Intermittent preventive treatment in infants (IPTi) with sulfadoxine-pyrimethamine (SP) is recommended by WHO where malaria incidence in infancy is high and SP resistance is low. The current delivery strategy is via routine Expanded Program on Immunisation contacts during infancy (EPI-IPTi). However, improvements to this approach may be possible where malaria transmission is seasonal, or where the malaria burden lies mainly outside infancy.A mathematical model was developed to estimate the protective efficacy (PE) of IPT against clinical malaria in children aged 2-24 months, using entomological and epidemiological data from an EPI-IPTi trial in Navrongo, Ghana to parameterise the model. The protection achieved by seasonally-targeted IPT in infants (sIPTi), seasonal IPT in children (sIPTc), and by case-management with long-acting artemisinin combination therapies (LA-ACTs) was predicted for Navrongo and for sites with different transmission intensity and seasonality. In Navrongo, the predicted PE of sIPTi was 26% by 24 months of age, compared to 16% with EPI-IPTi. sIPTc given to all children under 2 years would provide PE of 52% by 24 months of age. Seasonally-targeted IPT retained its advantages in a range of transmission patterns. Under certain circumstances, LA-ACTs for case-management may provide similar protection to EPI-IPTi. However, EPI-IPTi or sIPT combined with LA-ACTs would be substantially more protective than either strategy used alone.Delivery of IPT to infants via the EPI is sub-optimal because individuals are not protected by IPT at the time of highest malaria risk, and because older children are not protected. Alternative delivery strategies to the EPI are needed where transmission varies seasonally or the malaria burden extends beyond infancy. Long-acting ACTs may also make important reductions in malaria incidence. However, delivery systems must be developed to ensure that both forms of chemoprevention reach the individuals who are most exposed to malaria. Intermittent preventive treatment of malaria in infants (IPTi) consists of a full therapeutic course of antimalarial drugs given at the time of Expanded Program on Immunisation (EPI) vaccination in infancy. EPI-linked IPTi using sulphadoxine-pyrimethamine (SP) provides a protective efficacy (PE) of approximately 30% against clinical episodes of malaria during infancy One potential alternative to EPI-IPTi is seasonally-targeted intermittent preventive treatment, where IPT is delivered at the time of peak malaria transmission rather than at the time of age-based EPI vaccination contacts. Seasonal IPT could be restricted to infants alone, or extended to include children above one year of age. A number of studies have shown that seasonal IPT in young children is very effective in preventing malaria when given at the time of peak transmission Another possible means to prevent malaria through the use of antimalarials would be to use long-acting drugs for the treatment of malaria cases. Both malaria infection and clinical malaria are overdispersed, meaning that some children suffer multiple malaria episodes while others around them suffer no malaria. A clinical malaria episode is thus a strong predictor of subsequent episodes In this study, a mathematical model was developed to investigate the protective efficacy of IPTi delivered at EPI vaccination contacts in infancy, and three alternatives, namely seasonal IPT in infants (sIPTi), seasonal IPT in children (sIPTc) and the use of long-acting ACTs for case-management of malaria (LA-ACTs). This was undertaken first for a specific site in Ghana for which detailed contemporaneous data on malaria incidence in children <24 months of age, entomological inoculation rate (EIR), and SP-IPTi efficacy were available. The site represents an area where malaria transmission is highly seasonal, although there is some transmission throughout the year. Under such conditions, the advantages of seasonal targeting are less obvious than in areas with a very short transmission season each year, such as many areas of the Sahel This study used data from the Navrongo IPTi study, which took place in Kassena-Nankana District (KND), Ghana between 2000 and 2004. The IPTi trial is described in detail elsewhere A set of deterministic compartmental models was developed. These are specified as sets of differential equations which describe changes over calendar time in the number of individuals in each compartment. The model equations are described in detail in 1), previously asymptomatic/untreated incubating superinfection (E2), asymptomatic or untreated parasitaemia (A), symptomatic and treated (S) or protected by IPT (I) , or remain asymptomatic and return to A1. In the simplest case, the probability of developing symptoms was assumed to be the same for both newly infected children and children with superinfections; this assumption was relaxed in later versions of the model. Acquisition of clinical immunity with increasing age is accounted for by allowing the proportion of infected children that develop clinical symptoms to vary between age groups. To incorporate effects of maternally-derived immunity, the probability that an infectious bite results in infection is assumed to be lower early in infancy than later , previously uninfected incubating infection (E IPT (I) . ChildreBecause the intention was to validate the model with trial data, we assumed that all children who had symptomatic infections and who reported to trial staff were treated. In the model, treated children recover faster than untreated or asymptomatic children and receive a period of post-treatment prophylaxis (PTP) when they cannot be reinfected. This is modelled by delaying the return of treated children to the uninfected (and susceptible) compartment, U, via a series of sub-compartments as described above This model structure describes the transmission dynamics in the control group of the Navrongo trial. For the intervention group, an identical set of equations is used but with the addition of compartments representing intermittent preventive treatment. The model was stratified into two separate age groups , allowing the impact of IPT given to specific age groups to be explored. The model was further stratified to reflect heterogeneity in malaria exposure, assuming that 20% of individuals receive 80% of all infections P. falciparum, receipt of an insufficient dose of SP, vomiting of treatment, or other similar reasons. It is assumed that an efficacious course of IPT will clear parasitaemia. In addition to its curative effect, IPT is considered to provide prophylactic protection against new infections. Individuals protected by IPT move into the IPT component of the model and remain in this state for a variable period before returning to U. As for the symptomatic treatment component, the IPT component is modelled with multiple compartments such that the duration spent protected follows a gamma distribution. Implementation of IPT is modelled in one of two ways, reflecting two different delivery strategies: 1) EPI-linked IPT delivered to infants at specific age-based vaccination contacts (EPI-IPTi) and 2) IPT given outside the EPI programme to all infants, regardless of their exact age, as simultaneous mass treatments during the season of highest malaria transmission (sIPT). With IPT given via the EPI, the cohort of infants receives IPT throughout the year as members reach the appropriate age to attend vaccination clinics . With seasonal-IPT, all infants receive IPT at specific times of year according to the peak in transmission . Firstly, IPTi delivered through the EPI as in the Navrongo trial was compared to a seasonal IPT strategy in which the same number of courses of treatment (four) was delivered to all infants, regardless of their exact age, on a monthly basis during the peak transmission period. Secondly, the PE of different seasonal IPT strategies was investigated by varying the number of IPT courses per year, the timing of IPT courses, and the coverage of each IPT course. Thirdly, the effect of using a longer-acting drug for sIPT or EPI-IPTi on the relative benefit of seasonal targeting was investigated.In later versions of the model, seasonal IPT was extended to include children aged 12–23 months in addition to infants. Although previous trials of IPT in children have usually included children under 5 or 10 years of age, because the model is fitted to the Navrongo trial cohort we simulate only the impact of including one additional year of IPT and present protective efficacy at 24 months of age.Finally, the effect of a long-acting drug regimen for case management was investigated by varying the duration of protection obtained after treatment for symptomatic malaria. This was done both in the absence of IPT strategies, and alongside EPI-IPTi, sIPTi and sIPTc.A previous study defined ‘marked seasonality’ as the occurrence of more than 75% of the annual malaria incidence in six months of the year Malaria incidence by calendar month in Navrongo is shown in Cumulative incidence of clinical malaria observed during the Navrongo trial, and the corresponding values predicted by the model are shown in The model prediction of the efficacy of four courses of seasonal IPT given to infants during the peak transmission season was 26.1% at 24 months of age . Four coThe use of longer-acting drugs for IPT would increase protective efficacy and reduce the advantages of seasonal targeting. However, within the constraints of drugs that are currently available, targeting IPT at the peak in transmission remains necessary to maximise efficacy in seasonal settings. With a hypothetical IPT drug that provided a mean of 90 days prophylaxis, longer than any currently available antimalarial, four courses of EPI-IPTi would provide protective efficacy by 24 months of age of 36.1%; this would be 42.3% with four courses of monthly seasonal IPT in infants.The predicted PE of four monthly courses of sIPT given at the peak in transmission during both the first and second years of life was 51.7% between 2–24 months of age.Use of a long-acting drug regimen for case management would be expected to reduce the number of malaria cases in both the SP and placebo groups. The PE of IPT would also decrease with increasing post-treatment prophylaxis after curative treatment, because more children would already be protected by their most recent curative treatment at the time they receive IPT . In modeSeasonal targeting of IPT given only to infants does not improve efficacy in sites with completely uniform transmission over the whole year, as would be expected . In siteMalaria chemoprevention by intermittent administration of courses of antimalarials such as SP can provide personal protection against sensitive parasites for about one month. The impact of a particular strategy on the malaria burden will depend on the age distribution and seasonal pattern of malaria in a particular situation, the number and timing of treatment courses, the age groups targeted, and the coverage achieved. Our mathematical model helps to explain the likely impact of different chemoprevention strategies in a range of epidemiological settings.In Navrongo, an area with extended seasonal transmission, four monthly courses of sIPT given only to infants would provide a protective efficacy of 26.1% at 24 months of age. Seasonal IPT would provide similar efficacy to EPI-IPTi even if the coverage achieved by the alternative delivery system was markedly lower. When possible differences in coverage among highly exposed individuals were considered, seasonal IPT was predicted to be more protective in all but the most extreme scenarios . Bojang The model simulations predicted that the use of a long-acting regimen for malaria case management would lower overall malaria incidence by providing post-treatment prophylaxis to children who are treated for malaria. Reductions in transmission as a result of ACT use have been predicted by previous modelling studies However, an important caveat is that the impact