fig. showing the plasma sampling intervals. In the Results section, the authors use "age" in the first reference to Fig. 2. However, Fig. 2 plots use months from the 3rd vaccine dose on the X axes. "Age" should be changed to months after vaccine dose as the latter is more accurate. In the statistical analysis section, please indicate that deciles were determined on a per time point basis if this is the case. For better readability, semicolons should be used to separate the listed items . For the AMA1 in Fig. 2, the y axis could be re-scaled to better visualize the significant difference between groups at the circled time points. Currently, lines for both groups appear superimposed. Note that that there is a discrepancy in Fig. 2: Text legend states shaded regions are 95% CI whereas Fig. legend denotes these are IQRs. As such, Fig. 2 also needs clarification with regard to which comparisons were means and which were comparisons of medians. If means, then means with 95% CI's should be shown. If medians, then medians with IQR's should be shown.  For Fig. 2, Table I, and text, it appears that month 6 and 8.5 all represent that same time point, which is the March 2008 time point (variable number of days post vaccinations). If this is the case, please use consistent nomenclature to avoid confusion (I suggest March 2008 as this is probably the least ambiguous). For Fig. 3, the main text and legend mention deciles were used but only 5 categories are shown for AMA1 and 8 categories for the other 3 antigens. If the lowest deciles (5 for AMA1 and 2 for the others) were combined into a single reference group, please indicate in the legend. Also, it might be clearer if X axis was labeled as categorical deciles  as the current X axis can be assumed to be continuous. Typographical notes: For the last sentence of ELISA methods, extra "s" in “levelss” For Fig. 3 legend, "The analysis [was] adjusted for..."24” In first paragraph of Discussion, “MSP1 In Data availability, “Ha[r]vard”I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. In order to test the hypothesis that immunity to clinical malaria is delayed by RTS,S, this manuscript details the findings from a study of serum ELISA antibody levels of blood stage antigens found in blood from RTS,S trial participants or Rabies vaccine controls. As this cohort was studied over 7 years, and active surveillance data is available, it is an invaluable indication of the effects of this vaccine on infection, clinical episodes and immune parameters affected. IgG to MSP1-42, (3D7), MSP3 (FVO), PfEBA175RII (3D7), and AMA1 (3D7) were used as an indication of exposure and/or immune status and found to represent (recent) exposure best, though they do accumulate with time since vaccination/age. Please describe the extent of exposure of participants using averages or distribution of EIR or episodes. Perhaps the result would depend on this range. All I see is "Exposure Index, as previously described...based on geographical location".Fig 1: I suggest you break the y-axis to show the normal  part of the curve more clearly. You could label the number of patients represented in the negative fraction on the graph? Is it 500/1735? Is density the correct label for the y-axis, not sure what that means.Fig 1 result: I would say values below the "linear part of the sigmoid curve" of the hyperimmune standard curve, were considered unresponsive. Are these the data points marked as a concentration of 2 in Figure 1?Fig 2 text: The increase with age is apparent for AMA1 for participants with highest levels, the difference may be in the sensitivity of the ELISA.Fig 2: The legend could be more descriptive of the data and less of the method. Define IQR, CTl vs Vac in legend. Also, more could be done to make the figure print well in black and white-change shape of symbols and lines in one group. Could significance be indicated on the graph itself?Table 1: Text - the sentence describing the data is hard to understand. "Antibody levels for A, E, M diverged after vaccination. Levels were higher for Rabies vaccinees than RTS,S vaccinees for AMA1 at 24 months, for EBA at 24-72 months, and for MSP1 8.5, 24, 36 and 84 months. (Unless p<.05 is not the cutoff?) But as is, only two sets of dates are listed with three antigens listed "respectively". The table itself could be improved by adding stars for significance, and by separating antigens with a thicker line for easier readability.Figure 3: Great analysis, please state the meaning of IRR-Incident Rate Ratio in the legend, or on the graph. Can you tell from your data, if the larger error bars for MSP1 slide positive group, are due to the faster decline of these antibodies compared to other specificities? It will be great when robust  neutralizing assays have been developed that could distinguish effective blood stage antibody from ELISA positive/exposure induced. In the last sentence, maybe you mean among the children with asymptomatic parasitemia? or without symptomatic parasitemia?Discussion: Please check the run-on sentence "In the presence of asymptomatic infection, anti-EBA175 antibodies at all the deciles were higher than the lowest (i.e. non-reactive) decile , and some of the higher deciles for AMA1...". I would add a sentence about the positive side-reduced exposure leading to reduced clinical episodes. How does that balance out with the potential for increased episodes from delayed immunity, are they predicted to be worse episodes, as in later severe disease?