Enterovirus 71 (EV71) is a great disease burden across the whole world, particularly in Southeast Asia. However, in recent decades, the pathogenesis of severe EV71 infection was not well understood. This study was aimed to investigate the correlation between the presence of viremia and the clinical severity of EV71 infection.We organized a prospective cohort study and enrolled laboratory-confirmed EV71 cases in six tertiary care hospitals in Taiwan during the EV71 epidemic from 2011 to 2012. Blood samples were collected once in the acute stage, on the first day of admission. We used real-time RT-PCR to detect EV71 viremia. Demographical and clinical data were collected and the clinical severity was categorized into four grades. Data analysis was performed to identify the risk factors of viremia and the correlation between viremia and clinical severity of EV71 infection.p < 0.001) but the association between the presence of viremia and complicated EV71 infection was not found . In the viremia group, patients had significantly more severe complications if viremia was detected after the third day of disease onset .Of the total 224 enrolled patients, 59 (26%) patients were confirmed to have viremia. Two-thirds (68%) of viremic cases were detected within the first three days of infection. Viremia occurred more frequently in children under the age of one year old  contains supplementary material, which is available to authorized users. In recent decades, enterovirus infection has accounted for several outbreaks and has become a heavy burden of public health and socioeconomics in both developing and developed countries . Among tIn Southeast Asia, outbreaks of EV71 infection have become a serious public health problem , 8, 9. IAfter investigating the characteristics of the transmission of EV71, the professionals in Taiwan’s Enterovirus Ad Hoc Committee and the Taiwan Centers for Disease Control designed several health policies, such as both physician and virological laboratory surveillance systems and hand-washing education program to control the epidemics of EV71. The above policies limited the outbreak and the impact of EV71 infection and reduced the numbers of fatal EV71 infection , 17. As According to the laboratory-based surveillance system of Taiwan CDC, the baseline proportion of EV71 among all the enterovirus isolates from patients with enterovirus infections was about 2% but the proportion increased sharply between 2011 and 2012 and peaked in 2012 (45%) . As a reEV71 infection of clinically-suspected cases was confirmed on the basis of the following laboratory methods: positive virus isolation of EV71, and/or positive EV71 identification by pan-enterovirus real-time RT-PCR followed by molecular serotyping. We collected the basic demographic data including age and gender, the date of the onset of disease, the date of blood sampling for EV71 virologic test, and their clinical manifestations and outcomes. According to their clinical manifestation and outcome, the clinical severity of the patients with EV71 infection was classified according to the following four categories at the hospital discharge :Grade 1: Uncomplicated HFMD, herpangina or febrile illness,Grade 2: Mild central nervous system (CNS) involvement including myolonic jerk alone or aseptic meningitis,Grade 3: Severe CNS involvement, such as encephalitis, encephalomyelitis or polio-like syndrome, but without cardiopulmonary failure.Grade 4: CNS involvement complicated by cardiopulmonary failure.Blood samples for basic laboratory tests including complete blood count and virologic test were collected only once on the first day of admission. Real-time RT-PCR was used for detection of virus RNA in the blood sample to confirm the presence of viremia and its magnitude according to the protocol mentioned as below.Throat swabs were submitted for virus isolation. Samples were inoculated into human embryonic fibroblast (MRC-5), LLC-MK2, HEp-2, and RD cell cultures. If enteroviral cytopathic effect involved more than 50% of the cell monolayer, the cells were scraped and indirect fluorescent antibody staining with panenteroviral antibody  was performed to identify the enterovirus. These isolates would be further serotyped to be EV71 by an immunofluorescent assay using EV71-specific mono-clonal antibodies against viruses .Viral RNA extraction from throat swabs and sera was performed using an isolation kit , and reverse transcription was performed with first strand cDNA synthesis kit for RT-PCR  according to the manufacturer’s directions. Real time RT-PCR for pan-enterovirus was performed with the primers of 5′-TCCTCCGGCCCCTGAATG-3′ and 5′-AATTGTCACCATAAGCAGCCA-3′, the probe of 6FAM-AACCGACTACTTTGGGTGTCCGTGTTTCXT-PH. The detection limitation (sensitivity) was ≤ 10 copies of in vitro transcribed RNA for pan-enterovirus real-time RT-PCR .If the result of pan-enterovirus real-time RT-PCR was positive, molecular serotyping was subsequently performed. The cDNA was sent for semi-nested PCR with primers used in a previous report , the PCR10 copies/ml. In univariate analysis, Student’s t test and Mann–Whitney U test were used for continuous variables and chi-square test or Fisher’s exact test was used for categorical variables, when appropriate, to identify the factors associated with viremia and the risk factors of complicated EV71 infection. In multivariate analysis, multiple logistic regression analysis was used. A two-tailed p value of 0.05 was considered statistically significant. Data were maintained in Microsoft Excel, Mac, 2011  and analyzed by using Stata 12.0 .Descriptive analysis was performed for the demographic data and other characteristics of the EV71 patients. Viral load was presented as logFrom 2011 to 2012, we totally enrolled 224 patients with their sera collected in this study. The median age was 2.71 years old (range 0 to 15 years), and male to female ratio was 1.49 .p < 0.001). The viral load ranged from 0.88 log10 copies/mL to 6.99 log10 copies/mL (median 3.46 log10 copies/mL). The viral load was highest when the blood was sampled between the third and fourth day after disease onset, and the viral load was lower if the blood was sampled later .Fifty-nine of 224 cases (26%) had positive PCR results and viremia was confirmed. The percentage of confirmed viremia was highest on the first day of illness and viremia was lower in patients sampled after day 3 Figure . Two-thip < 0.001), while gender and other symptoms and signs, such as skin rash, oral ulcer, fever and the duration of fever, did not influence the presence or the magnitude of viremia . The risk factor for complicated EV71 diseases was fever  and fever over 3 days . Higher viral load did not correlate with more severe clinical disease. Those patients with complicated EV71 infection were younger than those with uncomplicated disease . The multivariate analysis adjusting age, gender, and clinical symptoms revealed similar results .For those who had severe complications grade 3 to 4), the percentage of viremic cases did not decrease beyond three days of illness compared to those who had milder EV71 diseases (grade 1 to 2)  remained similar after the third day of disease while the proportion of viremia decreased in those who had mild diseases (grade 1 to 2). The finding suggested the viremia might persist longer in those who had severe diseases and thus their risk of systemic involvement and complications increased. In addition, the proportion of patients with severe CNS involvement and cardiopulmonary failure increased in those patients whose viremia was detected after the third day of disease. That is to say, a much higher virus burden, prolonged viremic phase or the consequent immunologic reactions might be the principle mechanism of the severe complications of disease rather than viremia alone. Previous studies suggested that the most severe form of EV71 infection causing pulmonary edema might result from the overreaction of immune system and consequent cytokine storm , 42. BesNevertheless, the clinical severity of EV71 infection may be multifactorial and is not determined only by the presence of viremia. Either the different virulence of different genotypes, host factors and other socioeconomic factors , 19–22 mThere are some limitations in our study. First, we only collected blood samples for PCR test at one time point in each patient and it is difficult to describe the kinetics and the duration of viremia. A kinetic data could provide more information about the influence of viremia on the clinical manifestation, disease severity and the whole course of EV71 infection. It is also impossible to determine the peak viral load of individuals and this limitation makes it more difficult to clarify the definite relationship between the magnitude of viremia and the clinical manifestation. Second, most patients in our cohort had only uncomplicated disease or myoclonic jerk and the proportion of severe CNS complications or further cardiopulmonary failure was relatively low. Because the epidemics of non-polio enterovirus occurred almost every year in Taiwan, the decreased severity of EV71 infection may result from partial cross-protection from previous enterovirus infection by different serotypes or genotypes . Only 15In summary, our study found that viremia occurred more frequently in children under the age of one year and viremia detected beyond three days after the onset of disease correlated with more severe disease in EV71 patients. Our findings may shed a light on the identification of the role of viremia for the pathogenesis of severe EV71 infection, but further studies targeted at the interaction between the magnitude, the kinetics of viremia and other host factors in patients with severe EV71 infection will be required.