of long-acting drugs predicted by this model is based on the assumption that in a clinical trial, appropriate case management would be given to a high proportion of symptomatic malaria cases. In reality, a smaller proportion of the overall burden malaria cases are likely to be treated and the impact of long-acting drugs on protecting individuals who actually have malaria will be smaller. For this reason, our findings do not support the use of long-acting treatment drugs as an alternative to either IPT strategy, but rather as a complementary approach.Our results indicate that the use of a long-acting regimen for case management of malaria in conjunction with either form of IPT would be more effective than either form of IPT or long-acting ACTs as a lone strategy. This is biologically plausible given that case-management cannot prevent first episodes, but will prevent additional episodes in highly exposed children, whereas EPI-IPTi or sIPT can also prevent initial malaria episodes. Thus, while providing long-acting drugs for treatment may be of some value where no infrastructure exists to deliver IPT or other forms of chemoprevention (either via the EPI or via other approaches), this will not be as effective as use of long-acting drugs in addition to deliberate chemoprevention by IPT. These findings are in agreement with two studies in Ghana of seasonal IPT in addition to home management of malaria with long-acting drugs For most of the range of transmission patterns considered, IPT courses given in infancy during the part of the year when transmission peaks predicted higher efficacy than a four-course EPI-linked IPT program, with a clear advantage in settings with ‘marked seasonality’ The results presented show that if a delivery system can be developed outside the EPI for the delivery of seasonal IPT, inclusion of young children in addition to infants would offer important advantages. However, the challenges of developing an alternative delivery system could be substantial. In addition to requiring an alternative delivery mechanism, seasonal IPT given for the first few years of life will require more courses of IPT than EPI-IPTi (even if fewer courses are given per year), may achieve lower coverage, and would be more costly. However, the gains achieved by protecting older children who are at high risk of severe malaria could be substantial.We have limited our analyses to children less than two years old and focused on protective efficacy at 24 months of age for two reasons. Firstly, in order to model the Navrongo cohort in detail, which was only followed-up until two years of age, and secondly, to show the impact on extending for a single year beyond infancy. Our model suggests that with four monthly courses of seasonal IPT in each of the first two years of life, PE would have been 51.7% at 24 months of age. Equivalent protective efficacy to IPT strategies targeting only infants may be achievable with lower coverage or fewer courses. Where seasonal IPT has been evaluated in practice, older children up to 5 or 10 years have been included. The potential advantages of a delivery system capable of administering IPT to older children would be even greater than that shown here. Furthermore, the protective efficacy of seasonal IPT covering a broader age range would be less affected by any future shift in the age-specific disease burden as a consequence of reduced malaria transmission than would EPI-linked IPT given only to infants. In the context of reducing malaria transmission in some parts of Africa Despite the potential advantages of seasonal IPT in children, there are several important factors in addition to protective efficacy that must be considered when comparing seasonal IPT to EPI-IPTi. Several factors were not taken into account in the models, which focused on the direct protection against clinical malaria. This analysis does not consider potential differences in the cost of the alternative delivery strategies, which will weigh against the gains in protection since EPI-IPTi has been shown to be highly cost-effective. However, seasonal IPT using community based health-workers has been piloted in several countries, and can achieve high coverage at moderate cost Rebound effects were not modelled since the mechanisms by which such effects might be seen are only speculative at present. Nevertheless, this issue requires further study since a temporary increase in incidence of clinical malaria was observed in children given long-term chemoprophylaxis Our model was initially parameterised using data collected in Navrongo, Ghana. Some of the values of parameters are likely to differ in other settings, including the proportion of infections that are symptomatic, the recovery rate, and the proportion of infectious bites that lead to a successful infection In summary, while IPTi delivered through the EPI is likely to be a valuable malaria control tool in certain situations, particularly those with high transmission, seasonal IPT targeted at the peak transmission period may provide greater protection in other epidemiological settings. Long-acting regimens for case management may complement the protection provided by IPT, but for case-management to have an impact as large as that indicated here, it will be necessary to ensure that these antimalarials reach the children who are most exposed to malaria, typically the individuals who also have poorer access to treatment. Extending seasonal IPT to include young children as well as infants has important additional advantages, and may become increasingly relevant in the future if the reductions in malaria transmission seen in parts of Africa can be achieved more widely and can be sustained. Developing effective delivery mechanisms for these alternative strategies is crucial to ensure the potential advantages presented here can be achieved in practice.Supporting Information S1Extended Methods, Results and Sensitivity Analysis.(DOCX)Click here for additional data file.


Plasmodium vivax and Mansonella ozzardi was detected in a blood sample from a person who had shown symptoms of malaria and lived in a city that was close to the Argentina/Bolivia border. The case was detected during a random revision of thick and thin smears from patients diagnosed with malaria from various towns and cities located in north-western Argentina between 1983 and 2001. Trophozoites of P. vivax were observed in the thin blood smear along with M. ozzardi microfilaria , which presented a long, slender, pointed anucleate tail and the absence of the sheath. This last characteristic is shared with Mansonella perstans, Mansonella streptocerca and Onchocerca volvulus. More rigorously controlled studies to detect other co-infection cases in the area as well as the possibility of importation from Bolivia into Argentina are currently ongoing. The relationship between the malaria parasite and microfilaria, the potential effect of malaria treatment on the development of M. ozzardi, and the possible impact of this microfilaria on the immunity of a person against P. vivax are all still unknown. This contribution constitutes a point of focus for future studies involving the interaction between the parasites and the potential risk that humans are exposed to.A case of co-infection with  Plasmodium vivax spans the greatest geographic range [P. vivax are estimated between 130 and 390 million, with 2.6 billion individuals living at risk of infection [Plasmodium falciparum; however, an increasing number of P. vivax cases with severe manifestations have been reported recently [P. vivax becoming asymptomatic patients [Malaria caused by ic range . Worldwipatients -10.Plasmodium parasites in Anopheles mosquitoes [P. falciparum), a “tertian malaria” (produced by P. vivax) and a “quartan malaria” , with all types co-existing at the same time [The first cites of malarial disease in Argentina occurred between the end of the 19th Century and the beginning of the 20th Century and included reports on the geography of the disease within the country , the presquitoes , and theame time .Plasmodium vivax was the only malaria parasite reported in the north-west region of the country since the 1970s [P. vivax was due to active searches for sick people conducted by technicians of the Ministry of Health of Argentina.he 1970s -19. PosiAnopheles pseudopunctipennis is the main malaria vector in north-western Argentina [P. vivax and transmitted by A. pseudopunctipennis is much more benign compared to infections caused by other malarial parasites; commonly observed manifestations include intermediate episodes of fever and chills.rgentina -16. MalaPlasmodium parasites with the larval forms of the nematodes called microfilaria. The presence of microfilaria infection in Argentina was recognized by malaria surveys in the north-western region of the country [Microfilaria tucumana first described [Microfilaria dermaquayi[Mansonella ozzardi[et al.[Nematodes that cause filariasis have been reported throughout the tropical regions of the world -22. Gene country , with thescribed  followedermaquayi,25, a hoa ozzardi, being ddi[et al..Mansonella ozzardi is endemic to the subtropical mountainous rainforest in the north-west region of Argentina [Culicoides lahillei (main vector) and Culicoides paraensis (secondary vector) and black flies, Simulium exiguum (secondary vector) [M. ozzardi is considered a relatively non-pathogenic filarial parasite, its pathogenicity is still a controversial subject requiring further study [et al.[et al.  on the influence of ivermectin in patients and the epidemiology of the disease in Argentina, respectively. A high prevalence of filariasis (20.7%) was observed in one locality in the north-west region of the country [rgentina . In this vector) . Althouger study ,30. Thery [et al. and Vegg country . A simil country .Plasmodium nematodes co-infection was widely reported in America [et al.[Plasmodium microfilaria in the country; thus, the current study is the first report of the presence of P. vivax and M. ozzardi after several decades. America -37. Aráo America  and Rose America  have cita [et al. reportedP. vivax with others parasites is considered important.According to the World Health Organization (WHO) , ArgentiP. vivax and M. ozzardi in patients with a diagnosis of malaria who received anti-malaria treatment with primaquine-cloroquine in north-western Argentina from 1983 to 2001.The present study aimed to detect co-infection with From October to December 2012, it was performed a preliminary analysis of 166 thick and thin smears from patient blood samples from different towns and cities situated in north-western Argentina, at the border with Bolivia, where there are still reports of imported malaria cases. These towns and cities included Aguas Blancas , El Oculto , San Ramón de la Nueva Orán , Pichanal , Embarcación  General Ballivian , General Mosconi  Tartagal , Aguaray , Campo Durán  and Salvador Mazza  , the patient’s name and address, place where the disease was supposedly acquired, travel during the previous three months, type of work, nationality and case classification. The epidemiological investigation revealed that the man living in Tartagal City and working in a banana farm close to the city acquired the malarial disease in Yacuiba (city on the Bolivia border) where he travelled two weeks before the report. The patient did not suffer any type of pain, with the presence of disease revealed by intermediate fever episodes. After laboratory confirmation, the treatment of the patient proceeded with a combination of chloroquine/primaquine pills for two consecutive weeks. The doses used were consistent with WHO guidelines  as folloThe study methodology, including the ethical aspects, was approved by the Ministry of Health of Argentina, which has an Ethical Committee who revised these aspects. They are included in the Protocol of the Manual de Normas y Procedimientos de Vigilancia y Control de Enfermedades de Notificación Obligatoria, Ministerio de Salud Argentina.P. vivax as the most