Plasmodium falciparum, often requires intensive care unit (ICU) admission and can be complicated by cerebral malaria, respiratory distress, acute kidney injury, bleeding complications, and co-infection. The mortality from imported malaria remains significant. This article reviews the manifestations, complications and principles of management of severe malaria as relevant to critical care clinicians, incorporating recent studies of anti-malarial and adjunctive treatment. Effective management of severe malaria includes prompt diagnosis and early institution of effective anti-malarial therapy, recognition of complications, and appropriate supportive management in an ICU. All cases should be discussed with a specialist unit and transfer of the patient considered.The number of people travelling to malaria-endemic countries continues to increase, and malaria remains the commonest cause of serious imported infection in non-endemic areas. Severe malaria, mostly caused by  The authors describe the presentation and management of malaria on the ICU.They review the literature and provide management strategies for dealing with this life-threatening condition.1 Malaria is responsible for the death of at least three quarters of a million people worldwide every year2 and is the commonest cause of serious imported infection in non-endemic areas.5 Severe malaria is mostly caused by Plasmodium falciparum, although other species can cause severe disease.67The number of people who travel to malaria-endemic areas continues to increase Fig. .1 Malari8 This review is based on recent studies of anti-malarial and supportive therapies and outlines the epidemiology of malaria, clinical manifestations, and risk stratification of severe disease, and provides an update on the management of patients with imported malaria requiring intensive care unit (ICU) support.Recommendations for the management of severe imported malaria are largely derived from trials in endemic regions and retrospective series of imported malaria. In spite of advances in management, the mortality rate of severe malaria remains ∼10% and data from the UK suggest that the outcome may be worse for patients managed in centres with less experience of treating the disease.5 There were 6749 cases of imported malaria reported within the European Union in 2010 (0.99 cases per 100 000)9 and 1688 cases reported in the USA (0.55 cases per 100 000).3 In Europe, four countries  account for 80% of all cases. Surveillance from both Europe and the USA show that most cases of falciparum malaria are acquired in sub-Saharan Africa.312 Compared with malaria in endemic settings, where children are most commonly affected, imported malaria is predominantly a disease of young- and middle-aged adults—the median age of cases in the UK is 31 years.13Malaria is endemic throughout most of the tropics and sub-tropics and is one of the commonest causes of febrile illness in returning travellers.513 although these individuals may be at reduced risk of developing severe disease because of partial immunity.1114 Anti-malarial chemoprophylaxis is very effective but surveillance data consistently demonstrate that most travellers do not take it appropriately.31315 Reasons for poor adherence include an assumption of low risk, particularly among individuals who grew up in endemic regions, and concerns about potential drug side effects.Surveillance data demonstrate that individuals originating from endemic regions who travel to ‘visit friends and relatives’ are more likely to develop malaria than people who travel for other reasons (relative risk 3.65),316 The case fatality rate is ∼1%.313 UK surveillance data demonstrate significantly higher mortality (odds ratio 10.68) in patients aged >65 yr compared with adults aged 18–35 yr and among tourists compared with patients originally from endemic countries (odds ratio 8.2).8 Death from non-falciparum malaria is extremely rare with a case fatality rate of 0.05%.8Nearly all severe disease is caused by falciparum malaria; ∼10% of patients with imported falciparum malaria are reported to develop severe disease.Plasmodium and is transmitted by female Anopheline mosquitoes.17 Four species are classically considered to cause disease in humans  although a fifth, P. knowlesi, is now recognized as a zoonotic cause of malaria in parts of Malaysia.18 After the bite of an infected Anopheline mosquito  between return from a malaria-endemic area and hospital admission.P. falciparum results in the expression of P. falciparum erythrocyte membrane protein 1 (Pfemp1), an important virulence factor, on the surface of red blood cells. Pfemp1 mediates binding of infected red blood cells to endothelial surfaces and sequestration in capillary beds.21Pfemp1 is encoded by the var family of genes and the parasite regularly switches between ∼60 variants of this gene resulting in significant antigenic variation and an impaired immune response.21 Release of pro-inflammatory cytokines, including TNF-α and IL-1, in response to infection leads to an up-regulation of endothelial receptors including Intracellular-Adhesion Molecule 1 resulting in further sequestration.22 Occlusion of capillary beds leads to microvascular obstruction, tissue hypoperfusion, and lactic acidosis.23 Capillary sequestration also impairs splenic clearance of infected red blood cells. The severity of disease is associated with both a higher total body parasite biomass and a higher biomass of sequestered parasites.24Infection with 26 This allows speciation, quantification of parasitaemia, and detection of other markers of severity such as the presence of schizonts. In a non-endemic setting, a parasite count >2% of infected red cells is usually considered a marker of severe disease although lower counts do not exclude this.2627 Both thick and thin films should be examined. Thick films have a higher sensitivity for diagnosis while thin films allow more accurate speciation and quantification of parasitaemia.28 Rapid diagnostic tests (RDTs) are used in many settings. RDTs detect circulating parasite-associated proteins and enzymes. Most tests detect both a pan-species target and a falciparum-specific target. RDTs allow diagnosis of malaria without a trained microscopist but do not usually provide speciation nor quantification of parasitaemia. As such, these tests should be considered an adjunct rather than a replacement for blood film analysis.2627 The use of PCR to diagnose malaria remains a research tool, but may occasionally be useful, particularly when there is doubt about the infecting species, for example in the differentiation of P. knowlesii from