Malaria is one of the most severe global public health problems worldwide, particularly in Africa, where Nigeria has the greatest number of malaria cases. This community-based study was designed to investigate the prevalence and risk factors of malaria and to evaluate the knowledge, attitudes, and practices (KAP) regarding malaria among rural Hausa communities in Kano State, Nigeria.Plasmodium species by rapid diagnostic test (RDT), Giemsa-stained thin and thick blood films, and PCR. Moreover, demographic, socioeconomic, and environmental information as well as KAP data were collected using a pre-tested questionnaire.A cross-sectional community-based study was conducted on 551 participants from five local government areas in Kano State. Blood samples were collected and examined for the presence of Plasmodium falciparum. The prevalence differed significantly by age group (p < 0.01), but not by gender or location. A multivariate analysis showed that malaria was associated significantly with being aged 12 years or older, having a low household family income, not using insecticide treated nets (ITNs), and having no toilets in the house. Overall, 95.6 % of the respondents had prior knowledge about malaria, and 79.7, 87.6 and 95.7 % of them knew about the transmission, symptoms, and prevention of malaria, respectively. The majority (93.4 %) of the respondents considered malaria a serious disease. Although 79.5 % of the respondents had at least one ITN in their household, utilization rate of ITNs was 49.5 %. Significant associations between the respondents’ knowledge concerning malaria and their age, gender, education, and household monthly income were reported.A total of 334 (60.6 %) participants were found positive for Malaria is still highly prevalent among rural Hausa communities in Nigeria. Despite high levels of knowledge and attitudes in the study area, significant gaps persist in appropriate preventive practices, particularly the use of ITNs. Innovative and Integrated control measures to reduce the burden of malaria should be identified and implemented in these communities. Community mobilization and health education regarding the importance of using ITNs to prevent malaria and save lives should be considered. Malaria continues to be a major public health problem in 97 countries and territories in the tropics and subtropics. Globally, approximately 214 million cases of malaria occur annually and 3.2 billion people are at risk of infection [Plasmodium falciparum, and the mosquitoes Anopheles gambiae, Anopheles funestus, Anopheles arabiensis, and Anopheles moucheti are the major vectors that cause year-round transmission; artemether-lumefantrine (AL) or artesunate + amodiaquine (AS + AQ) is the treatment regime adopted in 2004 [Nigeria suffers the world’s greatest malaria burden, with approximately 51 million cases and 207,000 deaths reported annually , while 97 % of the total population (approximately 173 million) is at risk of infection . Malaria in 2004 , 9.Since 2008, the National Malaria Control Programme (NMCP) in Nigeria has adopted a specific plan, the goal of which is to reduce 50 % of the malaria burden by 2013 by achieving at least 80 % coverage of long-lasting impregnated mosquito nets (LLINs), together with other measures, such as 20 % of houses in targeted areas receiving indoor residual spraying (IRS), and treatment with two doses of intermittent preventative therapy (IPT) for 100 % of pregnant women who visit antenatal care clinics . BecauseA cross-sectional, community-based study was carried out between May and June 2013 among inhabitants aged 1–90 years in the five rural communities. Primary healthcare personnel and community leaders were involved in the study, and discussions were held with the households’ heads at the community leaders’ hall to explain the rationale and importance of the study.2  and has a total population of approximately 11 million. Kano State is a commercial and agricultural region known for the production of groundnuts and cotton. It is also the second largest industrial center in Nigeria, with textile, tanning, footwear, cosmetics, plastic, and other industries. The state consists primarily of Sudan savanna type vegetation, with an annual mean rainfall of 800-900 mm, a temperature that ranges between 25–40 °C (mean approximately 26 °C), and a relative humidity of 47.43 %. The climate of the study area is a tropical dry-and-wet season type typical of West African savannah. The wet season lasts from May to October, while the dry season extends from November to April [Kano State is located in the North Central part of the country between longitude 8.500°E and latitude 11.500°N; it occupies a total surface area of 20,131 kmThe ecology of Nigeria varies from mangrove swamps and tropical rain forest belts in the coastal areas to open savannah woodland on the low plateau—which extends through much of the central part of the country—to semi-arid plains and Sahel grassland in the north and the eastern highlands. The seasonality, intensity, and duration of malaria transmission vary according to the different ecological strata . The durIn collaboration with primary healthcare personnel and traditional rulers in each local government area, five districts were selected randomly from the available district list. The districts included were Kura , Bebeji , Gwarzo , Shanono , and Minjibir .Residents of Kano State are predominantly farmers and merchants, and the districts selected have a homogenous nature with respect to sociocultural and daily economic activities. In the selected areas, primary healthcare staff and leaders of the communities were informed about the objectives and procedures of the study in order to obtain their permission to conduct the study and established a station. Then, the community leaders asked all residents to gather at the station  where they received explanation about the objectives of the study and their involvement. All residents who agreed voluntarily to participate were included in this study . A total of 609 individuals volunteered to participate in this study; however, 551 (90.5 %) individuals, aged 1–90 years, met the inclusion criteria .Ethical approval for the study protocol was obtained from the Medical Ethics Committee of the University of Malaya Medical Centre, Kuala Lumpur as well as from Kano State’s Ministry of Health, Kano State Hospitals Management Board, the respective local government authorities, and the district heads of the communities. When seeking consent from the volunteers in each village, the objectives and procedures of the study were explained clearly to them in the local language, Hausa. Participants were also informed that they could withdraw from the study at any time without consequences. Thus, written and signed or thumb-printed informed consents were obtained from all adult participants and guardians/parents on behalf of their children before starting the survey; the ethics committees approved these procedures as well. All malaria-positive individuals were treated with the standard medication according to national malaria drug policy.A pre-tested questionnaire was administered to the participants in order to collect information on their demographic, socioeconomic, and environmental factors, and the health status, as well as their KAP towards malaria. Information was collected from the respondents via face-to-face interviews conducted by trained research assistants. Both assistants were aware of the purpose of the study and the way in which to administer the questionnaire.The survey gathered information offered both spontaneously and in response to specific questions that addressed their knowledge about malaria. Questions about knowledge were open-ended to avoid guessing about the answers to multiple-choice questions, which might give a false impression concerning the participants’ knowledge. However, questions pertaining to practices were multiple-choice to assess the frequency with which participants’ performed the various activities and actions. At the end of each interview, the interviewers probed for further knowledge related to malaria that the respondents did not mention spontaneously. However, this was done only for selected variables, specifically their source(s) of information about the disease, history, and reasons for previous visits to clinics or hospitals, as well as the availability, condition, and use of ITNs. During the survey, direct observations were made concerning the use of ITNs, as well as housing conditions and cleanliness , and status of windows.® filter paper  and kept in individual sealed plastic bags at room temperature until use. At Aminu Kano Teaching Hospital, Kano State, the blood films were stained with diluted Giemsa and then examined microscopically for the presence of malaria parasites; 200 fields under 1000× magnification were examined from the thick film before the slide was considered negative.Approximately 2–3 ml of venous blood was drawn from each participant into an EDTA tube. Thick and thin blood films were prepared and a drop of the blood was used with an RDT kit, the “CareStart Malaria HRP2 from Access Bio, Inc.” Moreover, 2–3 drops were collected on 3 MM WhatmanFor positive slides, parasite species and stages were assessed and parasitaemia (parasite density) was determined by counting only the asexual stages against 300 white blood cells (WBC) and then multiplying by 25, assuming the mean total WBC count of individuals is 7500 cells per μl of blood . The lev® Blood & Tissue Kit, Cat. no. 69506, Germany); the filter paper blood spot was cut using an ethanol flame–sterilized hole-punch, put into a new 1.5 ml microcentrifuge tube, and then extraction was conducted according to the manufacturer’s instructions. DNA was eluted using 50 µl AE  elution buffer (included in the kit) and maintained at −20 °C until PCR analysis. Plasmodium species were identified by 18s rRNA-based nested PCR using genus- and species-specific nucleotides primer sets, as described previously [All malaria microscopy-positive slides and a random selection of 20 % of the negative slides were re-examined by another senior specialist at the Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur. Afterwards, all those samples were confirmed by PCR. Genomic DNA was extracted using the Qiagen blood and tissue kit . Then, a third researcher crosschecked the two datasets for accuracy and created a single dataset for analysis. The demographic, socioeconomic, environmental, and behavioural characteristics of the respondents, as well as the KAP variables were treated as categorical variables and presented as frequencies and percentages.p ≤ 0.25 were used in the multiple logistic regression model, as suggested by Bendel and Afifi [p < 0.05 was considered statistically significant.Pearson’s Chi squared test and Fisher’s exact test were used to test the associations between malaria prevalence and KAP items as the dependent variables, with the demographic  and socioeconomic factors  as the explanatory variables. All variables were coded as binary dummy variables. For example, malaria ; gender , and so on. Odds ratios (OR) and 95 % confidence intervals (CI) were also computed for the explanatory variables. Moreover, participants