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al., 20111) on the effect of the RTS,S vaccine of the development of the natural immunity against blood stage malaria. The authors have previously reported some reduction in antibody titers against specificP. falciparum antigens. What did not come up clearly in this previous report was the longevity of this RTS,S-related effect. This recent report therefore was aimed at addressing that point.This is an extension of a previously published work  seems to have slightly different time points . This needs to be checked and the correct time points used in both sections and throughout the manuscript.P. falciparum antigens were lower in the RTS,S group compared to the group that had the Rabies vaccine administered. Going through Table 1 the trend seems to be different from this. The trend shown in Figure 2 does agree with the authors’ report but not with the trend observed in Table 1. The authors therefore need to clarify how the results presented in Table 1 differ from those presented in Figure 2.The authors state that the geometric means of antibodies against the fouret al. (20042) and say more about this proposed threshold of antibody titers above which they confer protection against malaria infection but below which they do not. They can then proceed by stating if the antibody titers detected in this study are either above or below that threshold level and then proceed to speculate if there is merit on the statement that the administration of the RTS,S vaccine directly (or indirectly) impairs the development of the P.f. specific antibodies. It’s worth noting that it is the same team who reported a 53% efficacy for the RTS,S vaccine with 38 episodes of clinical malaria in the RTS,S arm compared to 89 episodes observed in the control group vaccinated with a Rabies vaccine that was associated with higher anticircumsporozoite antibody titers . This detail needs to come in their discussion.The statement “antibodies to four specific merozoite antigens were not associated with clinical protection, rather anti-merozoite antibodies were positively associated with risk of clinical malaria for the group as a whole.” is a strong and potentially damaging report which does not augur well with the overall aims of out-rolling this vaccine candidate. It might be considered to water down the whole point of introducing this vaccine and may need to be revisited and presented in a more promising manner. More importantly, the authors seem to contradict this strong statement with the follow up statement: “it is possible that higher antibodies titers might have been protective (i.e. those above a protective threshold)." To marry these two rather disjointed/conflicting statements the authors may wish to start off by introducing the results and proposed concept emanating from this study reported by Kinyanjui Some major points:The authors need to justify the choice of Rabies vaccine in the control group either by referring to their previous works or other related works.There is need for a paragraph on the limitations of the study in the Discussion section.Their previous paper on the same Kenyan cohort combined data from Kenyan and Tanzanian participants. It’s worth mentioning why this time they have decided to report only on the Kenyan cohort.et al., 20104, Osieret al., 20085, Osieret al., 20146, Stanisicet al., 20157 and Cromptonet al., 20108). The authors might wish to refer to these in their introduction and discussion sections.The observation that antibody levels do not correlate positively with immunity against malaria has been discussed in detail in a number of good papers  might add weight to their argument. This is in light of their observation that RTS,S might actually be predisposing the recipient children to acquiring blood stage malaria infection presumably due to the impaired development of this stage-specific immunity.They only introduce about the four-dose in passing in the discussion section. Could they please expand on that and say more on what is being proposed to be implemented?The fact that seasonality was not entirely eliminated as a potential confounded in this second report needs to be expanded in the discussion section with appropriate reference to how seasonality was observed to affect the outcome of the vaccine in the previous study/report.In the introduction please consider change the sentence to “...based on the circumsporozoite protein (CSP) that targets the pre-erythrocytic STAGE (not cycle) of the P. falciparum life cycle in humans.”et al., 20111) and build on that in this report?The main objective was to determine the duration of the effect of the RTS,S vaccine. Has this been achieved and reported appropriately? Could they do this better by referring to what they had reported in their JID paper  needs to be written in full either in the legend or on