P. malariae.Microscopic examination of a blood film remains the gold standard for diagnosis of malaria Fig. .26 This 29 In imported malaria, the commonest reasons for ICU admission are cerebral malaria, acute respiratory distress syndrome (ARDS), and acute kidney injury (AKI), either alone or in combination.112030 As parasitized erythrocytes sequester in deep capillaries, the peripheral parasitaemia may not accurately reflect the true burden of infection. Hyperparasitaemia is therefore not a consistent finding in patients with severe malaria, and may not be a feature in a substantial proportion of patients requiring ICU admission.11Criteria for severe malaria in both endemic and non-endemic settings have been defined by the World Health Organisation (WHO) Table .29 In im31 Major considerations include the common causes of community-acquired Gram-positive and Gram-negative bacterial sepsis, enteric fever, severe rickettsial infections, and leptospirosis, and also arboviral infections (including dengue fever) and the viral haemorrhagic fevers. Consultation with a specialist is recommended if there is any doubt as to the diagnoses or in any patient considered to be at risk of viral haemorrhagic fever.The differential diagnosis of severe malaria is broad and varies depending on the patient's travel history.cinchonism, which comprises tinnitus, blurred vision, reversible hearing loss, headache, and nausea. Quinine may also cause hypoglycaemia and prolongation of the QTc interval on an electroencephalography (ECG) necessitating regular monitoring, but is usually well tolerated. Artemisinins, derived from the Chinese herb qinghausu or wormwood, have been used by Chinese traditional healers for many years but have become available to western practioners only in recent years.Since the emergence of chloroquine resistance in the 1970s, parenteral quinine has been the mainstay of treatment for severe malaria Table . The com3233 The SEAQUAMAT study randomized 1461 mostly adult patients with severe malaria in South and South-East Asia, and showed a significant reduction in mortality in patients treated with artesunate compared with quinine .32 The subsequent AQUAMAT study randomized 5425 children (<15 years) with severe malaria across eleven countries in sub-Saharan Africa and again showed a significant reduction in mortality with artesunate .33 Routine monitoring for hypoglycaemia and QT prolongation are unnecessary if an artemisinin derivative is used. Although extremely effective in treating malaria, they cannot be used as single agents as the rate of recrudescent infection is very high.The artemisinin derivative artesunate has recently been shown to be more effective than quinine for the treatment of severe falciparum malaria in two large randomized trials.34 Because of this, parenteral artesunate is only available in relatively few specialist centres. Treatment with quinine should not be delayed while artesunate is obtained. If there is any doubt about the infecting species, then treatment for falciparum should be prescribed. Artemisinin combination treatments (ACTs), which combine an artemisinin with another effective anti-malarial drug, are now recommended by the WHO as first-line treatment for non-severe malaria27 and should be used after initial treatment with parental artesunate. Early liaison with a specialist tropical medicine or infectious diseases unit is essential to ensure appropriate treatment as soon as possible. The treatment strategy used at the Hospital for Tropical Diseases, London, UK, is outlined in Table Availability of artesunate has been limited in many countries, including the UK, as the drug is not licenced and is only available on a named patient basis.35 However, in the context of malaria, resistance is a relative rather than an absolute phenomenon. Partial artemisinin resistance has also been seen in South-East Asia, especially Cambodia, and Thailand38 and results in a decreased rate of clearance of asexual parasites from the blood. Treatment failure with ACTs has not yet been reported, but its development and the potential for artemisinin resistance spreading to Africa is a major concern.Resistance to quinine has been widely reported from South-East Asia and occasionally from sub-Saharan Africa.26 The rationale is that exchange transfusion removes both infected red cells, lowering parasite burden and parasite-derived antigen load, and circulating pro-inflammatory cytokines. A recent report from a single-centre reported no deaths among 25 patients who had an exchange performed.39 However, a meta-analysis in 2002 found only eight quasi-experimental studies of the efficacy of exchange transfusion and demonstrated no overall survival benefit, a finding echoed in a recent study of US patients;40 the WHO states that no recommendation regarding exchange transfusion is possible based on the paucity of current evidence.2741 At our centre, exchange transfusion has not been used since the introduction of artemisinin-based therapy for all patients with severe malaria.Several anecdotal reports and some case series have supported the use of exchange transfusion in severe malaria, especially in patients with high parasitaemia; it continues to be recommended in some national guidelines.42 However, this restrictive fluid regimen commences only after initial resuscitation has been completed. No large trials have assessed fluid resuscitation strategies in patients with severe malaria in a developed world ICU setting; current data are derived only from trials in endemic areas and from small physiological studies.The traditional adage is that patients with severe malaria should be managed with relatively restricted fluid volumes because of concerns about the risk of capillary leak and lung and cerebral oedema. This practice is supported by the movement in critical care to a more fluid restrictive practice in patients with acute lung injury (ALI).43 Malaria was the reason for admission in 57% of these children. The trial was stopped early because of a significant increase in mortality in both groups who received fluid boluses compared with the group receiving maintenance fluids only . In a post hoc analysis, there was an increase in terminal cardiovascular events related to fluid resuscitation.44 There were no differences in mortality between the sub-groups of children with malarial and bacterial infections. There are obvious limitations in applying these findings to critical care settings in the developed world as the children could not be offered mechanical ventilation, renal replacement therapy, nor inotropic support. Furthermore, time to hospital