abundant parasite that appeared in blood smears, as well as the high prevalence of co-infection with M. ozzardi[In north-western Argentina, the diagnosis of malaria is directly related to intermediate fever episodes, but the diagnosis of filariasis is a consequence of blood smear analysis for malaria. The first reports of malaria in Argentina showed that the disease was the most important parasitic disease of the time, not only because of the number of cases reported but also because of their wide geographical distribution across the country. The majority of malaria reports cited . ozzardi.P. vivax. This species seems to be the only parasite incriminated in malaria in human beings, and the most tolerant to the climatic and environmental changes, enabling its survival during this time in north-western Argentina [M. ozzardi co-infection also decreased dramatically during the last few decades, with the latest studies reporting high prevalence of microfilaria only within isolated communities, but affecting both sexes and with increasing infection rates progressively with increasing patient age [After the malaria eradication programme in 1959, malaria cases decreased considerably before a resurgence in disease in 1967 . Since 1rgentina -19,39. Tient age ,40-42.P. vivax and M. ozzardi. These studies should try to establish the prevalence of co-infection and quantify the potential effects of malaria treatment on the development of M. ozzardi and the impact of this microfilaria on the immunity of humans infected with P. vivax in north-western Argentina. The results presented in this study could be used as the basis for future studies involving the interaction of these parasites and include other localities on the Bolivian border, considering that the parasites and their vectors do not recognize geographical barriers to their transmission.From the present report, it has concluded that further studies are necessary to search for cases of co-infection with The authors declare that they have no competing interests.MJDJ is a Research Assistant of CONICET and a Consultant of the Ministry of Health, this research is part of the studies that are ongoing in the country included in the Malaria Pre-elimination Phase according to the World Health Organization. She conceived the study and drafted the manuscript. CAVA checked all the human samples and also prepared the manuscript. ESO and GBG reviewed the literature and references. MOZ as a part of Ministry of Health of the Argentina contributed with the human samples to be analysed and participated in the edition of the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum disease was assessed in a cohort of 100 consecutive travellers with various species of imported malaria.Imported malaria occurs as a relatively rare event in developed countries. As a consequence, most clinicians have little experience in making clinical assessments of disease severity and decisions regarding the need for parenteral therapy or high-level monitoring. In this study, the diagnostic accuracy of procalcitonin (PCT) for severe P. falciparum malaria had significantly higher median PCT levels on admission as compared with patients with uncomplicated P. falciparum disease. In addition, PCT levels in patients with non-falciparum malaria were also higher compared with patients with non-severe falciparum malaria but lower compared with severe P. falciparum malaria. At a cut-off point of 10 ng/mL, PCT had a sensitivity of 0,67 and a specificity of 0,94 for severe falciparum disease. However, at lower cut-off points the specificity and positive predictive value were rather poor although the sensitivity and negative predictive value remained high.In all patients, PCT was measured on admission with a semi-quantitative 'point-of-care' test. Patients with severe falciparum species and by concomitant bacterial infections.Potential drawbacks in the interpretation of elevated PCT levels on admission may be caused by infections with non-Semi-quantitative determination of PCT on admission is of limited use in the initial clinical assessment of disease severity in travellers with imported malaria, especially in settings with limited experience with the treatment of malaria. Plasmodium species and quantification of the parasite load [Plasmodium falciparum infections. Therefore, well-validated, simple, laboratory-based prognostic markers would be of great value in assisting clinicians dealing with returned travellers with malaria. Unfortunately, many laboratory markers including parasitaemia have poor sensitivity, specificity and predictive values for estimating the severity of disease [Imported malaria occurs as a relatively rare event in developed countries. As a consequence, most clinicians have little experience in making clinical assessments of disease severity and decisions regarding the need for parenteral therapy or high-level monitoring. In addition, obtaining a correct diagnosis of malaria may be troublesome in centres where laboratory staff are less skilled in preparing malaria thick and thin smears and in the proper identification of the causative ite load . These c disease .P. falciparum disease was evaluated in a cohort of 100 consecutive travellers with various imported malaria species. For this purpose, the PCT-Q® test was used, a semi-quantitative immunochromatographic "point-of-care" test, which, in contrast to quantitative PCT assays, allows a rapid measurement of PCT without a need for further technical equipment or support and can be easily incorporated in the initial clinical assessment of travellers who return home ill.Procalcitonin (PCT), a precursor peptide from the hormone calcitonin, has been considered by some, but not all, to be a specific and useful indicator of systemic bacterial infections ,4. Inter® test (see below) in the initial assessment of travellers who return home sick after a stay in the tropics or sub-tropics. Of each traveller demographic, clinical and laboratory data were collected including a blood sample on admission for PCT testing. Patients were classified as having severe or complicated P. falciparum malaria if they met pre-specified criteria for severe malaria [The Harbour Hospital is a 161-bed general hospital located in Rotterdam, the Netherlands. It also harbours the Institute of Tropical Diseases, which serves as a national referral centre for tropical diseases. All patients reported herein were consecutive travellers diagnosed with imported malaria, who were admitted to the Institute of Tropical Diseases of the Harbour Hospital in the period January 2004 to December 2007 and participated in the PCTTM  observational survey. The PCTTM was an observational study that had the aim of evaluating the diagnostic accuracy of the semi-quantitative PCT-Q malaria .PfHRP-2 screening and conventional microscopy with subsequent specification of the Plasmodium species . In case of P. falciparum infections, parasite density was expressed as the number of P. falciparum trophozoites per 100 red cells in a thin film or the number of parasites per 100 white blood cells in a thick film. The parasite load was calculated from these figures using the actual number of white or red blood cells counted in the blood sample.Routine examinations included red blood cell count, haematocrit, white blood cell count, platelet count, serum electrolytes, total bilirubin, serum creatinine, liver enzymes, blood glucose and plasma lactate. Blood smears (thin and thick films), were obtained from finger pricks and stained with Giemsa and parasite counts were performed. Malaria was diagnosed by QBC analysis, ® test  according to the instructions of the manufacturer. PCT results were classified as follows: a negative result or a PCT < 0,5 ng/ml was classified as "normal"; a PCT of 0,5 ng/ml or a PCT between 0,5-2,0 ng/ml was classified as "low"; a PCT of 2,0 ng/ml or a PCT between 2,0-10,0 was classified as "moderate"; a PCT of 10,0 ng/ml and above 10 ng/ml was classified as "high" as recommended [PCT was measured with the BRAHMS PCT-Qommended .All values are presented as median (range). For comparison between groups, the Mann-Whitney U test, Kruskal-Wallis test or Chi square test were used as appropriate. Post-hoc analysis was performed using Mann-Whitney U test. P values at α < 0.05 were considered statistically significant.P. falciparum malaria infection , 65 travellers had an uncomplicated P. falciparum infection, whereas another six patients had severe P. falciparum disease. Thirty-one patients with uncomplicated P. falciparum disease, one patient with severe P. falciparum disease and eight patients with non-P. falciparum disease were considered to be semi-immune or partially immune, respectively. As depicted in Table P. falciparum infection had the highest serum CRP, lactate, creatinine, lactate dehydrogenase and bilirubin concentrations and the lowest haemoglobin concentrations and platelet counts on admission. As shown in Table P. falciparum infections, whereas patients with severe P. falciparum infections had the highest median PCT concentration  or high (n = 4), respectively. Depending on the selected cut-off point , the use of semi-quantitative PCT levels on admission to guide to treatment, would result in only very few patients with severe P. falciparum malaria being denied high-level monitoring and intensive treatment. However, the specificity and positive predictive value of the semi-quantive PCT test were rather poor. Applying semi-quantitative PCT levels on admission as a guide to therapy would therefore lead to a large proportion of patients receiving more intensive monitoring and treatment than strictly necessary. In our opinion, only at a PCT cut-off point of 10 ng/mL for severe P. falciparum disease, the test characteristics were clinically valuable. Moreover, in settings were the prevalence of malaria is lower than that reported herein, the positive predictive value of PCT will probably be even lower resulting in more false positive tests.In the present study, PCT had an acceptable to excellent sensitivity for severe Possibly, the overall sensitivity of PCT for the prediction of the course of malaria, could be improved if PCT was measured more precisely by use of an ultrasensitive PCT assay instead of the semi-quantitative assay used in this study. Several studies have directly compared the performance of both techniques and demonstrated satisfactory but not complete, concordance ,15. Howefalciparum malaria, 13 of 93 (14%) patients with severe imported malaria were diagnosed with a concomitant bacterial infection, which contributed to a poor outcome [Another drawback on the use of PCT for estimating malaria severity might be the occurrence of bacterial co-infection, which may also lead to elevated PCT levels on admission. In a large French intensive care unit study on imported severe  outcome . HoweverP. falciparum disease and non-falciparum malaria are associated with elevated PCT levels on admission. Although determination of PCT may be of some help in the initial clinical assessment of disease severity in travellers with falciparum malaria in hospitals with extensive experience with the treatment of malaria, the overall test characteristics were too poor to advocate a general use of this semi-quantitative PCT assay. Potential drawbacks in the interpretation of elevated PCT levels on admission may be caused by infections with non-falciparum Plasmodium species and by concomitant bacterial infections.Severe The authors declare that they have no competing interests.DAH participated in the data collection, performed the statistical analysis and data interpretation and contributed to the writing of the manuscript. JSB and HBT participated in the data collection and contributed to the writing of the manuscript. PP performed the procalcitonin assays, undertook the data collection and contributed to the manuscript. PVG conceived and designed the study, carried out the main analysis and wrote the manuscript. All authors approved the final version of the manuscript.