the axis. Minor comments:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Plasmodium cynomolgi in a tourist from Denmark who had visited forested areas in peninsular Malaysia and Thailand in August and September 2018. Because P. cynomolgi may go unnoticed by standard malaria diagnostics, this malaria species may be more common in humans than was previously thought.We report human infection with simian  Plasmodium parasites  have been considered to cause natural human malaria; however, several simian Plasmodium species have also been found to infect humans , slightly elevated S-alanine aminotransferase , and thrombocytopenia (104 × 109 platelets/L); platelet levels on the next day further dropped to 96 × 109 /L. Results of initial malaria testing using a malaria rapid diagnostic test (RDT)  and malaria microscopy were negative. Because her symptoms persisted, we tested an EDTA-blood sample by loop-mediated isothermal amplification ; results were positive for Plasmodium spp. Repeated microscopic examination of Giemsa-stained thin blood smear  revealed low-grade parasitemia. We observed different parasite stages that resembled P. vivax but with slightly different morphologic features  and suspected malaria; circulatory and respiratory systems were stable. Chest radiograph results were normal. Blood examination showed slightly elevated C-reactive protein (39 mg/L), leukocyte count within reference range (6.3 × 10features  (2).Plasmodium DNA, whereas the rapid test was again negative. In-house real-time PCRs were positive for Plasmodium  rRNA genes , and clone 2 had 100% similarity to GenBank accession no. AB287288 (sporozoite [S]–type SSU rDNA), both of which were isolates identified in a long-tailed macaque in Southeast Asia , followed by primaquine (26.4 mg/d for 14 d). Symptoms resolved on the second day of treatment, and the patient was discharged for outpatient follow-up. Within a week, platelet count normalized, and S-ALAT further increased to 135 U/L. Results of malaria microscopies repeated on days 9 and 37 of treatment were negative. The patient fully recovered.P. cynomolgi malaria during a trip to Southeast Asia. Exactly where the patient became infected is not known. The presence of nocturnal mosquitoes and macaques makes Khao Sok National Park in Thailand a likely site of infection. The time interval of 17 days between her arrival in Khao Sok and the onset of symptoms matches reported incubation periods for human P. cynomolgi infections (A short-term traveler contracted P. cynomolgi infection was only recently reported in a local resident (Surat Thani Province in Thailand is located ≈800 km north of Hulu Terengganu in peninsular Malaysia, where a natural human P. cynomolgi and the widespread occurrence of its natural host across Southeast Asia, it is likely that this simian Plasmodium sp. is underdiagnosed in both residents and visiting travelers.We obtained species identification by DNA sequencing only after negative species-specific real-time PCR. Given the challenge of diagnosing Urban development into forested areas leads to closer coexistence of human and monkey. The number of cases in which malaria is transmitted from monkeys to humans may therefore increase. Advanced detection and identification techniques may improve knowledge of the epidemiology of simian malaria in humans.


P < 0.01). Extent of anemia and admission during periods when blood was unavailable for transfusion were associated with early death (P ≤ 0.03). Delays in initiation of treatment of severe malaria contribute to the increased odds of death in children referred from more distant health centers, and might be mitigated by transportation improvements, capacity at rural health posts to administer treatment before transfer, hospital triage systems that minimize time to treatment, and reliable blood product stores at referral hospitals.Malaria remains a public health crisis in areas where it has resisted control efforts. In Nchelenge District, a high-transmission area in northern Zambia, malaria accounts for more than one-third of pediatric hospitalizations and nearly one-half of hospital deaths in children. To identify risk factors for death due to malaria, we conducted a retrospective, time-matched case-control study of 126 children hospitalized with malaria who died (cases) and 126 children who survived (controls). There were no differences in age, gender, hemoglobin concentration, or prevalence of severe anemia between cases and controls. Children who died were more likely to come from villages located at greater distances from the hospital than children who survived (median 13.5 versus 3.2 km). Each additional kilometer of distance from the hospital increased the odds of death by 4% (odds ratio 1.04, 95% confidence interval 1.01–1.07,  Plasmodium, is the leading parasitic cause of mortality worldwide. Plasmodium falciparum, the most lethal among human malaria parasites, predominates in sub-Saharan Africa where it causes a disconcerting 1,200 child deaths per day concentrated in regions where malaria has proven recalcitrant to control measures or where control measures are tenuous or absent.2 In Zambia, there were an