presentation, while not formally measured, was likely to be significantly longer than in a developed world setting, so the potential risk of an exaggerated reperfusion injury after aggressive resuscitation cannot be discounted.The fluid expansion as supportive therapy study randomized 3141 African children presenting with severe febrile illness to maintenance fluids with or without boluses of either crystalloid  or colloid .47 Intravascular haemolysis as a result of severe malaria has been shown in one small study to result in decreased levels of nitric oxide,47 increased pulmonary pressures, and myocardial wall stress. Further studies to understand the clinical importance of cardiac dysfunction in severe malaria are warranted.Although not considered a traditional manifestation of severe malaria, there is emerging evidence that cardiac dysfunction may complicate severe disease. A number of studies have found evidence of increased circulating levels of cardiac enzymes including BNP in individuals with severe malaria.48 The argument against aggressive fluid loading is strengthened by recent data on the physiological response of 28 adult ICU patients with severe malaria to fluid expansion guided by invasive cardiac monitoring.23 Despite trans-pulmonary thermodilution (PiCCO) guided therapy, acid–base status deteriorated in 68% and no improvement in renal function was observed after volume expansion. Significant increases in extravascular lung water occurred in 17 of 22 (77%) patients who were liberally resuscitated, with eight developing frank pulmonary oedema despite being hypo- or euvolaemic. Five patients died, all of whom developed pulmonary oedema. The authors found that the degree of lactataemia correlated with the degree of parasite microvascular sequestration, but not with hypovolaemia.Previous studies have demonstrated that traditional markers of fluid balance correlate poorly with acid–base status and respiratory function.49 Where vasopressor, inotrope, or both requirements persist, we maintain an acceptable cardiac output, avoiding fluid overload but targeting tissue perfusion and have a low threshold for diuresis in the haemodynamically stable patient in the postinflammatory phase of illness.It would appear that, in the absence of prospective, randomized trial data, liberal fluid therapy is best avoided in the context of severe malaria. Because of the propensity of these patients towards capillary leak and thus a greater risk of ARDS and cerebral oedema, our own management approach is to target significant hypovolaemia with concurrent tissue hypoperfusion, that is, clinical markers of organ dysfunction (e.g. oliguria) with biochemical markers . Fluid loading is guided by a goal-directed algorithm using both the stroke volume response to a fluid challenge  and markers of tissue perfusion as therapeutic endpoints. Otherwise, fluid balance is kept neutral, in line with studies in critically ill patients showing a strong association between positive fluid balance and worse outcomes.27 Cough is a common symptom,50 and tachypnoea may be caused by fever, anaemia, and a metabolic acidosis, and also primary lung pathology such as the ARDS and pneumonia. ARDS has clear diagnostic criteria that have been recently redefined,51 with the previous clinical distinction between ALI and ARDS being replaced by mild, moderate, and severe levels of ARDS. This condition is more common among adults than children.52 The reported incidence of ‘respiratory distress’ in severe malaria varies between 2 and 30%, with differences in definitions accounting for some of this variation.122054 ARDS and respiratory distress are poor prognostic signs in both endemic and imported malaria.205556The WHO defines respiratory manifestations of severe malaria in terms of deep breathing, respiratory distress, and pulmonary oedema.5758 However, ARDS can develop after apparently successful treatment and after the disappearance of parasites from the blood.1153 In these cases, ARDS may reflect persistence of inflammatory cytokines in the absence of any infected erythrocytes. There is emerging evidence that free parasite antigens may persist after treatment suggesting that these may represent a potential on-going stimuli for inflammation.52 Concurrent bacterial pneumonia and cardiogenic pulmonary oedema  are other important causes of respiratory distress.The mechanisms underlying ARDS are not entirely understood, but likely causes include endothelial dysfunction and altered capillary permeability because of parasitized erythrocyte adherence and sequestration and exaggerated host immune and inflammatory responses, particularly TNF-α, IL-1, IL-6, and IL-8.62 Fluid balance is kept neutral, or negative if the patient is considered to be volume overloaded. Cerebral oedema and raised intracranial pressure associated with cerebral malaria may limit permissive hypercapnia and the use of high PEEP strategies; however, pragmatic clinical decision-making should be used.63 Successful use of extracorporeal membrane oxygenation for severe respiratory failure has been reported in malaria.1264 Bacterial co-infection is relatively common suggesting that a low threshold for starting antibiotics, when supported by clinical and laboratory investigations, may be appropriate.112030As there are no specific trials addressing ARDS treatment in malaria, strategies are based on evidence-based ARDS management, including the use of low tidal volume ‘protective’ ventilation and moderate levels of PEEP.−1 (<40 mg dl−1),27 is a common complication of malaria and can be a marker of severe disease, particularly in children.6566 Case series of imported malaria report a prevalence of hypoglycaemia between 1 and 20% at admission, with a higher rate among those who die.122054 The pathogenesis is poorly understood but is thought to be related both to parasite glucose consumption and to impaired host gluconeogenesis rather than to malnutrition or hyper-insulinaemia.67 Hypoglycaemia may be exacerbated by parenteral quinine (an insulin secretagogue). A meta-analysis reported a significantly lower incidence during treatment with artemisinins compared with quinine [combined HR 0.55 (95% CI 0.41–0.74)].68 Clinical features include a reduced level of consciousness and seizures. Blood glucose should be routinely and regularly assessed and monitored, especially during treatment with quinine. Early enteral feeding has been established as beneficial in a wide range of patients requiring intensive care, and may mitigate against hypoglycaemia in severe malaria.6970Hypoglycaemia, defined as blood glucose values <2.2 mmol litre29 In clinical