Plasmodium falciparum) occur as a direct result of parasitemia following invasion of erythrocytes by post-liver blood-stage merozoites, and during subsequent cyto-adherence of infected erythrocytes to the vascular endothelium. However, the disproportionate epidemiologic clustering of severe malaria with aggressive forms of endemic diseases such as Kaposi’s sarcoma (KS), a neoplasm that is etiologically linked to infection with KS-associated herpesvirus (KSHV), underscores the significance of previously unexplored co-pathogenetic interactions that have the potential to modify the overall disease burden in co-infected individuals. Based on recent studies of the mechanisms that P. falciparum and KSHV have evolved to interact with their mutual human host, several new perspectives are emerging that highlight a surprising convergence of biological themes potentially underlying their associated co-morbidities. Against this background, ongoing studies are rapidly constructing a fascinating new paradigm in which the major host receptors that control parasite invasion (Basigin/CD147) and cyto-adherence (CD36) are, surprisingly, also important targets for exploitation by KSHV. In this article, we consider the major pathobiological implications of the co-option of Basigin/CD147 and CD36 signaling pathways by both P. falciparum and KSHV, not only as essential host factors for parasite persistence but also as important mediators of the pro-angiogenic phenotype within the virus-infected endothelial microenvironment. Consequently, the triangulation of interactions between P. falciparum, KSHV, and their mutual human host articulates a syndemic relationship that points to a conceptual framework for prevalence of aggressive forms of KS in malaria-endemic areas, with implications for the possibility of dual-use therapies against these debilitating infections in resource-limited parts of the world.The most severe manifestations of malaria (caused by  Plasmodium falciparum (Pf) malaria is one of the world’s leading health challenges, and at least two million people, mainly children below the age of 5 years, die each year from clinical complications of the disease  to the vascular endothelium and other host cells and tissues. Erythrocyte invasion is executed by a family of Pf reticulocyte-binding-like homolog (PfRh) ligands displayed on the surface of merozoites. Among at least five known members of this family, PfRh5 , extracellular matrix metalloproteinase inducer (EMMPRIN), and leukocyte activation antigen, M6 (hereafter CD147); PfRh5 pair is essential for invasion of all laboratory-adapted and field strains of Pf, a cross-strain dependency that reveals opportunities for new anti-malarial therapies based on targeting this receptor/ligand interaction. Following invasion, extensive replication within infected human red blood cells (RBCs) results in surface expression of the multi-domain Pf erythrocyte membrane protein-1 (PfEMP-1) family of genes ], with a variety of host molecules on the surface of capillary endothelial cells. A well-characterized host receptor that mediates cyto-adherence of most Pf isolates to the peripheral vasculature is human CD36 .The cyto-adhesive property of infected red blood cells to the microvasculature and sequestration within vital organ systems is an important survival strategy that allows the parasite to escape immune-mediated destruction . Cyto-adman CD36 , althougPf infection -associated herpesvirus (KSHV), an inherently persistent cancer-associated herpesvirus that is prevalent in malaria-endemic regions. We discuss a number of surprising nodes of pathogenetic interface between Pf and KSHV in this context, and evaluate the major implications of the apparent co-option, by both Pf and KSHV, of CD147 and CD36 signaling pathways as a means to promote Pf persistence on one hand, and virus-induced regulation of the angiogenic phenotype, on the other. We then provide a synthesis of how the triangulation of interactions between Pf, KSHV, and their mutual human host represents the basis for a venerable syndemic relationship that may explain the co-incidence of aggressive forms of KS in malaria-endemic regions.Recent studies have revealed that while erythrocyte invasion and cyto-adherence represent essential evolutionary strategies for parasite growth, survival, and persistence, they are also invariably associated with alteration of cellular physiology, which in turn may contribute directly to the defining clinical manifestations of nfection . Howeveriatrogenic KS is mostly associated with organ  transplantation and is mostly seen as localized skin lesions among people from areas where KSHV is endemic. Epidemic HIV/AIDS-associated KS is more commonly seen among HIV-infected individuals, while classical KS (cKS) manifests among older men of Mediterranean origin as red to purple skin plaques or nodules primarily on the lower extremities. Endemic KS (eKS), which is strikingly similar to cKS in its clinical disposition, is highly prevalent in East and Central Africa, where it affects children and young adults as a cutaneous disease invading soft tissue and bone, or as a fulminant lymphadenopathy that can rapidly disseminate to visceral organs  may result in molecular interactions between Pf and KSHV that could modify the overall disease burden exerted by both pathogens at a micro level. Based on this attribution, we refer to the putative co-morbidity of KS and Pf malaria (in settings for which the evidence for this linkage is strong), as representing an example of a classic syndemic relationship articulated by the display of co-incident clustering within defined geographic regions.Like Burkitt’s lymphoma, KS is one of the most prevalent childhood cancers in malaria-endemic areas. The incidence of KS displays a considerable degree of geographic variation that mirrors the prevalence of its causative agent, and may depend on etiologic mechanisms that are controlled by geographically restricted co-factors, including malaria endemicity . For exaPf malaria has not been rigorously examined at a micro level, and the correlation is even more difficult to establish at a population level because of the indeterminate nature of the mechanisms by which malaria might influence KS pathogenesis outside the known clusters of endemic disease co-incidence. On one hand, the role of malaria as a co-factor for eKS has been hypothesized based on the potential of the Anopheles mosquito vector to contribute to person-to-person spread of KSHV, and to establish an immunosuppressed state at the site of the mosquito bite, which would then create a local permissive environment for KSHV infection , sodium n-butyrate  and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), all of which can also be used to induce EBV lytic cycle  and related herpesviruses, KSHV establishes both lytic and long-term latent infections, the balance between which determines the timing and intensity of pathologic outcomes in specific organ systems. Interestingly, both KSHV and EBV are highly prevalent in malaria-endemic areas and also share many features in their life cycles, including the manner in which they regulate the molecular switch between latency and lytic replication. For example, expression of the major KSHV replication and transcription activator (RTA) is essential for virus replication and dissemination, and is dependent on activation of p38 and extracellular signal-related kinase 1/2 (ERK1/2) mitogen-activated protein kinase MAPK; . KSHV RTic cycle .c-myc oncogene as a result of chromosomal translocation, and although EBV is a necessary etiological agent for eBL, it is clearly not sufficient in absence of other essential co-factors. It is also worth noting that whereas nearly all African children in endemic areas suffer from several malaria episodes as a result of chronic exposure to Pf, only a fraction of them show signs of severe, life-threatening forms of the disease, implying that the biological processes underlying the progression of infection to disease are much more complex. Interestingly, EBV is ubiquitous in the general human population, and in children living in malaria endemic areas, primary infection can occur within a few months of birth  expressed on the surface of EBV-infected human memory B cells, stimulates B cell proliferation  but also reactivated the virus from latency through transcriptional activation of KSHV RTA , and that the molecular mechanisms that control this process overlap with those that putatively regulate PfEMP-1-dependent EBV reactivation from latently infected cells , suggesting that they bind a region on the exoplasmic face of CD36 that overlaps with MC179. It is also insightful that the KSHV lytic cycle was activated only by the PfEMP-1-CIDR1α-derived peptide from Pf Malayan Camp strain, but not by peptides derived from the Vietnam Oak Knoll (FVO) or A4tres strain [that preferentially binds intercellular adhesion molecule 1 (ICAM-1) and not CD36] , demonstrating that reactivation of KSHV by the parasite ligand displays some degree of strain specificity for Pf that is associated with severe malaria in Africa.Remarkably, we have also discovered that cross-linking of CD36 on the surface of KSHV-infected cells with MC179, a recombinant peptide derived from the CIDR1α domain of dherence , not onled cells . Remarkain vivo cross-linking of CD36 on the surface of KSHV-infected cells, either by its natural ligand(s) or upon conjugation with the CIDR1α domain of PfEMP-1 expressed on the surface of parasitized erythrocytes , represents a previously unrecognized mechanism by which KSHV lytic replication could be induced in the context of a Pf malaria co-infection. This model provides many new opportunities for experimental examination of the ability of parasite-derived antigens to reactivate KSHV during cyto-adherence on the surface of infected blood endothelial cells and within tissues and organs: (i) Biochemical analysis of structure/function relationships that control interactions of CIDR1α  with CD36, should reveal potential targets for small-molecule inhibition of parasite-induced sequestration or reactivation of KSHV in co-infected individuals. (ii) Elucidation of the signaling mechanisms that regulate CD36-dependent RTA activation will require multi-pronged approaches that employ dominant negative versions of the Src-like kinases such as yes, fyn, and lyn that control down-stream signaling events initiated by CD36 ligation . (iii) Deletional mutagenesis, domain-swapping, functional complementation, and loss-of-function analysis of genetic mimics of the recently identified CD36 polymorphisms that lack the signaling motif , a potent natural inhibitor of angiogenesis . However, our model also raises many significant questions: (a) Does CIDR1α-induced KSHV replication involve mechanisms that overlap with, or distinct from those that support CIDR1α-induced EBV replication (Src-like kinases and subsequent phosphorylation of p38 and ERK MAPK? (c) Does it directly result in downstream activation of a specific cellular transcription factor such as AP-1 that is known to activate KSHV RTA (or EBV ZTA) promoter(s) ? (d) Is it strictly dependent on interactions of CIDR1α with a distinct epitope on CD36, or does it overlap with, and is therefore