were categorized into three age groups ; however, they were re-categorized into two groups (children <18 years and adults 18 years and older) for analysis of the KAP variables. ORs at 95 % CIs were also computed. Multivariable logistic regression was conducted to identify the risk factors associated significantly with infection. To retain all possible significant associations, all variables that showed an association with nd Afifi . A p < 0A total of 551 individuals participated in the study; 340 (61.7 %) were male, 211 (38.3 %) were female, and 64.1 % were adults aged 18 years and older. Table P. falciparum and the majority (70.1 %) constituted low parasitaemia (<1000 parasites/μl of blood). Moreover, 10 (3.0 %) and 90 (26.9 %) cases were severe  and moderate (1000–9999 parasites/μl of blood), respectively. The asexual P. falciparum parasitaemia from the samples collected ranged from 50–12,500 parasites/μl of blood, with a geometric mean of 1023 parasites/μl. Overall, the prevalence of malaria was similar between males and females  = 0.12, p > 0.05). Similarly, there was no significant difference in prevalence according to location . However, the prevalence differed significantly  among age groups, with those aged 12–17 years having the highest prevalence (66.7 %). Among those aged less than 12 years, the overall prevalence was 42.9 % with those aged below 5 years having a prevalence of 37.5 %.The prevalence of malaria infection was 60.6 % ; Bebeji had the highest percentage (42.9 %) and Shanono the lowest (24.2 %). Approximately half of the respondents  reported that they used ITNs, and there was a significant difference in use between age groups (p < 0.01) and location (p < 0.001), with participants aged 12–17 years (37.0 %), and those from Gwarzo (25.8 %) having the lowest percentage of use; there was no significant difference between males and females (p > 0.5).With regard to history of infection, approximately one-third (33.2 %) of the participants had experienced a recent malaria episode. A significant association was found between this history of infection and location (χp < 0.01), those from families with low monthly income  compared to their counterparts , and those who did not use ITNs compared to those who did .Table p < 0.25 [p = 0.049).The multiple logistic regression showed that participants aged 12 years and older were approximately twice as likely to have malaria compared to those younger than 12 years . Similarly, low household monthly income  was retained as a significant risk factor and was found to increase the odds of infection by 1.61 times . Moreover, it was found that not using ITNs increased participants’ odds for malaria by 1.57 times  when compared to those who used them. Interestingly, the type of toilet was considered in the regression model, based on the p < 0.25 , and wasKnowledge of the cause, transmission, symptoms, and prevention of malaria, as well as the perception of its seriousness, is presented in Table With respect to attitudes, most subjects (93.4 %) regarded malaria as a serious infection. The distribution of ITNs was high in the study area, and 78.2 % of the respondents had at least one ITN in their homes. However, only 48.3 % of the 505 respondents used ITNs, and only one-third (37.4 %) of the respondents used insecticide. With regard to treatment seeking behaviour, a considerable number of the respondents  mentioned that they went to hospitals or clinics when they had an episode of fever, while 19.2 % of the respondents self-medicated as a first-line of treatment for fever; 3.8 % of the respondents did not treat malaria infection.Tables With respect to gender, male respondents showed significantly higher levels of knowledge about malaria, particularly mosquito bites (82.6 vs 63.6 %) as a cause; fever as a symptom (83.3 vs 69.0 %); using bed nets/insecticide (79.9 vs 52.7 %); improved personal hygiene (27.8 vs 10.9 %), and medication (10.4 vs 1.6 %) as preventive measures. Moreover, significantly higher levels of knowledge of the role of mosquitoes in malaria transmission (81.6 vs 50.8 %) as well as fever (79.7 vs 66.2 %) and vomiting (23.4 vs 9.2 %) as symptoms of malaria, and using bed nets/insecticide to prevent infection (73.2 vs 46.2 %) were reported among educated respondents compared to their non-educated counterparts , 59.6 % (118/198), and 58.0 % 233/400), respectively 0, 59.6 %, 38. Simectively , 40. Non0, respecectively –45. Moreectively . Similarectively , 47.P. falciparum [In contrast, other previous studies reported lower prevalence rates: 39.2 % of 360 antenatal clients who visit primary healthcare facilities in Kano State , 29.4 % lciparum .Findings of the present study showed a similar prevalence rate among the male and female participants, which is consistent with previous studies in Nigeria, Kenya, and Mozambique, which suggests that the distribution of malaria risk is heterogeneous , 53, 58.With respect to location, there were no significant differences in the prevalence of malaria among the districts studied; however, an obvious correspondence between a history of recent malaria and use of ITNs was observed, with the lowest percentage of history of recent malaria reported in Shanono (24.2 %), which also has the highest percentage of ITNs use (62.6 %). Due to the specific characteristics of populations that may facilitate human–mosquito contact, malaria risk can vary widely among districts, villages, and even households . A recenPlasmodium infection was associated significantly with age, with the highest prevalence among children 5–9 years [P. falciparum infection reduced with increasing age, and those aged 11–15 years had a 0.78 odds of infection compared to those aged 5–10 years [The present study demonstrated that age, household monthly income, ITNs use and type of toilets were the significant risk factors associated with malaria among these participants. The present findings showed that prevalence was significantly higher among those aged 12–17 years (66.7 %) and ≥18 years (61.5 %) compared to those younger than 12 years (42.9 %). Interestingly, this difference could be explained by the reported age-specific use of ITNs; 60.3 % of those younger than 12 years compared to only 37.0 % of those aged 12–17 years used ITNs. Moreover, individuals aged 12 years and older are expected to have a higher exposure to mosquito bites because they engage in more outdoor activities compared to younger children, particularly at night. These findings are consistent with recent reports that rates of ITNs use were significantly higher among the youngest children, particularly those less than 5 years, and heads of the family, and this might be attributed to the inadequate ITNs per household . Hence, –9 years . In cont10 years .The present study also showed that low household monthly income  increased the odds of malaria among these children by approximately 1.5 times. This is consistent with previous reports from other African countries that showed malaria is more common among people of lower socioeconomic status who often live in poor housing conditions that increase their exposure to infection , 66, 67.Use of ITNs is considered one of the most cost-effective interventions against malaria in endemic areas, and is associated with significant reductions in malaria morbidity and mortality, particularly among pregnant women and children less than 5 years . The curDespite the high number of participants who had ITNs (79.5 %), only half of the participants stated that they used them, and were found to do so (49.5 %). This unsatisfactory compliance is consistent with previous studies in Nigeria , 71–73 aInterestingly, the present study showed that approximately one-third (30 %) of those who had ITNs and stated that they used them were found to misuse the nets; hence, they were considered non-users. The improper uses of the ITNs observed included employing bed covers, blankets, window curtains, and door curtains rather than actual ITNs. Moreover, some participants declared that they washed the ITNs before using them to remove the chemicals, which either have an unpleasant smell or induce allergies in some individuals. In addition, some of the ITNs were old and were not re-treated, while others were in poor condition. Similar misuse has been reported in different malaria-endemic African countries, including Nigeria, Uganda, Tanzania, Kenya, Zambia, Sierra Leone, and the Democratic Republic of Congo –81. In aThe present study also found that the type of toilet in the house was a significant risk factor for malaria in the communities studied, as those living in houses with pit or ground dug latrines were 1.7 times more likely to be infected with malaria than were those who have toilets in their houses with a pour/flush system. The lack of availability of in-home toilets has been reported to be a significant predictor of malaria in Ethiopia and Yemen , 83.With respect to respondents’ KAP, findings the current study indicated that general awareness of malaria was high among Hausa communities in Kano State and almost all of them (95.6 %) had heard about malaria. This is expected, as malaria is considered the primary health problem in these communities –86. MostThe present study showed that the majority of the respondents (93.4 %) indicated that malaria is a serious disease, which is consistent with previous studies in Nigeria and other countries , 85, 88.Notwithstanding the high prevalence of attitudes that the disease is serious, approximately 20 % of the respondents tended to begin treatment at home and sought help from professionals only when it failed. Home treatment usually involved self-medication or unqualified prescription by family members, friends, and unauthorized shops. This behaviour did not different with malaria fever, wherein people began treatment with analgesics and then anti-malarial drugs if symptoms persisted. The same behaviour has been reported elsewhere in Nigeria , 93–95, This study found that age, gender, educational level, and household monthly income had significant influences on having adequate knowledge about malaria. Younger respondents showed a greater level of knowledge about malaria transmission and prevention than did adults (aged 18 and older), which could be attributed to schools or media, especially television. Similarly, males had substantially higher levels of knowledge about malaria transmission, symptoms, and prevention, which could be because of their higher exposure, as well as certain behavioural and cultural factors. In Nigeria, the prevalence of malaria is highest among pregnant women compared to other groups , 42, 46.Similar high levels of knowledge about malaria transmission, and prevention were also reported among those having household monthly incomes below NGN 32,000 compared to their counterparts, which is inconsistent with the higher prevalence reported among those respondents. This suggests that the higher prevalence rate among this group is more related to the practices but not the knowledge. This finding also is consistent with previous studies in Nigeria , 90, 91.falciparum malaria was reported among rural populations in Kano State. Hence, there is an urgent need to identify innovative and integrated control measures to reduce malaria prevalence significantly in these communities. Age, household monthly income, use of ITNs, and availability of in-house toilets were the significant predictors of malaria in this population. The study demonstrated that respondents had good levels of knowledge and attitudes regarding malaria transmission, symptoms, and prevention; however, these did not translate into improved preventive practices. The present study found that, despite the high level of ITNs ownership, their use still fell short of the national target. Hence, additional strategies, including community mobilization and more focused health education on the importance of using ITNs to control malaria and save lives, are required to increase use and improve current levels of ownership. That said, the number of ITNs distributed to households should be increased to ensure that populations living in malaria-risk areas have enough ITNs for all households members. Moreover, long-term interventions, such as providing job opportunities and improving the level of education to reduce poverty and improve the quality of life of these vulnerable communities also should be considered in order to achieve a breakthrough in the fight against malaria in Nigeria.This study revealed that Nigeria’s battle against malaria has a long way to go. A high prevalence of