practice, a decrease in GCS <11 or the occurrence of seizures should be taken to represent cerebral malaria once hypoglycaemia and other potential causes of reduced consciousness have been excluded. As with respiratory distress, cerebral malaria is associated with worse outcomes.71 The pathogenesis remains incompletely understood.72 Electroencephalography has previously found sub-clinical seizure activity in a proportion of patients with cerebral malaria73 which prompted three anti-epileptic trials.76 However, despite a reduction in seizure frequency, mortality was increased in patients receiving routine anti-convulsant therapy with phenobarbital.77 This increased mortality is postulated to occur as a result of respiratory depression. The extent to which these findings can be generalized to other classes of anti-epileptics, in particular those that cause less respiratory depression, is unknown. In view of these findings, there is currently no role for routine EEG monitoring and the use of anti-epileptics in patients with cerebral malaria should be limited to those with clinically overt seizure activity.Cerebral malaria is strictly defined as coma [Glasgow Coma Score (GCS) <9] in a patient with malaria in whom other aetiologies have been excluded.78 randomized patients with cerebral malaria to receive either dexamethasone or placebo. Mortality did not differ between the two groups (P=0.8) but coma was prolonged in those who received dexamethasone . This finding is consistent with other studies of corticosteroids in cerebral oedema because of other aetiologies such as head injury.79 Complications, including gastrointestinal bleeding and pneumonia, were also more common in patients receiving dexamethasone (P=0.004). Mohanty and colleagues80 randomized 61 adult patients with cerebral malaria and CT-confirmed cerebral oedema to adjunctive treatment with mannitol. There was a non-significant trend towards a higher mortality in those receiving mannitol . Mannitol was also associated with a significant increase in the duration of coma . Other adjunctive treatments including N-acetyl cysteine, heparin, aspirin, deferoxamine, anti-TNF therapy, and pentoxifylline have all been trialled but none has been shown to be of benefit.85 No adjunctive treatments are currently recommended for cerebral malaria.Cerebral oedema is a well-recognized component of cerebral malaria and strategies to reduce this have been assessed in controlled trials. Warrell and colleaguesP. falciparum, although it has been reported with other species.8687 The WHO uses a serum creatinine of >265 µmol litre−1 (or ≥3 mg dl−1) as a criterion for severe malaria,27 although this definition is at variance with commonly applied definitions of AKI.88 AKI is particularly common among individuals who did not grow up in endemic regions, suggesting that it may be more common in the malaria-naïve.91 The incidence of AKI in severe malaria varies from 1 to 5% in endemic areas,91 but the rate of AKI is much higher in series of imported malaria (ranging from 23 to >50%).12205354 Cytoadherence of parasitized erythrocytes to glomerular and tubular vascular beds, cytokine release, immune complex deposition, hypovolaemia, and haemolysis may all be contributory.5793 Histopathological findings of AKI in severe malaria include acute tubular necrosis, interstitial nephritis, and glomerulonephritis, although tubular changes are the most common findings.89AKI in malaria is usually caused by 91 Management is supportive with maintenance of fluid balance and electrolytes and renal replacement therapy as indicated. Trials of both dopamine and epinephrine have been performed in severe malaria, but neither has been shown to improve renal oxygen metabolism nor function.94 Artemisinin doses do not need adjusting in AKI; however, quinine may accumulate, so doses should be reduced by one-third after 48 h of established renal failure, unless renal replacement therapy has been initiated.27 The prognosis of AKI associated with severe malaria is usually good, and it inevitably resolves in days to weeks. A recent UK series found that even those patients with persisting renal impairment at discharge from ICU ultimately recovered their renal function.11All patients with falciparum malaria should be screened for AKI, which may not develop until several days after the onset of fever and can be non-oliguric.Salmonellae, has been shown to occur in 5–12% of children with malaria.97 Recent studies have suggested that almost two-thirds of cases of community bacteraemia in endemic regions may be the result of malaria. Furthermore, invasive bacterial disease is associated with a worse prognosis.97 One proposed mechanism is induction of heme oxygenase-1, which mediates tolerance to malaria-induced haemolysis, resulting in reduced resistance to infection with non-typhoidal Salmonellae.98 However, few data are available on the frequency of bacteraemia in adult patients or returned travellers.In endemic areas, concurrent community-acquired Gram-negative bacteraemia, in particular with non-typhoidal 112030 Pneumonia was the commonest co-infection. Community-acquired bacteraemia has been reported in 1.5–3% of cases requiring ICU admission. However, these studies were all retrospective and a failure to take blood cultures before antibiotics were administered may have resulted in an under-estimate of the true frequency of bacteraemia. The possibility of co-infection in returning travellers is an additional reason for early liaison with a specialist unit. High rates of co-infection with HIV have also been reported in some series.11Rates of microbiologically confirmed community-acquired bacterial infections have been reported as 5–10% in patients with imported malaria requiring ICU admission.27 albeit de-escalating quickly or stopping treatment if bacterial infection is subsequently not confirmed. Clinicians should also remain alert to the possibility of nosocomial infection. Ventilator-associated pneumonia and catheter-related sepsis are well recognized in this setting and frequently contribute to poor outcomes despite adequate anti-malarial treatment.1120The use of empiric antibiotics remains controversial, but bacterial co-infection should be suspected in any patient with focal signs or symptoms of sepsis or significant neutrophilia. In such cases, blood cultures should be taken and broad-spectrum antibiotics commenced,99 WHO defines severe anaemia as a haemoglobin concentration of <5 g dl−1.27 However, severe malarial anaemia is mostly seen in endemic areas, especially among children and pregnant women, and is likely to be multifactorial.99 Only