also inducible by, other CD36 ligands such as TSP-1? Investigation of the role of TSP-1 in this context is relevant, given that TSP-1 is present in high concentrations in human saliva  Doen saliva  that genn saliva . CriticaFigure 4). The mechanism(s) by which KSHV accomplishes these dichotomous, cell type-specific effects are not fully understood, but one study recently demonstrated that KSHV-encoded microRNAs can directly target TSP-1 mRNA for degradation  system. ET-1 is a pro-angiogenic peptide secreted by the vascular endothelium and its deregulation is implicated in the pathogenesis of many malignancies . ET-1 poth model . The intelevated  as a direlevated . There iPfEMP-1 with ICAM-1 but not CD36 (It was recently discovered that many people of African origin harbor a high frequency of mutations and single-nucleotide polymorphisms (SNPs) that cause a deficiency in the CD36 gene, yet they still suffer from severe  malaria . The SNPnot CD36 . This lePfEMP-1 interactions with CD36 result in induction of the viral lytic cycle, such a virus-induced genetic output would conceivably be designed to promote virus escape from immune surveillance by limiting virus reactivation that might result from interactions between PfEMP-1 and CD36 on latently infected cells. The impact of such a genetic influence, can only be measured against the host’s ability to restrict virus replication and dissemination, and it could be achieved by interrogating viral genomic variability or stability in a given population against a profile of polymorphisms within the genetic registries at the CD36 locus on a population basis. Availability of patient samples with known patho-status and disease severity from regions in which the distribution of malaria overlaps with the incidence of virus-associated endemic cancers would facilitate such a retrospective analysis.The fact that the mutations and their phenotypes occur at high frequency in malaria-endemic areas with a high prevalence of viruses associated with endemic cancers underscores a pathobiological paradigm whereby an inherently persistent tumor virus such as KSHV (or EBV) could provide the selective pressure for introduction or maintenance of such a mutation into the genetic registry of populations living in regions of high malaria endemicity. Given that PfRh5 interactions in parasite invasion unlocks new avenues for investigating the nodes of pathogenetic intersection between Pf malaria and other co-infecting agents such as KSHV that have evolved mechanisms to subvert the CD147 signaling pathway to promote their existential success.CD147 is a widely expressed, type I integral membrane receptor that belongs to the immunoglobulin (Ig) superfamily . It is oPf and KSHV have evolved to exploit CD147 and endothelial cell biology:Several layers of this pathogenetic intersection are revealed by new data on the manner in which both a.Whereas endothelial cells play a prominent role as a platform for cyto-adherence of pRBCs, KSHV displays profound tropism for this cell lineage that represents the basis for origination of the hyper-proliferating spindle cells characteristically found in KS lesions .b.de novo KSHV infection of human endothelial cells as well as oral and fore-skin-derived fibroblasts results in upregulated expression of CD147 and that this effect is directly associated with various virological outcomes consistent with a pro-invasive, migratory, and pro-angiogenic phenotypes .Recent studies demonstrated compelling evidence that enotypes ,b. We hac.Two recent reports showed that KSHV promotes endothelial-to-mesenchymal transition (endo-MT) through activation of Notch-dependent signaling events that culminate in stimulation of an invasive phenotype analogous to that orchestrated by CD147 . KSHV-ind.CD147 has been implicated in the entry processes of a number enveloped viruses including HIV-1 , measlese.PfRh5-mediated conjugation of merozoites with CD147 on normal or infected endothelial cell surfaces should result in so-called parasite-to-host “trans-signaling” events that may lead to untoward pathologic outcomes unrelated to parasite invasion itself. Given that PfRh5 has neither a transmembrane domain nor a cytoplasmic tail, the probability of trans-signaling is likely to be high since a soluble form of PfRh5 [existing either as a monomer, as part of a bioavailable complex with another pathogen molecule such as PfRipr , KS tumorigenesis, and malaria disease outcomes.Available evidence suggests that “outside-in” signaling may be required for the malaria parasite invasion , but it s PfRipr , or as bnfection . A first of KSHV . In FiguKaposi’s sarcoma is one of the most frequent neoplasia diagnosed in malaria-endemic regions of Africa, yet despite recent progress , the lanPf and KSHV in this case) can be blunted by targeting the molecular underpinnings that link the parasite with the disease-modifying influence of KSHV. In addition, it has been proposed that quinine and its chloroquine and hydroxychloroquine derivatives – drugs that have been used widely to treat malaria – are immunosuppressive and may, as such, serve as co-factors for KS by stimulating KSHV reactivation, which would not only promote virus dissemination but could also support KS histogenesis  exposed how alterations in the expression and function of the host receptors that support these processes could not only dramatically change the dynamics of malaria but may also influence the pathogenesis of other co-infections such as KSHV that exploit these pathways for existential benefit; and (c) generated new interest in the structural dispositions of these receptor/ligand pairs as potential multi-domain vaccine targets against specific stages of the Pf life cycle.Infectious agents have long been implicated in the etiology of a variety of illnesses, and although recent studies have examined the role of microbial co-infections in many disease settings, it is not known whether “cooperative pathogenesis” is sufficient to provide the driving force behind strategies in which co-pathogenic agents co-evolve and forge a state of forbearance with each other and with their shared host to advance mutually exclusive existential goals. Clearly, eing eBL , but recPf can bidirectionally influence the clinical course of each other at many physiological levels leading to a variety of clinical outcomes . For example, CD36-mediated cyto-adherence, by virtue of its ability to stimulate pathways that overlap with those required for KSHV reactivation, may contribute to a transient increase in KSHV viral load and dissemination, which in turn could increase the frequency of other defining correlates of KSHV-associated disease that rely on a viremic state. On the other hand, the pathogenic mechanisms of a latent KSHV infection, which include KSHV-induced downregulation of CD36, could effectively alter the overall disease burden by limiting peripheral sequestration; this could in turn increase parasitic access to the central nervous system, leading to a higher probability for cerebral malaria. Ultimately, the emerging picture supports a syndemic link which, however serendipitous, reveals a co-evolutionary paradigm centered at the putative dueling role of CD36 as a mediator of parasite sequestration on one hand and KSHV replication on the other. In this regard, more extensive molecular and genetic analysis is required in order to determine the extent to which KSHV  might provide the driving force for altering the genetic registry at the CD36 locus in regions of high malaria endemicity. It might also be necessary to analyze the CD36 locus in hematopoietic versus peripheral B cells that may serve as the vehicle for virus dissemination in vivo, in order to determine whether infection increases the propensity for a heritable lesion at this locus.Although malaria is not considered a typical opportunistic infection in the same way that KSHV is, recent advances have revealed a surprising node of intersection between KSHV and malaria pathogenesis at the level of the molecular controls that regulate the persistence of these two highly successful infectious agents. To the extent that such interactions can be measured at a micro level, the concept of co-pathogenesis establishes grounds for the expectation that KSHV and in vivo platforms  that could facilitate systematic, experimental integration of population studies with reductionist multi-component molecular data. Although we are still a long way from developing such a platform for studying the malaria and KSHV co-infection paradigm, attempts toward this goal are an essential step in elucidating the extent to which triangular interactions between Pf, KSHV, and their mutual human host might articulate a syndemic relationship that underlies the co-incidence of aggressive KS in parts of the world with endemic malaria. Once the correlates of co-pathogenesis are isolated, innovative research efforts oriented toward development of effective “combined” therapies can be launched.In conclusion, recent advances have revealed several nodes of pathobiological intersection between malaria and a variety of clinically significant infections, and although substantial progress has been made, we are still in the “embryonic stage” of understanding how co-infections interact with each other in their mutual host. Derivative new research emphasis that is inspired by these concepts should include the important goal of developing practical The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum malaria, especially among non-immune adults. It has recently been revealed that activation of transcription factor nuclear factor kappa B (NF-κB) induces pro-inflammatory gene expression involved in the development of progressive renal inflammatory diseases. The aim of this study was to determine whether urinary sediment NF-κB p65 can act as a biomarker for AKI in patients with P. falciparum malaria.Acute kidney injury (AKI) is one of the major complications of Plasmodium vivax malaria, uncomplicated P. falciparum malaria, complicated P. falciparum malaria without AKI (serum creatinine-Cr <3 mg/dl) and complicated P. falciparum malaria with AKI (Cr ≥3 mg/dl) were used to determine NF-κB p65 level by sandwich enzyme-linked immunosorbent assay (ELISA). Urinary sediments obtained from healthy controls were used as a normal baseline. Correlations between levels of urinary sediment NF-κB p65 and pertinent clinical data were analysed.Urinary sediments from malaria patients, including P. falciparum malaria patients with AKI, compared with those without AKI , P. vivax patients  and healthy controls . NF-κB p65 levels in urinary sediment cells showed a significant positive correlation with serum Cr  and blood urea nitrogen (BUN) .Urinary sediment NF-κB p65 levels were significantly increased on the day of admission (day 0) and on day 7 post-treatment in complicated P. falciparum malaria.Urinary sediment NF-κB p65 level is a useful indicator for estimating renal tubular epithelial cell damage and subsequent development of AKI among patients with  Plasmodium falciparum infection, with mortality rates as high as 70% among untreated patients [Plasmodium vivax, occasionally contributing to renal impairment [Acute kidney injury (AKI) is one of the serious complications of