estimated 3.1 million cases of malaria (severe and uncomplicated) in 2016 among its population of 16.7 million and it was most prevalent in northern Zambia where it persists year round despite the recent scale-up of vector control efforts.3Severe malaria, caused by the protozoan 4 Only a small proportion (∼2%) of P. falciparum infections progress to severe malaria, but in highly malarious regions where children may experience several infections a year, the absolute number of cases is large.5 Prompt initiation of treatment is vital. Rapid disease progression is fueled by an exponential expansion of parasites and an exuberant host immune response, and hospital deaths occur most often within 24 hours of admission.6 The preferred treatment is intravenous artesunate and supportive care as indicated , but even with appropriate care, mortality can reach 20%.5 Delayed presentation to allopathic health providers on account of logistical constraints or health-seeking behaviors compounds the danger.7Severe malaria can feature one or more of severe anemia (hemoglobin < 5 g/dL), cerebral edema, lactic acidosis, respiratory failure, or shock. In high-transmission, resource-limited settings, malaria evades easy case definition and is often clinically indistinguishable from bacterial meningitis, pneumonia, bacteremia, and other febrile diseases which can conflate or confuse diagnosis.Distinguishing factors that contribute to poor outcomes for children with malaria can inform clinical and programmatic strategies to lessen its toll. However, research is limited by the inherent challenges of conducting intrahospital epidemiologic studies in rural centers where malaria prevalence is greatest. We present results of a retrospective, time-matched, case-control study of hospitalized children with malaria who died (cases) and those who survived (controls) at a rural, district-level hospital in a high-transmission region of northern Zambia to identify and evaluate demographic and clinical factors associated with increased risk of death.3 Community prevalence by rapid diagnostic tests is estimated to be ∼50%.8 In 2016, there were 1,128 adult and child hospital admissions for malaria, accounting for 35% of all pediatric hospital admissions and 38% of all pediatric deaths in the hospital. The malaria case fatality rate was 80 and 65 deaths per thousand adult and pediatric hospitalizations for malaria in 2016 and 2017, respectively.The study was conducted in Nchelenge District, a high malaria transmission area of northern Zambia. Since 2012, the National Malaria Elimination Program under the Ministry of Health have overseen bed net distributions and indoor residual spray campaigns in the study area, although little decrease in malaria burden has followed.5 Children judged by health center providers to require care for severe malaria are transferred to the hospital. Transportation from health centers to the hospital is challenging: patients’ families must arrange transport for a fee, and road conditions are poor.A single 175-bed hospital staffed by three physicians and one midlevel provider serves the district and surrounding areas. The hospital is a referral center for 11 rural health centers that lie 1.5 to > 40 km away, including an island community 30 km away. The rural health centers are neither outfitted with the means to initiate specific therapy for severe malaria, which includes intravenous artesunate, nor are alternatives such as rectal artesunate and intramuscular artemether available.Some patients do not survive transfer from the rural health center to the hospital or from the hospital outpatient department to the ward, and are recorded in ward registers as dead on arrival. Administration of intravenous artesunate, blood transfusion, and other supportive care can only occur once a child has been transferred to the ward.This was a retrospective, time-matched, case-control study of children with severe malaria admitted to the hospital who died (cases) or survived (controls). Cases were matched 1:1 with controls admitted on or near the same date to account for differences in health-care providers, stockouts of blood products or other hospital resources, and other unmeasured temporal confounders such as road conditions, which vary between the rainy and dry seasons and were hypothesized to impact time to treatment. If more than one control was identified for a given day, the patient admitted closest in time to the case was selected. The study was approved by the Johns Hopkins Bloomberg School of Public Health Institutional Review Board as part of the International Centers of Excellence for Malaria Research study and locally by the Tropical Diseases Research Centre Ethics Review Committee located in Ndola, Zambia.Case-control data were collected from hospital registers and laboratory logbooks for children admitted to the hospital between January 2016 and October 2017. Severe malaria was determined by the discharge diagnosis in the register, and the primary outcome of the study  was determined by the recorded disposition in the register. All patients admitted to the children’s ward with severe malaria who died were included. Those