one series of imported malaria reported patients presenting with severe anaemia as defined by the WHO.20 Transfusion is recommended in severe anaemia, although no specific studies have addressed transfusion targets in malaria. Current critical care practice supports a restrictive use of red cell transfusions, and this is supported by WHO recommendations of a haemoglobin threshold of 7 g dl−1, taking into account individual clinical circumstances.27100101Malarial anaemia is caused by a combination of haemolysis, dyserythropoiesis, and removal of infected erythrocytes from the circulation by the spleen. Parasite antigens, antibody activation, and subtle alterations in red cell membranes may also result in a similar fate for uninfected cells.2054102103 to 20% or more.1253 Profound thrombocytopenia is common in both severe and non-severe falciparum malaria and is probably caused by increased platelet consumption, sequestration within the spleen, or both. Disseminated intravascular coagulation, which occurs in about 5–10% of severe imported malaria, should be treated conventionally with transfusion of screened blood products  and guided by haematological expertise, but there is no evidence to support empirical platelet transfusion.111220Clinically apparent abnormal bleeding and coagulopathy is commonly seen in severe imported malaria, with a reported frequency ranging from <5%55 In the UK, the overall case fatality rate from falciparum malaria is ∼1%.8 Increased age and management at a centre with less experience of managing malaria have both been identified as risk factors for increased mortality, while individuals born in endemic countries had a lower mortality.8 In studies of artemisinins, overall mortality was 10–19%.3233 Case series of patients with imported malaria requiring ICU admission have reported mortality rates between 5 and 29%.11104 Older age, reduced GCS, and higher parasitaemia at ICU admission were significantly associated with an increased mortality in the largest cohort,20 albeit not consistently replicated in other studies.1130Mortality from severe malaria varies enormously by setting and clinical context. A mortality of >30% has been reported in children with respiratory distress and impaired consciousness.105106 Hanson and colleagues105 derived a simple score  using arterial base deficit and GCS derived from SEAQUAMAT trial data.32 Patients score 0–2 points for their GCS level and 0–2 points for base deficit. A total score of <2 accurately identified patients who survived . However, the positive predictive value of CAM scores for mortality is more limited.Two scoring systems have been proposed for the stratification of adult patients with severe malaria.106 derived the malaria score for adults (MSA) based on the presence or absence of severe anaemia (1 point), AKI (2 points), respiratory distress (3 points), and cerebral malaria (4 points). Mortality increased steadily with an increasing MSA, from 2% for MSA scores of 0–2 to 90% for those scoring ≥7. Taking MSA scores of 5 as a cut-off, they reported a sensitivity of 89.9% for mortality and a positive predictive value of 94.1%. As with the CAM score, patients with imported malaria having a low score (<5) had good predictive power for survival, whereas high scores had a limited predictive power for death.11 The utility of both these scores is likely to vary significantly between resource-rich and resource-scarce settings.Mishra and colleaguesRates of international travel continue to increase and the ‘febrile returned traveller’ is an increasingly common clinical problem. Malaria remains the most important cause of imported fever and cases requiring ICU admission continue to be associated with a high mortality. While there have been significant advances in our understanding of the management of malaria in the last decade, high-quality data to guide management of imported malaria remain scarce, with most derived from endemic settings or retrospective series. The emergence of artemisinin-based therapy has translated into a significant improvement in outcomes in endemic countries and is likely to improve outcomes in imported malaria in the future. Despite numerous studies, no adjunctive therapy has been shown yet to confer a survival advantage and several have proved harmful. ICU management remains supportive and improved outcomes may be attributable more to advances in multi-disciplinary team working, mechanical ventilation strategies, careful fluid management, and infection control. Despite these advances, the mortality from imported malaria remains significant; all cases should be discussed with a specialist unit and transfer of the patient considered.M.M. and A.G.-W. wrote the first and subsequent drafts of the paper. M.S. reviewed and redrafted the paper. J.F.D. and D.W. conceived of the article, reviewed, and redrafted the paper.The authors declare that they have no relevant conflicts of interest.Wellcome Trust Clinical Research Fellow (WT102807) at the London School of Hygiene and Tropical Medicine. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This study was supported by the Special Trustees of the Hospital for Tropical Diseases. All the authors are supported by the UCLH/UCL Biomedical Research Centre Infection, Immunity and Inflammation Theme. Michael Marks is a


In sub-Saharan Africa, malaria continues to cause over 10,000 maternal deaths and 75,000 to 200,000 infant deaths. Successful control of malaria in pregnancy could save lives of mothers and babies and is an essential part of antenatal care in endemic areas. The primary objective is to determine the protective efficacy of community-scheduled screening and treatment (CSST) using community health workers (CHW) against the primary outcome of prevalence of placental malaria. The secondary objectives are to determine the protective efficacy of CSST on maternal anaemia, maternal peripheral infection, low birth weight, selection of sulfadoxine-pyrimethamine (SP) resistance markers, and on antenatal clinic (ANC) attendance and coverage of intermittent preventive treatment during pregnancy (IPTp-SP).This is a multi-centre cluster-randomised controlled trial involving three countries with varying malaria endemicity; low (The Gambia) versus high transmission (Burkina Faso and Benin), and varying degrees of SP resistance (high in Benin and moderate in Gambia and Burkina Faso). CHW and their related catchment population who are randomised into the intervention arm will receive specific training on community-based case management of malaria in pregnancy. All women in both study arms will be enrolled at their first ANC