patients . The rarpairment . Malariapairment . Clinicapairment . Severalpairment . Studiespairment ,6.P. falciparum malaria varies from 0.14-0.18% at the Hospital for Tropical Diseases, Bangkok [AKI is generally characterized by the sudden loss of the kidneys’ ability to concentrate urine, excrete waste, conserve electrolytes, and maintain fluid balance. The disease is associated with a mortality rate of between 50 and 80% . The los Bangkok  and up t Bangkok . Existin Bangkok ,13. Seve Bangkok  and that Bangkok . NF-κB i Bangkok . The inc Bangkok .P. falciparum malaria patients.This study investigated NF-κB p65 levels in urinary sediments from malaria patients to assess whether urinary sediments NF-κB p65 can serve as a sensitive tool for detecting AKI in P. vivax malaria (n = 11), 2) uncomplicated P. falciparum malaria (n = 8), 3) complicated P. falciparum malaria without AKI (Cr <3 mg/dl) (n = 10), and, 4) complicated P. falciparum malaria with AKI (Cr ≥3 mg/dl) (n = 10). Classification of complicated P. falciparum malaria was based on WHO criteria [Thirty-nine malaria patients admitted to the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Thailand were enrolled into this study. Classification of malaria species was based on microscopic identification. The patients were divided into four groups: 1) Five ml of whole blood were obtained from malaria patients on day 0 (pre-treatment) and day 7 (post-treatment). Samples of clotted blood were centrifuged at 1,700 g for 10 min and the serum was harvested and stored in an aliquoted state at -70°C. The serum was used to measure Cr and BUN levels to investigate kidney function. Urinary sediment NF-κB p65 was determined by enzyme-linked immunosorbent assay (ELISA). Early morning whole-stream urine specimens were collected from all subjects on day 0 (pre-treatment) and day 7 (post-treatment). For urine sediment preparation, urine samples were centrifuged at 3,000 g for 15 min and urinary cell pellets were collected and stored at -70°C until use. The proteins from urinary cell pellets were extracted by lysis buffer  containing protease inhibitors  according to the manufacturers’ protocol. In summary, the pellets were sonicated on ice, centrifuged at 14,000 g for 10 min at 4°C and the supernatants were harvested and used for ELISA. Protein concentrations were determined by Bradford assay , using bovine serum albumin (BSA) as the standard. Fifteen ml of urine sample were sent to the laboratory immediately or stored at 4°C overnight for urine analysis.The NF-κB p65 levels in urinary sediments were measured by sandwich ELISA kit  according to the manufacturer’s protocol. Phospho-NF-κB p65 mouse monoclonal antibody diluted to 1: 100 in PBS was coated on a 96-well microplate and stored overnight. Protein samples (45 μg/well) were added and incubated for two hours at 37°C. The plate was then washed with PBS containing 0.05% (w/v) Tween-20 (PBS-T). Phospho-NF-κB p65 rabbit monoclonal antibody was added to the wells and incubated for one hour at 37°C to detect the captured phospho-NF-κB p65 protein. After washing with PBS-T, anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase (HRP) was added and incubated for 30 min at 37°C. The colour reaction was developed by incubation with 3,3′,5,5′ tetramethylbenzidine (TMB) substrate solution . Finally, the reaction was stopped with 0.18 M sulphuric acid. Optical density (OD) was measured at 450 nm. All assays were carried out in duplicate.rs). A p value <0.05 was considered statistically significant.Statistical analysis was performed using SPSS version 17.0 software . All results were presented as mean ± standard error of the mean (SEM). The normality of distribution was determined by the Kolmogorov-Smirnov test. Differences in levels of urinary sediment NF-κB p65 between groups were compared by Mann-Whitney U-test. Differences in levels of urinary sediment NF-κB p65 within groups between day 0 and day 7 were tested by Wilcoxon signed-rank test. In addition, the correlation between levels of NF-κB p65 and pertinent clinical data including age, malaria parasite density, haemoglobin, haematocrit, white blood cells (WBC) and urinary laboratory parameters were calculated using Spearman’s rank correlation   was similar to those with AKI (Cr ≥3mg/dl)  (p = 0.602). At day 7 post-treatment, no asexual form of malaria parasite was found in the peripheral blood of any malaria patient. A significant increase in the level of serum Cr was found in the group of complicated P. falciparum malaria patients with AKI (6.3 ± 0.5 mg/dl), compared with complicated P. falciparum malaria patients without AKI (1.8 ± 0.2 mg/dl) (p = 0.009). The Cr level was 3.5 times higher in malaria patients with AKI. No significant difference in serum Cr was found in P. vivax patients (0.8 ± 0.1 mg/dl) and uncomplicated P. falciparum patients (0.9 ± 0.1 mg/dl), compared with the healthy controls upon admission and day 7 post-treatment . Serum BUN was also significantly higher in P. falciparum malaria with AKI than in P. vivax, uncomplicated P. falciparum and P. falciparum malaria patients without AKI .Clinical data of the malaria patients and healthy controls are shown in Table p <0.05). Proteinuria was significantly increased in complicated P. falciparum (both with and without AKI groups), compared with P. vivax and uncomplicated P. falciparum  (Table p <0.05). Significantly lower RBC numbers were observed in P. vivax and uncomplicated P. falciparum, compared with complicated P. falciparum (both with and without AKI groups) . In addition, the mean number of WBC in the urine sediment was highest in the group of complicated P. falciparum malaria with AKI compared with the healthy controls (p <0.001), P. vivax, uncomplicated P. falciparum and P. falciparum malaria patients without AKI . At day 7, the microscopic levels of RBC, WBC and epithelial cells remained significantly elevated in the urine samples from complicated P. falciparum malaria with AKI patients, compared to the healthy controls   Table . The numP. falciparum malaria with AKI (0.47 ± 0.02), compared with P. vivax malaria (0.21 ± 0.02), uncomplicated P. falciparum malaria (0.27 ± 0.02) and complicated P. falciparum malaria without AKI (0.36 ± 0.01), . Compared with the healthy controls (0.26 ± 0.01), the level of urinary sediment NF-κB p65 increased significantly in the groups of complicated P. falciparum malaria with and without AKI . However, P. vivax malaria patients showed a significant decrease in urinary sediment NF-κB p65 level compared with healthy controls (p = 0.019), while NF-κB p65 levels in uncomplicated P. falciparum malaria patients were similar to the healthy controls (p >0.05).NF-κB p65 levels measured by ELISA in the urinary sediment cells of malaria patients and healthy controls are shown in Figure P. falciparum malaria patients , compared with the healthy controls . Among the complicated P. falciparum malaria, AKI group showed significantly higher urine NF-κB p65 levels, compared to those without AKI (p <0.05). In addition, the level of urinary sediment NF-κB p65 remained significantly decreased in patients infected with P. vivax malaria (0.19 ± 0.02), compared with the healthy controls (p = 0.014). The lowest levels of urinary sediment NF-κB p65 were found in P. vivax malaria patients. However, no significant differences in the levels of urine NF-κB p65 were observed between day 0 and day 7 in all malaria patient groups .On day 7 post-treatment, the NF-κB p65 levels in the urinary sediment cells were significantly increased in both groups of complicated rs = 0.792; p <0.001), BUN , parasite density  and microscopic WBC  , BUN , microscopic WBC  and urinary epithelial cells   and on day 7 post-treatment . There was no significant correlation between NF-κ B p65 levels in the urinary sediment cells and other urinary parameters including specific gravity, pH and urine protein in any malaria patient group.Within the complicated P. falciparum malaria has not been fully understood. Several hypotheses have been demonstrated including mechanical obstruction by infected PRBCs, immune mediated glomerular pathology, fluid loss due to multiple mechanisms and alterations in renal microcirculation [P. falciparum malaria associated with AKI (Cr ≥3 mg/dl), compared with healthy controls and other malaria groups. It is possible that activation of NF-κB p65 in the urine sediment containing renal epithelial cells and WBC is triggered by various ligands or proteins of malaria parasites that induce up-regulation of the NF-κB p65 signalling pathway. This eventually leads to nuclear translocation of NF-κB and regulation of gene expression involved in inflammation and apoptosis. The inflammation in the interstitium of kidney tissue has been proposed to play a major role in the pathophysiology of AKI [The precise mechanism of AKI in culation ,17,18. Tculation . An ultrculation . With reculation . In the y of AKI ,20. A sty of AKI .P. falciparum malaria patients [P. falciparum malaria without AKI also showed a rise in NF-κB p65 levels, compared to healthy controls, but was significantly lower than AKI group  measurement, where it was undetectable in the urine supernatant . Activatpatients . The souP. falciparum malaria associated with AKI, a significant positive correlation was found between NF-κB p65 in the urinary sediment cells and serum Cr on the day of admission  and on day 7 post-treatment . However, lower urinary NF-κB p65 levels were detected in P. vivax and uncomplicated P. falciparum patients. Therefore, NF-κB p65 may be a valuable indicator of renal cell damage and the subsequent development of AKI in malaria patients. Although, the results show clear statistical significance, a larger sample size and serial determination of urinary NF-κB p65 levels can further address its benefit over serum Cr level.AKI is usually diagnosed by increased serum Cr or BUN levels. Newer biomarkers have been used for the early diagnosis of AKI, such as IL-18, KIM-1 and NGAL . This stNF-κB p65 levels in urinary sediment cells showed a significant positive correlation with serum Cr on the day of admission and day 7 post-treatment. It has been reported that increased serum Cr was the major factor and an important predictor for mortality among malaria patients with AKI, and that early diagnosis and prompt management including dialysis can reduce mortality . A positP. falciparum malaria patients. It will be useful to investigate the NF-κB p65 expression level in specific cell types in urinary sediments, to further explore the exact precursor cells of NF-κB and establish its role in the pathogenesis of AKI in malaria.The levels of NF-κB p65 in urinary sediment cells reflect the degree of damage in renal tubular epithelial cells in malaria patients. Therefore, the urinary level of NF-κB p65 has a potential role as a disease biomarker in estimating damage to renal tubular epithelial cells and subsequent progression of AKI among complicated The authors declare that they have no competing interests.CP performed the laboratory work, obtained clinical data and drafted the manuscript. PV designed the experiments, recruited the patients, analysed the data, and revised the final manuscript. Both authors have approved the final version of this manuscript.