All suspected cases of malaria should receive a diagnostic test prior to treatment with artemisinin-based combinations based on the new WHO malaria treatment guidelines. This study compared the accuracy and some operational characteristics of 22 different immunochromatographic antigen capture point-of- malaria tests (RDTs) in Cameroon to inform test procurement prior to deployment of artemisinin-based combinations for malaria treatment.One hundred human blood samples (50 positive and 50 negative) collected from consenting febrile patients in two health centres at Yaoundé were used for evaluation of the 22 RDTs categorized as “Pf Only” (9) or “Pf + PAN” (13) based on parasite antigen captured [histidine rich protein II (HRP2) or lactate dehydrogenase (pLDH) or aldolase]. RDTs were coded to blind technicians performing the tests. The sensitivity, specificity, and predictive values of the positive and negative tests (PPV and NPV) as well as the likelihood ratios were assessed. The reliability and some operational characteristics were determined as the mean values from two assessors, and the Cohen’s kappa statistic was then used to compare agreement. Light microscopy was the referent.Of all RDTs tested, 94.2 % (21/22) had sensitivity values greater than 90 % among which 14 (63.6 %) were ‘Pf + PAN’ RDTs. The specificity was generally lower than the sensitivity for all RDTs and poorer for “Pf Only” RDTs. The predictive values and likelihood ratios were better for non-HRP2 analytes for “Pf + PAN” RDTs. The Kappa value for most of the tests obtained was around 67 % (95 % CI 50–69 %), corresponding to a moderate agreement.Overall, 94.2 % (21/22) of RDTs tested had accuracy within the range recommended by the WHO, while one performed poorly, below acceptable levels. Seven “Pf + PAN” and 3 “Pf Only” RDTs were selected for further assessment based on performance characteristics. Harmonizing RDT test presentation and procedures would prevent mistakes of test performance and interpretation.The online version of this article (doi:10.1186/s12936-016-1085-0) contains supplementary material, which is available to authorized users. Plasmodium species by microscopy depends on factors, such as the experience of the microscopists, proper staining of the slides, good quality reagents, appropriate maintenance of the microscope and the time spent reading a slide. Due to these limitations, physicians are often reluctant to accept results of microscopy in such operational settings. Therefore, the reliance of diagnosis on clinical grounds alone has resulted in over diagnosis of malaria in many clinical settings in developing countries including Cameroon [The challenge of controlling malaria is a continuous reality in most sub-Saharan countries. Despite increasing efforts in prevention and treatment, malaria has remained a major cause of morbidity and mortality, with an estimated 451 million clinical cases of malaria in 2007 alone, mostly in sub-Saharan Africa [Cameroon –5. RapidCameroon . Due to There is increasing interest in introducing RDTs for diagnosing malaria and improving malaria case management with artemisinin-based combination therapy (ACT), as studies show significant improvements in malaria control following implementation . In 2010The study population was made up of male and female febrile patients aged 1 year to 16 years suspected of a malaria infection in six busy peripheral health centres in Yaoundé. A patient was considered for enrolment into the study if he/she fulfilled the following criteria: willingness to donate 5 mls of blood after informed consent. If the patient sample was positive by microscopy, consent/assent was requested to participate. If the patient’s blood sample was negative by microscopy, the patient was still requested to consent. This procedure was followed consecutively until the required number of malaria positive and negative samples was obtained. Those performing the microscopy at the health centres were trained to collect, prepare, read and quantify malaria smears by light microscopy.Malaria transmission in Yaoundé is year round with four climatically distinct seasons composed of 2 short and two long dry and raining seasons respectively. Peak transmission occurs in the beginning of the rainy season. This assessment was conducted in April when transmission of malaria was expected to be at its peak.Plasmodium infection. The thin smears were considered negative if no parasite was seen in 100 oil-immersion fields from two independent readings. A third reading was performed in case of positive/negative discordance for asexual stages.In each centre, 5 ml of whole blood was collected from consenting febrile patients by venous puncture into pre-labelled EDTA tubes. For each blood specimen, about 200 μl was used to prepare a thick film and a thin blood smear for malaria parasite density determination and speciation. Microscopy was used as the reference test. This test was chosen because it is considered as the reference method for malaria diagnosis by the WHO. Blood smears were stained with freshly prepared 3 % Giemsa solution according to standard procedures. To confirm microscopy diagnosis at the health centre, a new diagnosis was performed by two experienced and certified microscopists from the Biotechnology Centre and the Yaoundé University Teaching Hospital. They were blinded to the health centre diagnosis. The thick smears were used to detect the presence of The RDTs in the study were obtained from major local distributors of clinical rapid diagnostic tests and other clinical laboratory suppliers in Cameroon. In total, twenty-two different RDTs used for malaria diagnosis and present in the market in 2010 were obtained. Selection of the RDTs was based on their presence in the Cameroon market and distributor’s willingness to participate in the assessments. The test cassettes or strips were of traceable quality . History of proper storage and transport conditions from manufacturers could not be guaranteed.Plasmodium falciparum, while the rest had detection analytes for P. falciparum and PAN representing other Plasmodium species. The test devices were mostly plastic lateral flow cassettes (18) and dipsticks (4). Most of the RDTs kits contained specimen transfer/reagent transfer devices except stated in the instructions to use a specific transfer device like a micropipette. Test antigens used in the kits were P. falciparum specific histidine rich protein II (HRP2), lactate dehydrogenase (LDH) and aldolase in order of frequency of occurrence in RDTs.The technical specifications of the various RDTs are presented as supplemental material  to 3(good) to 5(best): Ease and safety of taking blood; ease of adding reagents including the complexity of material/handling/transfer technique and device required and ease of interpretation of insert instructions. In addition, assessors of RDT results were asked to rank the tests independently based ease of interpretation defined by the position of bands, labeling, band intensity, background and time to interpretation of results.P1 and P2 are sensitivity and (1-specificity) respectively and n1 = n2 = sample size. In this way the sample size was estimated at 100.This assessment was a passive case detection survey. For malaria RDT under the study setting to perform well, the sensitivity should be at least 90 % and the specificity 80 %. expert opinion was sought that indicated a likelihood ratio of the positive test of at least 2 was clinically useful in ruling in malaria diagnosis with a good RDT. The likelihood ratio was used as our as our diagnostic test index because it does not change with pre-test probability. Therefore the 95 % confidence interval to exclude the likelihood ratio estimate using the values of the parameters provided above (Sensitivity/1-specificity) was calculated. This is given by the expressionThe data was checked for inconsistencies in data entry such as missing values and then used to calculate the sensitivity, specificity, the predictive values of the negative and positive RDT tests as well as the likelihood ratios and their 95 % confidence intervals. Inter-observer variations in the RDT test assessments were estimated by calculating for each operational parameter the Cohen’s kappa (κ) statistic as follows:A 95 % confidence interval (95 % CI) was calculated for each κ value and the overall agreement was evaluated as the weighted κ value with its 95 % confidence interval.This study was carried out under the auspices of the National Malaria Control Program with ethical clearance from the national ethics committee on health research in humans. Febrile patients whose left over blood specimen from routine medical exam were used provided verbal consent by phone. Information about the research was explained to each of one hundred participants/guardians by the consultant of the health centre either by phone or face to face for those who could come to the clinics. Participation was entirely voluntary.Overall, each of 22 different RDTs were tested on a total of ninety-seven venous blood samples. Three of the RDT test types were dipsticks while the rest were strips in rectangular cassettes.P. falciparum compared to pfHRP2 on the same RDTs.Results of the RDT evaluation are presented in Additional file The majority of RDTs with good sensitivity had specificity less than 75 %. Only 6/14 or 42 % had sensitivity and specificity values greater than 80 %. Even though the sensitivity of an RDT is most often considered as the key parameter in clinical case management in malaria, it is desirable that the test analyte detects the antigens for which it is intended. One of the challenges associated with the use of RDTs is cross reactivity with antigens of a similar nature but from a different source . ArguablP. falciparum demonstrated higher parasite detection rates compared to other analytes among all RDTs in this evaluation. However, tests