visits in their second trimester where they will receive their first dose of IPTp-SP. Thereafter, CHW in the intervention arm will perform scheduled monthly screening and treatment in the womens homes. At time of delivery, a placental biopsy will be collected from all women to determine placental malaria. At each contact point, filter paper and blood slides will be collected for detection of malaria infection and SP resistance markers.To reach successful global malaria control, there is an urgent need to access those at greatest risk of malaria infection. The project is designed to develop a low-cost intervention in pregnant women which will have an immediate impact on the malaria burden in resource-limited countries. This will be done by adding to the standard IPTp-SP delivered through the health facilities: an “extension” strategy to the communities in rural areas thus bringing health services closer to where women live.ISRCTN37259296 (5 July 2013), and clinicaltrials.gov: NCT01941264 (10 September 2013).Current Controlled Trials:  Malaria infection remains a major public health problem in (sub) tropical regions throughout the world. Approximately half of the world’s population is at risk of malaria and every year this leads to about 207 million clinical malaria cases .Plasmodium falciparum infection in pregnancy is often asymptomatic, yet it is an important contributor to maternal morbidity, preterm delivery, perinatal morbidity and mortality [P. falciparum in the placenta due to its affinity with the chondroitin sulphate A receptor. Placental malaria causes intrauterine growth retardation and the delivery of low birth weight babies who have a significantly higher risk of dying before their first birthday due to the reduced transfer of essential nutrients to the developing foetus as a result of the placental infection [In endemic countries, pregnant women have a higher risk of malaria compared to other adults due to the physiological hormonal changes and reduced immunity. In areas of stable, moderate to intense transmission, ortality , 3 and mortality . In areaortality , in HIV-ortality  or in thortality . The incnfection .per se is associated with an increased risk of malaria infection and anaemia in the first and second year of life of the infant [in utero and the acquisition of a tolerant phenotype that persists into childhood [In sub-Saharan Africa, over 30 million women living in malaria endemic areas become pregnant each year , with mae infant , 11, poshildhood . Importahildhood . If thesPrompt and effective management of uncomplicated malaria in the second and third trimester with artemisinin-based combination therapy. For the first trimester, it is recommended to combine quinine with clindamycin .Long-lasting insecticidal bed nets for all pregnant women in malaria-endemic countries .Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) at regular intervals during pregnancy . This isSuccessful control of malaria in pregnancy could save lives of mothers and babies and is an essential part of antenatal care in endemic areas , 3. For Unfortunately, the coverage of long-lasting insecticidal bed nets and IPTp-SP in many sub-Saharan African countries remains low , 16, parThe project is designed to develop a low-cost intervention for pregnant women that will have an immediate impact on the malaria burden in resource-limited countries. In this study, it is proposed to add an “extension” strategy to the communities in rural areas in addition to the standard IPTp-SP delivered through health facilities. The novelty of this study is to bring health services closer to where women live, using community health workers (CHWs) to provide an antimalarial intervention to women with difficult access to the formal health system. The study will build on the use of CHWs who are already managing malaria cases within the framework of community case management of malaria (CCMm) .The main aim is to establish whether maternal and infant health can be improved by adding to existing IPTp-SP at antenatal clinics (ANCs) community-scheduled screening and treatment (CSST) for malaria among pregnant women through CHWs. The study will test the primary hypothesis that CSST plus IPTp-SP will reduce placental malaria compared to IPTp-SP alone.Primary objective: To determine the protective efficacy of CSST using CHWs against the primary outcome of prevalence of placental malaria.Secondary objectives: To determine the protective efficacy of CSST on maternal anaemia, maternal peripheral infection, low birth weight, selection of SP resistance markers, and on ANC attendance and coverage of intermittent preventive treatment during pregnancy.http://www.cosmicmalaria.eu).These project objectives will be achieved through multidisciplinary research comprising clinical and bio-medical research, health system research, socio-economic and anthropological studies with a focus on CHW, involving policy makers right from the start of the project as full project partners  versus high transmission (Burkina Faso and Benin), and varying degrees of SP resistance . It will be centred around the Basse Health Centre and satellite health facilities. This is an area of seasonal malaria transmission (July-December) that has recently experienced a significant decline in malaria infections. Nevertheless, malaria remains a significant problem with about 15% of pregnant women with placental malaria at delivery as detected by histopathology .In Burkina Faso, the study is situated in the centre-west of the country in the Nanoro health district catchment area . Malaria transmission is high and extremely seasonal and overlaps with the rainy season (June-October).In Benin, the study is situated at the Atlantic Region in the southern part of the country, in the Glo-Djigbé, Zinvié and Zè district communities . Malaria transmission is perennial and the main burden of the disease occurs during the two rainy seasons (April-July and October-November).The current treatment policy of uncomplicated malaria in pregnancy is oral quinine but artemisinin-based combination therapy (artemether-lumefantrine (AL)) may be used in the second and third trimester of pregnancy for all three countries, or amodiaquine-artesunate in Burkina Faso and Benin –23.All pregnant women resident in the study area and willing to remain so until delivery will be invited to participate. Women who have a history of sensitivity to sulphonamides and vulnerable persons  will be excluded. In The Gambia, pregnant adolescents younger than 16 years will not be enrolled unless consent is given by a responsible adult. In Burkina Faso and Benin, a pregnant