who did not survive were further stratified by those who died before treatment could be administered  and those who died after they were admitted and underwent treatment. Patient charts of children who died were frequently unavailable, precluding chart extraction of data.Hospital epidemiologic data were collected from the hospital Health Management Information Systems database (current to April 2017) and hospital registers. Distances from villages to the hospital were provided by the district office of the Zambian Ministry of Health.All hemoglobin values were presumed to be pretransfusion values. Hospital practice is to require documented hemoglobin level before transfusion, and patients infrequently undergo more than a single laboratory evaluation. We use the term initial hemoglobin because not all children were subsequently transfused .P value of 0.05 and two-sided α. Analyses were carried out using Stata 14 .Matched case-control data were analyzed by conditional logistic regression. Linear and logistic regression models were applied for unmatched adjusted analyses of early death. We hypothesized that the relationship between village-to-hospital distance and survival would be partially mediated by the degree of anemia as patients with delayed presentation are more likely to have low mean hemoglobin concentrations due to disease progression. Mediation analysis was carried out to estimate the percent contribution of hemoglobin concentration to the total indirect effect of distance from the hospital on survival. Sensitivity analyses were performed, one excluding patients from the two most remote villages and their matched controls to account for outlying distances and another introducing a covariate designating admission during a blood stockout period to account for the five study pairs mismatched on that variable. A subgroup analysis of patients who were dead on arrival to the children’s ward was performed to examine risk factors of early death. In a cross-sectional design, these patients were compared with cases who survived the initial admission period but subsequently died. The frequencies of severe malaria hospitalization and death outcomes in relation to blood stockout periods were analyzed using Poisson regression. Statistical significance was prespecified to a threshold We identified 126 children admitted to the children’s ward with a diagnosis of severe malaria who died during the period January 2016 through October 2017. Most (89%) matched control patients were admitted within 1 day of the case patients, and of those, more than half (54%) were admitted the same day. Eleven were admitted within 2 days of the matched control and three within 3 days. Five of the 126 case-control pairs were mismatched on whether there was a blood stockout on the day of admission. One case was missing age and gender, but hemoglobin and village data were available. Data on village distance to the hospital were available for all except nine control and 18 case patients. Hemoglobin concentrations were unavailable for 49 (39%) control and 41 (33%) case children. Missingness was mainly due to hemoglobin measurements done at rural health centers prior to hospital transfer, hence results were not recorded in laboratory logbooks. Children whose hemoglobin measurements were not available were similar in age, gender, and distance from the hospital to those for whom hemoglobin data were available.P = 0.10).There were no statistically significant differences in age, gender, initial hemoglobin concentration, or prevalence of severe anemia between children who survived and those who died . The medP < 0.01). Mediation analyses estimated that hemoglobin concentration accounted for only 4% of the contribution of distance from the hospital to death among all case patients, and 28% among those recorded as dead on arrival to the ward. In a sensitivity analysis excluding cases from the two most outlying villages and their matched controls, the association between distance and death remained statistically significant . The same was true when introducing a covariate for admission during a stockout period to account for the five mismatched pairs.Children came from 73 villages situated between 1.5 and 89.6 km from the hospital. One child (control) lived within the hospital compound and one child (case) was from the island community located 30 km away. Those with severe malaria who did not survive were more likely to come from distant villages than those who survived . AdjusteP = 0.05), anemia was more severe , and a higher proportion were admitted when blood was unavailable for transfusion   were dead on arrival to the ward. Patients dead on arrival did not differ in age or gender from those who died after arrival to the ward . However = 0.03) .P < 0.01). During stockout periods, the incidence of death among children with malaria was once in 3 days, compared with one death every 6 days during periods when blood was in supply , but the number of deaths as a proportion of admissions did not differ .Within the study period, there were 12 whole blood stockouts ranging from 3 days to 3 weeks, totaling 68 days. Stockouts were more common during months