that target LDH performed better when comparing both sensitivity and specificity for “Pf + PAN” RDTs. These findings are in agreement with the WHO based evaluation of RDTs for informed recommendations [Plasmodium falciparum is the dominant species in our geo-ecological settings, but mixed parasite populations have been observed previously by our group to be circulating in some regions. In Bangolan, NW Cameroon where this observation was made, infections either consisted of P. falciparum coexisting with Plasmodium malariae (in 70 % of cases) or non-P. falciparum  [Tests that target HRP of ndations . Therefof cases) . Therefof cases) , althougThe weighted agreement between the two observers involved in reading the RDT results was quantified as the weighted Kappa value calculated for all test results irrespective of whether the test was a “Pf Only” or “Pf + PAN” RDT type. The weighted Kappa value obtained was 67.5 % (95 % CI 64.6–70.3 %), corresponding to a moderately agreement between the assessors under our field condition. However, the agreements were better for some tests over others. For example, good agreement was obtained for more “Pf + PAN” RDTs than for “Pf Only” RDTs. SD Bioline Pf + PAN test cassette had the best agreement , based on the detection of both Pf and PAN while Clearview Malaria Combo was worst .Based on the assessment of operational and technical characteristics of the 22 RDTs, differences in the test presentation, package contents, blood lancing and transfer devices, variations in insert instructions of use and safety disposal, procedures for test performance and results interpretation were observed. Additional file Because of the free distribution of long-lasting insecticide-treated bed nets since 2011 to almost every household in Cameroon, a rapid decrease in the prevalence of malaria in many areas in Cameroon is expected. Low intensity infections may pose a diagnostic challenge for malaria surveillance purposes in the future due to shifting disease control priority, necessitating nucleic acid based tests.This study has several potential limitations. Firstly, the results of rapid diagnostic tests vary as a function of parasite density . It woul®pf HRPII the most preferred among “Pf Only” RDTs. Among the dipsticks, “Pf + PAN” ParaHIT Total Dipstick was preferred. Harmonizing RDT test presentation and procedures would prevent mistakes of test performance and interpretation.Of all RDTs tested, 94.2 % (21/22) had sensitivity values greater than 90 % among which 14 (63.6 %) were ‘Pf + PAN’ RDTs. The specificity was generally lower than the sensitivity for all RDTs, and poorer for “Pf Only” RDTs. The predictive values and likelihood ratios were better for non-HRP2 analytes for “Pf + PAN” RDTs. Rapid diagnostic tests which incorporate histidine rich protein II and lactate dehydrogenase are appropriate for settings. There was moderate agreement that SD Bioline Malaria Antigen P.f/Pan, ACON Malaria P.f/pan and Parascreen pf + PAN and CareStart™ Malaria HRP2/pLDH Combo Test were most preferred in order of merit among “Pf + PAN” RDTs while SD Bioline Malaria Antigen P.f and ParaHIT


To assess the prevalence of malaria among illegal gold miners in the French Guiana rainforest, we screened 205 miners during May–June 2014. Malaria prevalence was 48.3%; 48.5% of cases were asymptomatic. Patients reported self-medication with artemisinin-based combination therapy. Risk for emergence and spread of artemisinin resistance among gold miners in the rainforest is high. Malaria control programs on the Guiana Shield, a region of South America, are challenged by migrant populations looking for gold. Since 2008, the “Harpie” operation to control and reduce illegal gold mining activities has been conducted by French Armed Forces in French Guiana. Military deployments at illegal gold mining sites have resulted in several outbreaks and increased incidence of malaria in French forces pdm09, um vivax . Given tBecause of violence and insecure conditions at illegal mining sites, French military health services conducted the mission. Twelve soldiers and military policemen accompanied the medical team to ensure their protection but also that of the miners’ community. A primary care clinic and laboratory were set up under tents at the Eau Claire gold mining camp from May 28, 2014, through June 6, 2014. Active malaria screening was offered to every person who sought care for any reason. Pf/Pan; Standard Diagnostics, Inc., Giheung-gu, South Korea) and thin blood films were performed in the field. Patients who had positive results of an RDT, thin blood film, or both received treatment. Diagnostic tests associating the malaria rapid diagnosis test (RDT)  and/or >2 of the following: nausea, vomiting, diarrhea, abdominal pain, anorexia, headache, or jaundice. Other Plasmodium infections were classified as asymptomatic.We defined>1 area outside Eau Claire: 54.1% (111/205) to Suriname ; 22.4% (46/205) to Brazil; and 18.5% (38/205) to the main cities in the malaria-free area along the French Guiana coast . Median age was 39 years . The workers had been gold panning for a median time of 7 years  and on illegal gold mining sites in French Guiana for a median time of 4 years . Most  patients came from Brazil, 4 (2.0%) came from Suriname, and 1 (0.5%) came from Guyana. Before their arrival at Eau Claire, patients had lived in Brazil , Suriname , or at another illegal gold mining site (19/69 [33.7%] different sites throughout inland French Guiana and 1 unknown site) . During na coast .>1 fever episode and 131 (63.9%) reported several (2–>4) episodes; 121 (59.0%) reported >1 malaria episode and 93 (45.4%) several episodes  reported episodes . Self-meP. falciparum and P. vivax single infections accounted for 44.4% (44/99) and 29.3% (29/99) cases, respectively, and mixed infection with P. falciparum and P. vivax for 26.3% (26/99) .  (26/99) . RDTs, tPlasmodium infections observed confirms that malaria is hyperendemic there (During the 10 days of field work, almost all inhabitants of the gold mining site sought medical care. The high prevalence of malaria and asymptomatic pfK13 C580Y mutants obtained from samples in 2010 were recently reported (Systematic self-medication by patients using ACTs without following a full course of treatment is a serious risk for emergence of resistance to artemisinin and associated drugs (


Periostin levels are associated with airway eosinophilia and are suppressed by corticosteroid treatment in asthma. This study sought to determine the relationship between serum and sputum periostin, airway inflammatory phenotype and asthma control.n = 83) underwent a clinical assessment, sputum induction and blood sampling. Dispersed sputum was used for a differential cell count and periostin assessment (ELISA). Serum periostin was determined by the Elecsys® immunoassay.Adults with poorly-controlled asthma (p < 0.001). Serum and sputum periostin were significantly higher in patients with eosinophilic asthma (n = 37) compared with non-eosinophilic asthma. Both serum and sputum periostin levels were significantly associated with proportion of sputum eosinophils  but were not associated with asthma control. In receiver operator characteristic curve analysis, the area under the curve (AUC) for serum periostin (n = 83) was 0.679, p = 0.007. Peripheral blood eosinophils assessed in 67 matched samples, had a numerically greater AUC of 0.820 compared with serum periostin, p = 0.086 for the detection of eosinophilic asthma.Periostin levels were significantly higher in serum (median (IQR) of 51.6  ng/mL) than in sputum  ng/mL)  contains supplementary material, which is available to authorized users. Asthma is a chronic heterogeneous inflammatory disorder of the airways where approximately 50 % of adults have eosinophilic airway inflammation which is referred to as eosinophilic asthma. Sputum eosinophils are an effective monitoring tool in asthma when used in therapeutic decision making and there is good evidence that this approach can significantly reduce severe exacerbations , 2. The We have recently shown that blood eosinophil counts are a suitable surrogate for sputum eosinophil proportion in identifying patients with eosinophilic asthma, being simple and readily available and thus may provide a suitable asthma management tool . SimilarThis study sought to examine the ability of serum periostin to predict the presence of eosinophilic asthma and to determine the relationship between airway and serum periostin levels, inflammatory subtype and asthma control in a group of adults with poorly-controlled asthma.n = 83) who were participants in a randomised controlled trial of macrolides in asthma. The diagnosis of asthma was established using the American Thoracic Society guidelines based upon the presence of current episodic respiratory symptoms (past 12 months), doctor-confirmed diagnosis and evidence of variable airflow obstruction [1 < 40 % predicted, current smokers, ex-smokers who had ceased smoking in the previous year and those with an exacerbation or respiratory infection in the past four weeks were excluded. Those with significant smoking-related emphysema (ex-smokers >10 pack year history and carbon monoxide transfer coefficient <70 % predicted) OR smoking history >10 pack years and exhaled carbon monoxide >10 ppm were also excluded.Eligible adults with asthma n = 3 who wertruction . ParticiParticipants underwent a clinical assessment, allergy skin test, spirometry with bronchodilator response, sputum induction and blood sampling in turn. All tests were performed on the same day prior to the commencement of study medication. Measurements were carried out by observers blinded to other results.Subjects gave written informed consent , Royal Adelaide Hospital Human Research Ethics Committee (081108f), Hunter New England Research Ethics Committee (08/11/19/3.03), The Prince Charles Hospital Metro North Hospital and Health Services (HREC/08/QPCH/4), Metro South Health Service District Human Research Ethics Committee (HREC/09/QPAH/015).This cross-sectional study characterised participants during a single visit where symptoms, atopy, medication use and smoking status were assessed, and the spirometry and sputum induction and blood collection were undertaken. Sputum was processed for inflammatory cell counts and sputum supernatant and serum were stored for periostin analysis.Spirometry  and induced sputum were performed. Sputum was induced by the inhalation of hypertonic saline (4.5 %) as described by Gibson et al. . SubjectSputum was processed as described . SelecteAsthma subtypes were defined by percentages of sputum eosinophils and neutrophils  and were classified as eosinophilic asthma if sputum eosinophils were ≥3 % and sputum neutrophils were <61 %, as previously defined .Venous blood samples were collected without anticoagulant. Periostin levels in serum were measured using the clinical trial version of the Elecsys® Periostin immunoassay  intended for use on the cobas e 601 immunology analyzer, an automated electrochemiluminescence immunoassay based on the sandwich principle . PeriphePeriostin levels in sputum were measured using the Genentech proprietary ELISA using the same antibody pair as the Elecsys® assay . The limp < 0.05. The performance characteristics of the periostin variables were examined by receiver operating characteristic (ROC) curves to determine the concentrations which best defined eosinophilic asthma based on the sputum cell count. Participants were assigned to the high periostin group if their serum periostin levels were ≥50 ng/mL [The results were expressed as mean ± SD for continuous variables and median with interquartile range (IQR) when data were not normally distributed. Categorical data were reported using frequencies and percentages. A Kruskal-Wallis test was performed in the different subgroups of subjects with asthma. Spearman’s rank correlation coefficient was used to assess the associations between non-parametric data. Results were reported as significant when 50 ng/mL .n = 28) had a paucigranulocytic phenotype (34 %); 17 had neutrophilic phenotype (21 %) and five participants had a mixed eosinophilic and neutrophilic phenotype (6 %).Participants consisted of 83 adults with a mean age of 61 years who were predominantly atopic (73 %) and poorly-controlled (mean ACQ6 1.83) on treatment (Table p < 0.001).For all subjects, periostin levels were significantly higher in serum  ng/mL) than in sputum  ng/mL) (n = 59) had sputum periostin concentrations within the assay dynamic quantitative range. Six samples had periostin levels that were beyond the maximum of quantitative range at 1:2 and 17 samples had levels that were below the detection level. Alternately, using the 1:10 dilution, sputum periostin was detectable in 35/83 (42 %) samples; this is significantly fewer when compared with the proportion of samples detected using the 1:2 dilution (78 % vs. 42 % p = 0.045). Subsequent analysis of sputum periostin was undertaken using the results from the 1:2 dilution.Sputum periostin was assessed at three dilutions, 1 in 2, 1 in 10 and 1 in 50. A matrix effect  was observed on assay linearity with increased concentrations of periostin measured when the sample was more diluted  we generated ROC curves. The area under the curve (AUC) for sputum periostin was 0.754, p = 0.086.Peripheral blood eosinophil counts were available in only 67 participants. We compared the ability of blood eosinophils and serum periostin as a biomarker to predict the presence of eosinophilic asthma in the same 67 samples. Peripheral blood eosinophils had a more favourable AUC of 0.820 compared with an AUC of 0.704 for serum periostin, r = 0.038 and r = −0.062, p > 0.05 respectively). Similarly, there was no difference in ACQ6 in those with high serum periostin (≥50 ng/mL) or low serum periostin (<50 ng/mL) levels . Both sputum and serum periostin were significantly and inversely associated with body mass index (BMI)  but not associated with ICS dose, smoking pack years, lung function or atopy. There was no difference in sputum or serum periostin levels in never smokers compared with ex-smokers (data not shown). Using logistic regression there was no improvement in the model to explain the presence of eosinophilic asthma by the addition of BMI to serum periostin levels.There was no association between mean asthma control score (ACQ6) and either sputum or serum periostin levels  greater than those derived by ELISA (unpublished data)In vitro tissue culture experiments using an air/liquid interface, representing the airway lumen and bronchial tissue respectively, support this hypothesis, as periostin was abundantly present in the basal medium, but could not be detected in the apical washes [There may also be a biological explanation for the observed lower levels of periostin in sputum. It has been shown that periostin is strongly expressed at the RNA level in epithelial cells but protein expression could not be detected in these cells , 7. HoweThe utility of identifying eosinophilic asthma is well established with improved management and reduced exacerbations as key outcomes , 15. HowThere are limited studies supporting the utility of periostin in the management of asthma. There is evidence that periostin levels can indicate better treatment outcomes in studies involving the IL-13 monoclonal antibody lebrikizumab or anti-IgE antobody omalizumab , 13, 18.In poorly-controlled asthma treated with inhaled corticosteroids, periostin levels are low in airway secretions, but can be detected. Although sputum and serum periostin levels are significantly related to sputum eosinophil proportions, their ability to predict eosinophilic asthma in poorly-controlled asthma appears relatively modest.Informed consent was obtained from the participants at the time of recruitment for publication of patient data where their identity was remained confidential.The detailed method describing validation of periostin assay has been included in Additional file The dataset(s) supporting the conclusions of this article are shown as individual data points in Figs.


Plasmodium falciparum malaria when travelling back from Cameroun. No antimalarial chemoprophylaxis had been observed. The patient was immediately admitted to the intensive care unit after evidence of multiple organ failure . However, initial parasitemia was less than 1%. The patient was managed by intravenous quinine and norepinephrine infusion due to refractory shock. The patient developed as an early complication ischemic lesions of both arms and feet. In addition to laboratory changes consistent with disseminated intravascular coagulation, there was also evidence for a low activity of the von Willebrand factor (VWF) cleaving protease ADAMTS13. Later complications included repeated candidemia and bacteraemia despite appropriate therapy; the origin appeared to be diffuse ischemic injury of the gastrointestinal tract. The patient ultimately recovered, but quadriamputation was necessary to treat symmetrical peripheral gangrene (SPG). In severe Plasmodium falciparum malaria, ischemic changes may be due to microvascular obstruction, but, in patients with low parasitemia, other endothelial factors may also be involved as observed in other groups of thrombotic microangiopathies.A 63-year-old Caucasian woman developed severe  Plasmodium falciparum malaria [Symmetrical peripheral gangrene (SPG) is a possible but still infrequent complication of malaria –13. The  malaria . More re malaria . Early c malaria . We desc Plasmodium falciparum malaria was rapidly made on the blood smear and treatment was started with intravenous quinine and doxycycline (as artesunate was not available). Parasitemia was estimated less than 1%. Admission coagulation tests revealed activated partial thromboplastin time 37 sec, international normalized ratio 1.2, fibrinogen 299 mg/dL, D-dimers >8000 ng/mL , ADAMTS13 activity undetectable , von Willebrand factor (VWF) antigen 340% , and platelet count 21,000/mm³. Early complications included refractory hypotension and hypoxemia and neurological worsening. Intubation was required for mechanical ventilation; norepinephrine infusion was progressively increased to the maximal infusion rate of 46 μg/min. An acute left ventricular dysfunction could be demonstrated at echocardiography. Continuous venovenous hemofiltration was initiated for acute renal failure. Due to the evidence of multiple organ failure and despite relatively low parasitemia, exchange transfusion therapy was performed. Only one session of 87 minutes was performed. The patient's theoretical total blood volume was 4407 mL; on the whole, 1409 mL of red blood cells was removed and replaced by 1277 mL of packed red blood cells. Blood coagulation tests remained disturbed, with epistaxis and rectal bleeding. The nadir of platelet count  and plasma fibrinogen (66 mg/dL) was noted on day 4, but there was only a mild increase in activated partial thromboplastin time and prothrombin time. The activity of ADAMTS13 slightly increased to 32%; no inhibitor or ADAMTS13 autoantibodies were found. From day 2, blackish discoloration of both hands and feet was noted and by the following days a definite line of demarcation was observed between gangrenous and normal skin , despite appropriate antifungal and antimicrobial therapy. The abdomen computed tomography (CT) failed to reveal bowel ischemia, but diffuse thickening of the jejunal wall was found. Endoscopy on day 11 disclosed multiple duodenal ulcerations from ischemic origin . After that, the patient became unable to ingest any food or drug and a transfer to Europe was rapidly decided. She became seriously ill during the flight back. On arrival at the airport, she was found stuporous and hypotensive and was rapidly transferred to the intensive care unit (ICU) of the nearest hospital. The diagnosis ofmal skin . Ultrasoc origin ; colonosc origin . NorepinThe decision to perform quadriamputation was discussed after recovery from the critical period, when she became able to give her consent. Surgery was performed two months after the first signs of peripheral gangrene. Renal function recovered and the patient was further transferred for rehabilitation. Plasmodium falciparum malaria but remains rare in travelers returning from endemic areas [ Plasmodium falciparum infection and by the release of inflammatory cytokines leading to thrombin formation [ Plasmodium falciparum infection was associated with a significant increase in VWF and active VWF levels and a decrease in ADAMTS13 activity, resulting in the presence of circulating ultra-large VWF multimers [Symmetrical peripheral gangrene (SPG) may be encountered afteric areas –13. It iic areas . In mostormation . There iultimers . The pre Plasmodium falciparum malaria associated with DIC and SPG, 17 had received quinine as antimalarial therapy [The role of concomitant therapy in the pathogenesis of SPG has also to be discussed. In a review of 23 cases ofThere is no evidence that blood exchange transfusions or fresh frozen plasma administration could be effective treatment options for patients with DIC and SPG. Heparin or streptokinase is not indicated. Surgery is often performed after some delay, when the delimitation of dry necrotic areas is evident. Plasmodium falciparum malaria are also profoundly immunosuppressed and susceptible to opportunistic fungal infections [ Plasmodium falciparum malaria with a high parasite count (24%), shock, and multiple organ failure [ Candida albicans from admission. The patient had a favourable outcome after amphotericin B plus flucytosine therapy. Several factors may be involved in this acquired immunosuppression: free iron overload as a result of massive hemolysis, immunosuppressive effects of some antimalarial drugs, interference of the organism with innate cellular and specific cell-mediated immunity… [Severely ill patients with complicatedfections , 16. Inv failure . Blood cmmunity… , 18. Plasmodium falciparum malaria may mimic the evolution of other thrombotic microangiopathies. The limitation of this observation is that some specific coagulation tests  could not be obtained.In conclusion, by some aspects, the clinical course of severeCytoadherence of parasitized erythrocytes causes not only mechanical obstruction but also endothelial activation. Further investigations of the coagulation cascade could be helpful to better investigate microcirculatory disorders and subsequent ischemic injuries.