woman is considered an adult if married (regardless of age). Study participants are free to withdraw from the study at any time without giving a reason.Prior to the trial start, community sensitization and involvement of village leaders will be carried out at each study site. Appropriate community meetings will be held where the trial will be explained to all community members and consent at the community level to participate from community leaders will be obtained. Individual signed informed consent will also be obtained from all participants.This is a multicenter, cluster-randomised controlled trial in which the CHWs and related catchment population (village/community) will be randomised to receive either the intervention (IPTp-SP plus CSST) or continue with current activities, that is IPTp-SP alone at health facilities during ANC visits Figure .Figure 1In principle, the first point of contact with pregnant women is at the first ANC visit. Here, the study will be explained to the woman and informed consent will be obtained. If the woman is in the early stage of pregnancy, she will be encouraged to return during her second trimester for IPTp-SP. If the woman is in the second or third trimester stage, health staff will give the first IPTp-SP dose and plan the second one onto the ANC card at least 1 month after the first dose. A data collection form on health and socio-demographic factors and a physical examination will be carried out, and a blood slide and a blood sample on filter paper will be collected for detection of malaria infection and SP resistance markers Figures . All womCHWs allocated to the intervention arm will follow specific training on community-based case management of malaria, the need for monthly testing of pregnant women using a rapid diagnostic test (RDT), and on issues related to malaria in pregnancy, including the benefit of IPTp-SP and administering SP as early as possible in the second trimester of pregnancy.The CHWs will continuously identify all pregnant women in their own catchment area, visit eligible women in their home and encourage them to attend the ANC as early as possible in their pregnancy for enrolment. The CHW will check after a week if the ANC was visited. For women who do not attend the ANC, the CHW will further encourage and discuss reasons for non-attendance.Thereafter, at monthly intervals up to the last week of gestation, the CHW will revisit the study participant’s house Figure . The CHWIn control communities, CHW will not be trained to administer RDT and AL to pregnant women. The women will be mainly followed through their attendance to the health facilities at the ANC.The final study visit will occur at the time of delivery. A blood sample will be collected just before delivery for haemoglobin measurement as well for detection of malaria infection and SP resistance markers and a short data collection form on their current health status will be administered. A placenta biopsy will be collected and all newborns will be physically examined and weighed as soon as possible after birth Figures  and 2. Ghttp://www.stata.com).The allocation of the two trial arms will be carried out separately but consistently for each country. Prior to the trial start, maps and population size of the study site in all three countries will be obtained. CHWs who work in villages with a population size of approximately 1,000 to 2,000 will be identified. If more clusters than required are identified, the appropriate number of representative clusters will be selected at random. Distance from the centre of each village to the nearest IPTp-SP-providing health facility will be calculated and used to group the clusters into three categories. Stratification by these categories will ensure that the villages with the larger distances are evenly distributed between trial arms. The balanced allocation of each cluster to a trial arm will be done using a computer-based randomisation , a RDT test will be performed and, if positive, AL administered.RDT will be performed on a monthly basis for women enrolled in the intervention arm regardless of the presence of malaria symptoms. If positive for malaria, treatment will be given with AL. If the woman is severely ill at time of visit  she will be referred to the health facility for further care.®  and anaemia (haemoglobin <11 g/dL) diagnosed at the time of delivery.Maternal haemoglobin will be measured using Hb301 HemocueBlood samples will be collected by finger pricks at specified time points during the trial for blood slides (thick blood film) and blood spots on filter paper. Thick blood smears will be stained with 3% Giemsa for 30 minutes and read by trained microscopists at each site. Parasite densities will be calculated by counting the number of asexual parasites per 200 leukocytes , assuming a leukocyte count of 8,000/μl. A blood smear will be considered negative when the examination of 100 high-power fields does not reveal asexual parasites. Each slide will be read separately by two experienced microscopists and discrepancies resolved by a third reader.P. falciparum (to detect sub-patent infections) and the identification of SP resistance markers.Blood spots on filter paper will be used for the molecular diagnosis of 2 biopsy specimen will be obtained from the maternal-facing side of the placenta as soon as possible after delivery. Biopsy specimens will be preserved in 10% neutral buffered formalin which will be processed and embedded in paraffin wax by standard techniques. Pending histological evaluation, all biopsies will be kept at 4°C. Paraffin sections 4 millimeters thick will be stained with hematoxylin-eosin stain.A 1 cmAcute infection Chronic infection Past infection (no parasites but pigment present)No infection Placental biopsies will be classified according to the following definitions :Acute inThe risk of participating in this cluster randomized trial is low. The World Health Organization recommends the use of artemisinin-based combination therapy, in this case AL, during the second and third trimester of pregnancy , 27. TheCHWs in the intervention arm will be trained on safety reporting requirements and on the recognition of danger signs and serious adverse events (SAEs). In case of any SAE of the pregnant woman, deterioration of her condition or reaction after the administration of the drug, the CHW will report this immediately to the study staff who will perform the necessary further assessments and referrals to the appropriate health facilities if required.During the course of the study, any important medical events which occur will be appropriately recorded, managed and