with greater-than-average daily pediatric admissions for malaria  relative to previously studied populations.18 Most children (85%) were younger than 5 years and age-related factors  may have been attenuated because of the narrow age range of the study children.The young ages of the patients, and the prevalence and profundity of anemia, were commensurate with a high malaria transmission environment.Blood stockouts were more common during peaks in hospital admissions for malaria. The number of deaths among hospitalized children with malaria was higher during stockout periods, but modeling showed this to be explained wholly by the increased number of admissions and not the depletion of blood stores.19 hemoglobin appears to influence survival only at concentrations < 3 g/dL.20 Twice as many cases as controls in our sample had such low hemoglobin concentrations, though the difference did not reach the preset significance threshold (P = 0.20).There were two unexpected findings related to the role of anemia. First, we detected no significant difference in hemoglobin concentrations between those who survived and those who died. Sample size limitations imposed by missingness in hemoglobin data and potential instances of misclassification of the cause of death leading to artificial inflation of the mean hemoglobin relative to controls are possible explanations for this finding. Based on data from a pivotal trial of artesunate versus quinine for severe malaria,The second unexpected finding was that hemoglobin concentration explained only a small portion (4%) of the effect of distance on malaria death. We hypothesized that greater distance from the hospital would correspond with delay in presentation and thereby more profound anemia, and ultimately a higher risk of death, as the illness progressed unchecked. Although we did not observe this in the overall sample, the association was observed among children brought in dead to the ward. In these children, hemoglobin concentration accounted for > 25% of the estimated effect of distance on death, consistent with the additional finding of an association between children who were dead on arrival to the ward and admission during a blood stockout period. Hemoglobin testing and subsequent blood transfusion, if indicated and possible, are often done between the time the child arrives to the hospital and is registered on the ward. Our findings suggest that those who did not survive the interval between presentation to the hospital and registration on the ward often died for lack of transfusion. These represent children who never had the opportunity to receive treatment and supportive care. Had they survived long enough to undergo treatment, it is likely that some would have recovered and survived to discharge, implying that ensuring reliable blood inventories would improve malaria survival.21 Similarly, we were unable to ascertain clinical syndromes  nor could we account for children with coinfections or incidental parasitemia unrelated to their illness. A hospital-based study such as this is unable to account for children who die at home from complications of malaria, die at rural health centers or healers, or die en route to the hospital, who constitute most severe malaria deaths in some settings.23 There are potential unmeasured confounders and mediators of the effect of distance on malaria death, including factors related to delays in care such as health-seeking behavior and knowledge, attitudes and practices around malaria, time to first dose of artesunate, and time to blood transfusion.There are limitations to this study. Reliance on hospital registers precluded a rigorous case definition of severe malaria and death due to malaria, introducing potential misclassification bias. However, given the high background prevalence of malaria in the catchment area, ready availability of malaria diagnostics, and experienced hospital staff, we believe this is unlikely to have significantly influenced the study’s results. Furthermore, there is precedent for our case definition; for example, the Severe Malaria in African Children network enrolled any child with malaria sick enough to be hospitalized.24 In the meantime, investments in transportation infrastructure  to reduce transfer times to the hospital, hospital triage systems that expedite treatment on arrival for patients with severe malaria, and maintenance of responsive supply chains for blood inventories to avert stockouts during periods of high malaria incidence would temper case fatality. Stationing ambulances at remote health posts and furnishing them with the capacity to initiate pre-transfer treatment of severe malaria, such as intramuscular or rectal artesunate, would be lifesaving.A precept of malaria control is prompt diagnosis and treatment to reduce deaths from severe malaria and to diminish the infectious reservoir. National and local malaria control programs prioritize surveillance of and response to severe malaria morbidity and mortality, but these priorities are often detrimentally overlooked by the research community where clinical trials of interventions for severe malaria are sparse and progress thereby slow.