Preventive measures and treatment guidelines are needed to address the sizeable prevalence of disease in this population. <6 months of age was 11.8% . Seroprevalence ranged from 5.7% in The Gambia to 41.6% in Guinea. Mean parasite densities in infants were significantly lower than those in children 1–9 years of age in The Gambia (p<0.0001) and Benin (p = 0.0021). Malaria in infants was significantly associated with fever or recent history of fever (p = 0.007) and anemia (p = 0.001). Targeted preventive interventions, adequate drug formulations, and treatment guidelines are needed to address the sizeable prevalence of malaria among young infants in malaria-endemic countries.The prevalence and consequences of malaria among infants are not well characterized and may be underestimated. A better understanding of the risk for malaria in early infancy is critical for drug development and informed decision making. In a cross-sectional survey in Guinea, The Gambia, and Benin, countries with different malaria transmission intensities, the overall prevalence of malaria among infants  Infants are thought to be protected against malaria during the first 6 months of life, largely due to the transfer of maternal antibodies , representing areas of low, moderate, and high malaria transmission, respectively. In each country, regions were selected to represent overall malaria transmission trends and surveys were conducted in the catchment areas of 3 health facilities selected by using simple random sampling. In The Gambia, the Essau and the Soma Major Health Centres and the AFPRC General Hospital in Farafenni were selected out of 6 available sentinel surveillance sites. In the southern part of Benin, Bethesda Hospital and the Dodji-Bata and Golo-Djigbe health facilities were selected from a total of 12 available facilities, and in the Farannah district in Guinea, the Nalia, Tiro, and Banian health centers were also selected from 12 available facilities .>2 eligible infants, 1 infant was selected by using simple random sampling. Once the index infant was selected, 2 older children (1–9 and 10–15 years of age) living in the same household were also selected by simple random sampling and included in the study with the objective of estimating the force of transmission and differences in the local risk for infection between infants and older children. If children of the required age group were not available within the infant’s household, the nearest households were visited consecutively until eligible children were identified and enrolled.Malaria transmission in The Gambia and Benin is seasonal, occurring during the rainy season, whereas transmission in Guinea occurs year-round. Surveys were timed to coincide with the peak of malaria transmission in each country: October–November 2011 in The Gambia and Guinea; July–August 2012 in Benin. Before conducting the surveys, we explained the study objectives to community members in the catchment areas and obtained community approval. We identified households with infants by reviewing delivery records to detect births in the 6 months before the survey; traditional birth attendants assisted with the reviews. An information sheet explaining the objectives of the survey and the study procedures was then distributed to the parents of the infants. After written informed consent was obtained from parents, identified infants were enrolled in the study. In households with <6 months of age). A rapid malaria diagnostic test (RDT)  was performed, and children who tested positive were immediately treated according to treatment guidelines for the country in which they lived. Hemoglobin concentration was measured by using a HemoCue Hb 301 System  according to the manufacturer’s instructions. Thick-film blood slides were stained with 10% Giemsa for 10 min, and the presence of Plasmodium falciparum parasites was determined by reading 100 high-power fields under oil immersion. Slides were read independently by 2 microscopists, and parasite density was estimated by counting the numbers of asexual parasites per 200 leukocytes. Results were expressed as the number of parasites per microliter, assuming a total leukocyte count of 8,000 cells/μL. A 20% error check was used to identify discrepancies between slide readers. All discordant results were read by a senior microscopist, and the result was used as the final read. Blood slides from Guinea and The Gambia were read at the Medical Research Council (MRC) Unit in The Gambia; blood slides from Benin were read, following the same protocol, at Entomological Research Centre of Cotonou. The first 99 slides from Benin were read again in The Gambia; results were comparable.Study participants underwent a physical examination; axillary temperature and weight were recorded for each child. Information on the use of bed nets, including long-lasting insecticidal nets, and history of fever in the previous 24 hours was collected by using a structured questionnaire. All study participants had a blood sample collected by finger prick (children 1–15 years of age) or heel prick (infants Plasmodium species were conducted by using dry blood-spot samples (DBSs) collected on filter paper . DNA was extracted from 3 disks (6-mm diameter), which had been punched from DBSs by using a QIA X-tractor robot  according to the manufacturer’s protocol, and analyzed by using nested PCR as previously described  by indirect ELISA, as previously described .To determine the prevalence of malaria antibodies, we punched disks (6-mm diameter) from DBSs and placed them in 96-well plates. Serum that had been eluted after overnight (18 h) incubation at room temperature in 150 μL of reconstitution buffer  was used to determine antibodies against the 19-kDa merozoite surface protein 1 . After being cleaned, the data were analyzed by using Stata Statistical Software, release 12.1 . Baseline data were analyzed by descriptive methods, and summary statistics were presented as means ±SDs for continuous data and frequencies and proportions for categorical data. The χ2 test was used to analyze differences in proportions. Two-tailed p values and a 5% significance level were used. Results for infants <6 months of age from all 3 countries were pooled together, and univariate and multivariate logistic regression analyses were performed to determine features associated with malaria in this age group. A forward fitting logistic regression model was used to account for confounders and interaction. The odds for malaria with increasing age within the 0- to 6-month-old age group was determined and presented by country. Data from the case record forms were double-entered into an OpenClinica database  +2 SDs was considered the seropositivity cutoff , and the National Committee of Ethics for Health Research (Guinea). Written informed consent was obtained from the parents of each participant by a signature or thumbprint.A total of 6,761 children were included in the survey: 2,270 from The Gambia, 2,276 from Benin, and 2,215 from Guinea. The number of children categorized by age group, sex, and mean weight by age was comparable between countries . Almost By all 3 diagnostic methods, malaria prevalence was lowest in The Gambia and highest in Guinea; Benin had intermediate values. In all 3 countries, malaria prevalence was generally lower in infants 0–6 months of age . ResultsP. falciparum parasites. In Benin and Guinea, P. malariae and P. ovale parasite infections were also identified, predominantly as mixed infections. In Guinea, the prevalence of P. malariae parasite infections was 0.3% (2/724) in young infants, 12.0% (90/748) in children 1–9 years of age, and 5.8% (43/743) in children 10–15 years of age. Of these infections, 97% (131/135) were mixed infections with P. falciparum parasites. The prevalence of P. ovale parasite infection in Guinea was 3.1% (23/748) in children 1–9 years of age and 0.9% (7/743) in children 10–15 years of age; no cases were detected among young infants. In Benin, the prevalence of P. malariae parasite infection was 0.1% (1/761) in young infants, 1.7% (13/759) in children 1–9 years of age, and 2.8% (21/756) in children 10–15 years of age. In Benin, 34% (12/35) of the infections were mixed P. malariae and P. falciparum parasite infections. Overall, the mean parasite density per microliter of blood was 371.5 in The Gambia, 1,688.3 in Benin, and 2,037.9 in Guinea.By microscopy, all malaria cases identified in children from The Gambia were determined to be caused by infection with Plasmodium spp.–specific PCR also showed a higher prevalence of malaria in all age groups with increasing malaria transmission intensity  and Guinea  . Gametocyte prevalence by microscopy was lower in infants and increased with age and transmission intensity across the 3 countries  . Howeverountries .19 antibodies varied from 5.7% among young infants in The Gambia to 45.9% among 1- to 9-year-old children in Guinea  as determined by microscopy and 11.8%  as determined by PCR; the prevalence was substantially higher in Guinea than in Benin or The Gambia . Mean pa>37.5°C , and anemia  , axillary temperature = 0.001) . InfantsIn The Gambia and Benin, the odds for having malaria remained almost stable across the 0- to 6-month-old age group . In contThe prevalence of malaria among infants 0- to 6-months of age was not trivial  and increased with transmission intensity, as documented by the prevalence among older children. This variability in prevalence may be due to differences in transmission intensity but may also be due to differences in the use of preventive measures, as illustrated by the extremely low use of bed nets in Guinea. Such low intervention coverage may be an indicator of weak health systems with limited access to other malaria control interventions , which enable an efficient cycle of malaria transmission in local populations, including infants.In the low-transmission setting in The Gambia, the risk for malaria did not vary substantially between age groups. This finding was in obvious contrast to those in Benin and Guinea, where prevalence among infants was substantially lower than that among older children. This suggests that in areas where transmission has decreased substantially to low levels, the risk for infection may be shared by the entire population, including infants, and in high-transmission settings, infection in infants may be relatively limited by passively transferred maternal antibodies or possibly by lower attractiveness of infants to mosquitoes  were found among infants in Guinea and Benin. Therefore, currently available ACTs should suffice for the management of these cases, although failed parasite clearance has been reported in some P. malariae and P. ovale parasite–infected persons treated with ACTs (>37.5°C at the time of the survey. Although parasite densities in infants were lower than those in older children, about half of the infants with malaria were symptomatic. This finding contrasts with the long-held belief that malaria in young infants is not associated with clinical symptoms (Malaria in infants was significantly associated with fever or with a history of fever in the 24 hours before the survey, but only 10% of infants with malaria had an axillary temperature We have shown that malaria in young infants is not rare, can be symptomatic, and has major health consequences, most notably anemia. Current World Health Organization guidelines recommend the use of ACTs in infants, but they specify that for young infants weighing <5 kg, the available evidence is insufficient to confidently recommend this treatment. Thus, many of the ACTs carry label restrictions saying they should not be used for infants weighing <5 kg (The tools used in our study provided comparable estimates of the prevalence of malaria in young infants, with the exception of RDTs, which greatly underestimated the prevalence of malaria in The Gambia, possibly because of the low parasite densities (The overall dynamics of infection across the 3 countries suggests that the period of protection against malaria may be the first 3 months of life, and thereafter the odds for malaria rise increasingly by age. Our findings therefore provide evidence that the period of perinatal protection may be shorter than 6 months and that the 0- to 6-month-old age group is not a homogenous group in terms of malaria susceptibility. Thus, the challenge is that young infants are not adequately protected against malaria because of their limited coverage by current preventive strategies, such as seasonal malaria chemoprevention and intermittent preventive treatment during infancy, which are not widely implemented. This inadequate coverage is critical because our findings show that young infants can be affected by malaria and subsequently become anemic, which would also potentially increase their vulnerability to other pathogens (In conclusion, the prevalence of malaria is sizeable among young infants living in malaria-endemic countries. This problem must be addressed through the development of adequate pediatric drug formulations, targeted preventive interventions, and treatment guidelines for young infants.