A candidate malaria vaccine was tested in a Phase 2 study in Mali. Based on results from the first eight months of follow-up, the vaccine appeared well-tolerated and immunogenic. It had no significant efficacy based on the primary endpoint, clinical malaria, but marginal efficacy against clinical malaria in secondary analyses, and high allele-specific efficacy. Extended follow-up was conducted to evaluate extended safety, immunogenicity and efficacy.The FMP2.1/AS02Plasmodium falciparum apical membrane antigen 1 (AMA1) vaccine FMP2.1/AS02A was conducted in Bandiagara, Mali. Children aged 1–6 years were randomized in a 1∶1 ratio to receive FMP2.1/AS02A or control rabies vaccine on days 0, 30 and 60. Using active and passive surveillance, clinical malaria and adverse events as well as antibodies against P. falciparum AMA1 were monitored for 24 months after the first vaccination, spanning two malaria seasons.A randomized, double-blinded trial of safety, immunogenicity and efficacy of the candidate A group and three in the control group; none was considered related to study vaccination. After two years, anti-AMA1 immune responses remained significantly higher in the FMP2.1/AS02A group than in the control group. For the entire 24-month follow-up period, vaccine efficacy was 7.6% (p = 0.51) against first clinical malaria episodes and 9.9% (p = 0.19) against all malaria episodes. For the final 16-month follow-up period, vaccine efficacy was 0.9% (p = 0.98) against all malaria episodes. Allele-specific efficacy seen in the first malaria season did not extend into the second season of follow-up.400 children were enrolled. Serious adverse events occurred in nine participants in the FMP2.1/AS02Allele-specific vaccine efficacy was not sustained in the second malaria season, despite continued high levels of anti-AMA1 antibodies. This study presents an opportunity to evaluate correlates of partial protection against clinical malaria that waned during the second malaria season.NCT00460525Clinicaltrials.gov NCT00460525  A was developed for potential use as a stand-alone vaccine and/or as a component of a multi-antigen vaccine to improve on the efficacy of RTS,S/AS01 P. falciparum that is produced in and purified from Escherichia coliA, due to the strong immunogenicity and favorable safety profile of this adjuvant system in infants and children A highly efficacious malaria vaccine that would reduce morbidity and mortality worldwide and advance the eradication agenda continues to be a research priority in vitro analysis AMA1 is an 83-kilodalton surface protein expressed by mature intra-erythrocytic malaria parasites that is processed to a 66-kilodalton protein before being exported to the merozoite surface around the time of rupture of the infected erythrocyte A candidate vaccine conducted at the study site. Phase 1 dose-escalation testing in semi-immune adults found that the vaccine was well-tolerated and highly immunogenic, with evidence of enhanced parasite growth inhibition activity in sera from high dose vaccinees compared to controls A candidate vaccine over the entire 24-month follow-up period are reported here.The current report follows previous studies of the FMP2.1/AS02The study protocol was approved by institutional review boards of the University of Sciences, Techniques and Technology Faculty of Medicine, Pharmacy and Dentistry in Bamako, Mali; the University of Maryland, Baltimore; the Walter Reed Army Institute of Research; and the United States Army Surgeon General. Written informed consent was obtained before starting screening and enrollment. Verbal consent of illiterate parents or guardians was administered and then documented using their thumbprints, a process verified by signatures of independent witnesses. The trial was monitored by the National Institute of Allergy and Infectious Diseases and the United States Army Medical Material Development Activity. An independent data and safety monitoring board and a local safety monitor were appointed. The trial was conducted in compliance with the International Conference on Harmonization of Good Clinical Practices, the Declaration of Helsinki and regulatory requirements of Mali.A vaccine or rabies vaccine (control). The study population included children aged 1–6 years at the time of enrollment (first vaccination) after informed consent was obtained from a parent or guardian. Participants were screened for acute and chronic illnesses by medical history, physical examination, laboratory testing . Active and passive surveillance for malaria began at the first vaccination and continued throughout the follow-up period. Venous blood samples were obtained for safety analyses on the days of vaccination, one week after each vaccination, and one month after the last vaccination. Venous blood samples were obtained for immunogenicity analyses on the days of vaccination and 1, 3, 6, 10, 16 and 22 months after the last vaccination. Information on bed net use was obtained monthly for the first 7 months of follow-up, and at 12, 18 and at 24 months after randomization.Details of the study design and enrollment including the study protocol and statistical analysis plan were published previously for this double-blinded, randomized, controlled Phase 2 clinical trial A  A vaccine were offered immunization with three doses of rabies vaccine.Participants were given either 50 µg of lyophilized FMP2.1  resuspended shortly before vaccination in 0.5 mL of AS02A or control vaccine groups according to a computer-generated predefined block randomization scheme by age stratum . Study pharmacists were unblinded to treatment assignment at vaccination, and senior study investigators were unblinded after the primary study endpoint was reached (study day 240). Study staff at the field site, participants and guardians remained blinded to treatment assignment until the end of the study (study day 730). Vaccines were prepared in identical syringes and covered to conceal their contents. Study vaccinators were not involved in post-vaccination participant assessments of safety or reactogenicity.Participants were randomly assigned in a 1∶1 ratio to the FMP2.1/AS02Parents and guardians were asked to return to the project clinic on the grounds of the district hospital any time a participant became ill. Study physicians were available at all times for evaluation, treatment and documentation of all adverse events, including serious adverse events. The study paid for laboratory testing and medication for participants during the study follow-up period.P. falciparum density of at least 2500 parasites/ µl. Severe malaria was recorded as a serious adverse event and was diagnosed based on WHO diagnostic criteria modified to include two additional criteria based on a previous study of severe malaria at the site Clinical malaria was defined as fever (axillary temperature of 37.5°C or higher) with an asexual To assess immunogenicity, serum was collected at baseline and at study months 1, 2, 3, 5, 8, 12, 18, and 24 to measure anti-AMA1 antibody by a standard, optimized ELISA Hemoglobin was measured at each vaccination, 7 days after each vaccination, and at study months 3, 4, 5, 6, 7, 8, 12, 18 and 24. Other hematologic and biochemical parameters were monitored up to 30 days after the last vaccination.P. falciparum AMA1. QiaAmp manufacturer’s instructions for the 96 DNA blood kit  were followed to extract malaria parasite DNA. The entire 1861 base pair ectodomain coding sequence of the ama1 gene was amplified using a previously described nested PCR Parasite DNA extracted from dried blood filter paper samples was used to sequence the gene encoding The study statistical analysis plan was agreed upon by the study sponsor, investigators and statistical consultant before analyses commenced. Statistical analyses of secondary safety and efficacy endpoints were done with SAS 9.2 statistical software . The sample size was calculated based on the primary study endpoint–the time to first clinical malaria episode occurring between randomization and six months after the assigned date of the third vaccination.Analyses of safety were done on data collected from all vaccinated children. The percentage of those with a serious adverse event, as classified by the MedDRA preferred-term level, reported from 0–24 months was tabulated. The frequency distributions of biochemical  and hematological  laboratory values beyond predefined reference ranges were compared using predefined severity grades. Analysis of immunogenicity was conducted on all samples available from participants according to their treatment assignment. Anti-AMA1 antibody titers were summarized by geometric mean titers with 95% confidence intervals.P. falciparum parasitemia; vaccine efficacy against episodes of clinical malaria due to parasites that had AMA1 genotypes identical to the 3D7 vaccine strain with respect to designated immunologically important AMA1 polymorphisms  in the cluster 1 loop of domain I; vaccine efficacy against cumulative asexual P. falciparum parasite density measured for each child as the total AUC for parasitemia in both clinical malaria episodes and asymptomatic infections detected in monthly surveys; vaccine efficacy against anemia measured at active  and passive follow-up timepoints; and vaccine efficacy against the incidence of severe malaria in the intention-to-treat cohort.The study was designed to have 90% power to detect 20% efficacy based on a 75% incidence of clinical malaria in the control group during the first eight months of follow-up. Primary analysis of vaccine efficacy was conducted in the intention-to-treat cohort  for the first eight months after randomization. Analyses of vaccine efficacy were also calculated for the per-protocol cohort . Predefined secondary analyses of vaccine efficacy included efficacy against all clinical episodes during the first eight months of follow-up, and efficacy against first clinical malaria episode and all clinical episodes using a range of parasitemia thresholds occurring in the 24 months of follow-up in the intention-to-treat cohort. Protocol-defined exploratory analyses included vaccine efficacy against malaria infection using asexual P. falciparum