Malaria rapid diagnostic tests (RDTs) are generally considered as point-of-care tests. However, most of the studies assessing the performance of malaria RDTs are conducted by research teams that are not representative of the classical end-users, who are typically unskilled in traditional laboratory techniques for diagnosing malaria. To evaluate the performance of a malaria RDT by end-users in a malaria-endemic area, a study protocol was designed and the VIKIA® Malaria Ag Pf/Pan test, previously evaluated in 2013, was re-evaluated by representative end-users.Plasmodium falciparum and Plasmodium vivax) and high positive samples  was used. Testing was performed directly by the end-users without any practical training on the VIKIA® Malaria Ag Pf/Pan kit.Twenty end-users with four different profiles in seven communes in Kampot Province (Cambodia) were selected. A set of 20 calibrated aliquots, including negative samples, low positive samples . No significant difference was observed between the different end-users. End-user interviews evaluating ease-of-use and ease-of-reading of the VIKIA® Malaria Ag Pf/Pan kit recorded 159 positive answers and only one negative answer. Out of 20 end-users, only one considered the test was not easy to perform with the support of the quick guide.All results obtained by the end-users were consistent with the expected results, except for the low positive (200 parasites/μL) The data presented in this study clearly demonstrate that the performance of the VIKIA® Malaria Ag Pf/Pan test when performed by traditional end-users in field conditions is similar to that obtained by a research team and that this RDT can be considered as a point-of-care tool/assay. Furthermore, the protocol designed for this study could be used systematically in parallel to conventional evaluation studies to determine the performance of malaria RDTs in field conditions.The online version of this article (doi:10.1186/s12936-015-0633-3) contains supplementary material, which is available to authorized users. Malaria remains one of the most important infectious diseases in tropical area, affecting over 300 million people every year, mainly in sub-Saharan Africa ,2. As rePlasmodium falciparum but often combined with the detection of other antigens common to all species, such as lactate dehydrogenase or aldolase. These combined RDTs can detect both P. falciparum and non-P. falciparum  infections [Malaria diagnosis has long been based on the microscopy examination of Giemsa-stained blood film, despite the requirement for highly qualified microscopists and reliable equipment, which are often lacking in remote areas where malaria is most prevalent . For ovefections .Over the past few years, the number of malaria RDTs and the scale of their use have rapidly increased. With over 100 commercially produced RDTs, this market has led to a proliferation in product availability against a background of relatively low capacity for regulation and quality control in many low-resourced, malaria-endemic countries. The variable quality of malaria RDTs, and consequently their diagnostic performance, have made it difficult for policy makers to determine which tests are the most suitable , even wiAt present, malaria RDTs are considered as point-of-care tests because they are mainly used by health care volunteers at community level, in remote malaria areas. Their use in field conditions allows an early diagnosis of malaria in any febrile patient and prompt treatment of confirmed cases. However, most of the studies aiming to assess the performance of malaria RDTs are conducted by research teams that are not representative of the classical end-users, who are typically unskilled in traditional laboratory techniques for diagnosing malaria. Consequently, only the ‘intrinsic’ performance of malaria RDTs is evaluated and information regarding their ‘global’ performance  when they are used by the traditional end-users is lacking.In this context, a protocol for evaluating the global performance of malaria RDTs by end-users in malaria-endemic areas was developed. As an example, the performance of the VIKIA® Malaria Ag Pf/Pan test, previously assessed in 2013 , was re-P. falciparum and other species of Plasmodium . This test is a ready-to-use cassette device, requiring the operator to transfer 5 μL of blood in the sample well with an appropriate device supplied in the kit, add five drops of the lysis buffer in the buffer well and read the results visually after 20 minutes  is a test based on immunochromatographic technology for detecting Seven communes in Kampot Province (Cambodia) were selected for this multisite, blinded study. Four different end-user profiles were considered as representative of the typical malaria RDTs end-users found in malaria-endemic areas: health medical centre staff, private physician, community health worker ) and private pharmacist. Five individuals of each end-user profiles were enrolled in the study, as presented in Figure P. falciparum/μL, one low positive sample at 200 parasites of P. vivax/μL, one high positive sample at 2,000 parasites of P. falciparum/μL and one high positive sample at 2,000 parasites of P. vivax/μL.Three types of sample  were tested in replicates of four, corresponding to a panel of 20 aliquots, as followed: one negative sample, one low positive sample at 200 parasites of The set of 20 coded aliquots was prepared according to the Standard Operating Procedures (SOP) developed in the Methods Manual for Laboratory QC Testing of Malaria RDTs  and codified as described in Table Frozen calibrated blood aliquots were transported at −80°C in a portable freezer running on 12 V (works with a cigarette lighter in a car). Defrosting was performed by the research team just before its use by the end-user , to confirm the good quality of the aliquots distributed to the users and to verify that no degradation occurred during transport or storage that could affect the expected results.Data were recorded and analysed using Excel software and MedCalc . The Chi-squared test was used to compare the proportion of positive results among end-users. A P-value <0.05 was considered to indicate statistical significance.Three successive quality assessments were performed by the research team: firstly in Phnom Penh at Institut Pasteur in Cambodia before going into the field, secondly and thirdly, seven and 14 days later, respectively, in the field. Results are presented in Table P. vivax at 200 parasites/μL. For this reason, aliquots ID 4, ID15 and ID19 were excluded from the final analysis of the end-user testing results.Quality assessments 2 and 3 showed a significant degradation of the quality of three out of four aliquots containing P. vivax aliquot with 35% of concordant results  ts Table .Table 4P. vivax aliquot, the proportion of positive results obtained by the VMW end-users (80%) was higher than other end-users (0-40%), but the difference was not significant (P = 0.22), and was probably due to the low number of aliquots tests (five per end-user).No significant difference was observed between the different end-users. For the low positive (200 parasites/μL) After performing the test, all end-users were interviewed and completed a questionnaire (‘use and usability’) to evaluate ease-of-use and ease-of-reading of the VIKIA® Malaria test. Results are presented in Table Out of the 20 end-users, only one considered the test was not easy to perform with the support of the quick guide. However, with the help of the package insert, all end-users considered that the operating instructions were clear enough to perform the test without having to participate in practical training. Moreover, all end-users claimed that the package insert allowed an easy interpretation of the results.P. falciparum parasites, an important issue in Southeast Asia [In endemic areas, health facility staff and community workers are in the front line to manage malaria cases, which mostly affect rural inhabitants, for achieving the laudable goal of elimination. To this end, early diagnosis of malaria by using malaria RDT and prompt treatment with effective anti-malarial drugs are the cornerstone of the management of suspect malaria cases . Therefoast Asia . Accordiast Asia .et al. that despite their apparent simplicity of use, the accuracy of malaria RDTs also depends on the accuracy of their preparation and interpretation [In this context, the availability of accurate, high performance malaria RDTs in terms of sensitivity, specificity, positive, and negative predictive values is crucial. However, most of the studies aiming to evaluate the performance of commercialized malaria RDTs are conducted by research teams, that are not representative of the traditional end-users in remote areas, whereas several studies have clearly shown that the accuracy of malaria RDTs is highly user-dependent -20. Inderetation . In mostretation .et al. [In the present study, a dedicated protocol was designed to re-evaluate the performance of the VIKIA® Malaria Ag Pf/Pan test, previously assessed in 2013 , by repret al. . No signP. vivax aliquot (200 parasites/μL) with a sensitivity of 35% (95% CI: 15.4-59.2%) were similar to the sensitivity reported for P. vivax samples containing 101–500 parasitaemia/μL of blood by Chou et al.  [P. vivax samples at low temperatures had probably accelerated the deterioration of antigen activity  [The results observed with the low positive egative) . The 200egative) ,24. The egative) , taking P. vivax aliquots and the use of frozen samples, although no other option was available. Indeed, it has been claimed by some RDT manufacturers that frozen blood samples are not optimum for RDT testing. They argue that malaria RDTs are optimized and recommended to be used with fresh finger-prick blood and that frozen blood samples could lead to aggregation, precipitation and leeching out of artefacts into the thawed blood. As all RDTs use a porous nitrocellulose membrane, the presence of artefacts clogging the membrane can alter the flow properties of the reagents and affect the completion of the test run in specified time. As a future option, WHO, TDR and FIND, along with other partners, have started the development of a generation of lot-testing samples based on the use of recombinant proteins, which could be used by community health workers to confirm quality of RDTs and significantly increase the confidence of national malaria control programmes in the results of RDTs.The main limitations of the study were the limited number of samples tested, especially the number of low positive The data presented in this study clearly demonstrate that the performance of the VIKIA® Malaria AgPf/Pan rapid test performed by traditional end-users in field conditions is similar to those obtained by a research team, and that VIKIA® Malaria AgPf/Pan can be considered as a point-of-care test. In addition, the protocol designed for this study, using well-characterized and calibrated blood samples at 2,000 and 200 parasites/μL could be used systematically in parallel to conventional evaluation studies  to deter