clinical malaria. For analysis of first or only episodes, hazard ratios were estimated using a standard Cox regression model unadjusted for covariates unless otherwise indicated. Efficacy against multiple clinical episodes was assessed using Poisson regression, which was not adjusted for other covariates. Cumulative incidence of clinical malaria for 22 months after the third dose was estimated by the Kaplan Meier method to account for loss to follow-up. Fisher’s exact test was used to compare proportions. All p-values presented are two-sided.Efficacy estimates were obtained for the hazard ratio for first or only episode and for all episodes of Ama1 sequences were defined as collected from single/predominant infections or from multiple infections based on nucleotide base peak heights on the sequencing chromatogram. Multiple-allele infections were defined as those with a secondary base peak height of 50% or more of the primary peak height at any polymorphic site. Haplotypes were defined only for sequences determined to correspond to single/predominant infections. Polymorphic codons in c1L were used to define haplotypes for exploratory measures of extended allele-specific efficacy Sequencher 4.8 software  was used to align and edit parasite DNA sequences obtained from infections causing clinical malaria episodes. A, we used SAS 9.2 statistical software  to assess the time to the first clinical malaria episode with a vaccine-type 3D7 (DERHFDKY) and non-3D7 haplotypes of AMA1 c1L. To evaluate the cross-protective efficacy of the malaria vaccine, we modeled the time to first clinical malaria episode with alleles matching the most frequently observed non-vaccine-type AMA1 c1L haplotypes during the second malaria transmission season (day 241 to 730): Fab9 (DQRHFDKY), Dd2 (DRRLLDED), M5 (NGRDLNEY) and FVO (NGRDFNEY). A Cox proportional hazards regression model was used to assess the association between treatment arms and risk of a clinical episode with parasites having an AMA1 c1L haplotype identical to 3D7 or any of the most common non-vaccine alleles, while a χ2 test was used to compare the frequency of c1L haplotypes in the two treatment arms.To assess the extended strain-specific efficacy of FMP2.1/AS02A group and 58% in the control group at the end of follow-up.Four hundred children were enrolled from May 28 to July 4, 2007 at the Bandiagara Malaria Project research clinic on the grounds of the Bandiagara District Hospital in Bandiagara, Mali. All received at least one vaccination . The maiA compared to the control group . The incidence of Grade 2 anemia was higher in the control group (p = 0.02).No cases of severe anemia occurred during the study follow-up period. Anemia experienced in each group by severity is shown in A group increased to levels similar to those measured in semi-immune adults at the site A group with 84.4% and 83.2% demonstrating an eight-fold increase at months 12 and 24 compared to 7.6% and 11.9% in the control group. Control group participants showed smaller increases in anti-AMA1 antibody levels, corresponding to peaks of natural exposure during the malaria transmission season  in the FMP2.1/AS02A group compared to 0.85 per PYAR in the control group. Vaccine efficacy against malaria in the FMP2.1/AS02A group for months 0–24 was 7.6% (p = 0.51) for first or only malaria episodes and 9.9% (p = 0.19) for all malaria episodes  compared to 175 episodes in the control group (0.69/PYAR). Vaccine efficacy against all clinical episodes was 0.9% (95% CI: -22.2 to 19.5). Vaccine efficacy analyses are reported for the per-protocol dataset in the Supporting Information  of these episodes were observed in the malaria vaccine group, and twenty (48%) in the control group. Seven of the 42 clinical episodes observed during the second malaria season were clinical episodes with ama1 c1L sequences identical to 3D7 for participants who previously had clinical malaria with a 3D7 AMA1 c1L allele during the initial 240 days of follow-up, with three episodes diagnosed in malaria vaccine participants and four in control participants. The cumulative proportion of participants with at least one clinical episode due to an infecting 3D7 AMA1 c1L allele strain was comparable between the two treatment arms (p = 0.81) during the second period of follow-up. The overall allele-specific efficacy against first episodes of clinical malaria with vaccine-type AMA1 c1L alleles from the entire follow-up period from days 0–730 was 35%  (data not shown). The estimated vaccine efficacy against clinical malaria episodes showed no difference between FMP2.1/AS02A and control vaccine . Moreover, the incidence of clinical malaria with 3D7 AMA1 c1L was similarly distributed between the FMP2.1/AS02A and control vaccine groups (p = 0.81). Finally, when we compared the incidence of 3D7 AMA1 c1L type clinical episodes observed during the first 240 days in all participants to 3D7 c1L alleles identified during days 241–270 of follow-up in all participants, the frequencies were similar (p = 0.63), signifying a stable incidence of 3D7 AMA1 c1L type infections during the entire follow-up period. These results indicate that the allele-specific efficacy of the malaria vaccine did not extend into the second malaria season and that the incidence of 3D7 AMA1 c1L allele was stable during the two transmission seasons.Although not part of the statistical analysis plan and not intended to estimate vaccine efficacy, to explore the possibility of a “rebound” increase in risk of malaria in the second season, we estimated the time without clinical malaria due to a 3D7 AMA1 c1L allele by setting the starting time to day 241, corresponding to the end of the first transmission season after vaccination. While the FMP2.1/AS02A according to a 0, 1, 2-month schedule did not show result in significant efficacy against the primary endpoint, but secondary analyses showed approximately 20% efficacy against first and multiple clinical malaria episodes defined using different parasite density thresholds. The vaccine had 64.3% efficacy (p = 0.03) against clinical malaria caused by parasites with AMA1 corresponding to the vaccine strain at pre-defined polymorphic amino acids sites. The safety profile was favorable based on comparison of reactogenicity, laboratory values and adverse event rates in FMP2.1/AS02A vaccinees compared to controls. High anti-AMA1 antibody titers were induced and sustained during follow-up, and cumulative parasitemia as measured by AUC was reduced in FMP2.1/AS02A vaccinees compared to controls During the first 240 days of follow-up, vaccination of malaria-exposed Malian children aged 1–6 years with FMP2.1/AS02A vaccine did not demonstrate allele-specific efficacy as seen during the first malaria season. The vaccine likewise provided no efficacy against first clinical malaria episodes, all malaria episodes, or episodes due to parasites matching the vaccine strain in the extended efficacy surveillance period for our study population. The rate of Grade 2 anemia during the entire follow-up period was slightly higher in the control group, although this event was uncommon in the study population . Although more serious adverse events occurred in the FMP2.1/AS02A group compared to controls, this difference was not statistically significant and none of the severe events was deemed related to vaccination. Overall, the extended follow-up showed favorable safety data, with no concerning findings.During the entire follow-up period from study days 0–730, the FMP2.1/AS02A group during months 9–24 of follow-up. Neither the overall incidence of clinical malaria nor incidence of clinical malaria due to the vaccine strain was increased in FMP2.1/AS02A vaccinees during this interval after the initial efficacy follow-up period. This is reassuring as a previous study at the site found that after an initial period of protection, those given a single dose of sulfadoxine-pyrimethamine at the start of the malaria transmission season experienced delayed but then subsequently increased incidence of clinical malaria compared to controls who did not receive sulfadoxine-pyrimethamine No evidence of a “rebound”–a period of increased risk of malaria following a period of protection by vaccines, drugs, nets or other interventions A group. This low number of cases is likely related to early diagnosis and treatment of uncomplicated malaria in this study population with close active and passive surveillance that included continuous access to and frequent contact with study physicians and malaria diagnostics.Severe malaria occurred in three participants, all in the FMP2.1/AS02A vaccine induced high levels of anti-AMA1 antibody that peaked at study day 90 and persisted at high levels throughout the entire 24 months of follow-up. The increase in antibody level from baseline to the measurement just before the first episode of clinical malaria correlated with protection in the first eight months of the study. The results of the extended follow-up found no evidence of overall or strain-specific vaccine efficacy in the second malaria season of follow-up. The waning of strain-specific efficacy occurred despite a sustained high level of anti-AMA1 antibody in FMP2.1/AS02A vaccinees, but this level may have been insufficient to protect against clinical disease in the second malaria season. As no efficacy was observed in the second malaria season, change in anti-AMA1 antibody level cannot be correlated with extended protection, but it may be possible to identify a marker of strain-specific protective immune response that was not measured and that did not persist through the second malaria season. Planned analyses of antibody avidity and subclass, cell-mediated immunity and growth inhibition analysis may help to better understand the strain-specific protection observed in the first malaria season following vaccination and provide insights into how to develop more broadly efficacious next generation malaria vaccines.The FMP2.1/AS02Table S1Vaccine efficacy against P. falciparum malaria, per-protocol cohort.(DOCX)Click here for additional data file.Checklist S1CONSORT checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(PDF)Click here for additional data file.