reported. Midwives and/or research staff will record all occurrences of adverse pregnancy outcomes such as a miscarriage or spontaneous abortion (<28 weeks of gestation), stillbirth (>28 weeks) or the occurrence of any congenital malformation. All reported SAEs and important medical events will be reported to the study site investigator or deputy. The local principal investigator will inform the sponsor and the coordinating investigator. The sponsor will inform the ethics committee and the Data Safety and Monitoring Board.Prevalence of placental malaria  determined by a placental biopsy taken soon after delivery at a health facility.Maternal anaemia (haemoglobin <11 g/dL) at deliveryP. falciparum peripheral infection (microscopy and PCR) at delivery and during pregnancyMaternal Low birth weight IPTp-SP coverage by village/clusterSP resistance markers profilesNumber of ANC visits during pregnancyNumber of doses of IPTp-SP taken during pregnancyThe study will test the primary hypothesis that CSST plus IPTp-SP will reduce placenta malaria compared to IPTp-SP alone. Sample size calculation was performed according to Hayes and Bennett . Based oAll data handling and record keeping will be standardised across the three study sites. At the health facility, all pregnant women will be recorded on a study register held at each clinic. Women who are enrolled onto the study will also be given a unique identity number.http://www.openclinica.com) databases at each site. Consistency checks will be performed and any outliers and missing data points will be checked against the original forms and subsequently amended in the dataset. Errors including missing data that may have occurred during the data collection will be reported with a minimum delay for further investigation.A standardised data collection form on the health status, medical history of the women, and socio-demographic characteristics will be administered by health staff at the time of enrolment at the health facility. A standardised data collection form on current health status will then be administered at subsequent ANC visits (intervention and control arm) and at each monthly visit by the CHWs in the intervention arm only. All data will be double entered using OpenClinica®  will be used to describe the baseline data.The primary analysis will compare the prevalence of placental malaria  between the intervention and the control group.P. falciparum peripheral infection (microscopy and PCR) at delivery and during pregnancy, (iii) mean birth weight and proportion of newborns with low birth weight  between the two trial arms, (iv) level of ANC attendance by village/cluster and coverage of IPTp-SP (defined by number of doses completed during pregnancy), and (v) SP resistance markers profiles.Secondary analysis will compare the following variables between trial arms: (i) mean haemoglobin levels and prevalence of anaemia (haemoglobin <11 g/dL) at delivery, (ii) prevalence of For the primary and secondary analysis both linear regression (for continuous outcomes such as haemoglobin levels) and logistic regression  will be used to quantify the difference between trial arms. The regression models will allow for the multi-level structure of the data  using random effects and for all relevant co-variates to be taken into account. Model assumptions will be checked to confirm reliable estimates.The recruitment of study subjects will last for 18 months. The expected duration for each study subject is approximately 6 months - from enrolment during pregnancy up to time of delivery. It is expected that the total duration of the trial will be 24 months to allow the delivery of the last recruited pregnant woman.o_0126/MS /DC/SGM /DFR /CNERS/SA).Ethical approval by the Gambia Government/MRC laboratories Joint Ethics Committee was obtained on 25 June 2013 (ref SCC1336). Ethical approval by the Institutional Ethics Committee of Centre Muraz in Burkina Faso was obtained on 19 September 2013 (ref A20-2013/CE-CM). Ethical approval by the Comité National d’Ethique pour la Recherche en Santé in Benin was obtained on 9 December 2013 (NTo reach successful global malaria control, there is an urgent need to access those at greatest risk of malaria infection. IPTp-SP if given at least twice after the first trimester has been shown to be effective in preventing malaria infection and its consequences during pregnancy. However, in the majority of sub-Saharan African countries, IPTp-SP coverage is low, with only 22% of pregnant women receiving IPTp-SP . SystemaIn this study, it is proposed to add an “extension” strategy to the communities in rural areas to the standard IPTp-SP delivered through the health facilities and thus bring health services closer to where women live. The study will build on the use of CHWs who are already managing malaria cases within the framework of CCMm . At thisThe study will extend the CCMm which at present is primarily aimed at children under the age of 5 years to pregnant women. The aim is to improve both IPTp-SP coverage as CHWs encourage women to attend the ANC for the IPTp at the right time, allow systematic screening for malaria infection and provide an opportunity to treat malaria when the prophylactic effect of IPTp-SP has waned. The trial will also investigate the prevalence of SP resistance markers in the positive malaria samples obtained during the study as it could be hypothesized that the beneficial effects of the intervention might be more pronounced in areas with high SP resistance, because women could be more prone to placental malaria when IPTp-SP has a reduced effect and, although not yet observed in West Africa, extremely high levels of SP resistance markers in the parasite population may even lead to adverse effects of SP .At time of submission of this paper, ethical approval has been obtained, and recruitment is ongoing in all three study sites.


Anopheles gambiae salivary peptide (gSG6-P1) and merozoite surface protein one (MSP-119) reflect human exposure to malaria vectors and parasites. This study estimated malaria transmission dynamics using serological evidence of vector and parasite exposure in southern Ghana.Seroepidemiology provides robust estimates for tracking malaria transmission when intensity is low and useful when there is no baseline entomological data. Serological evidence of exposure to malaria vectors and parasite contribute to our understanding of the risk of pathogen transmission, and facilitates implementation of targeted interventions. Ab to χ2 test with p < 0.05 statistically significant. Non-parametric repeated ANOVA – Friedman’s test was used to test differences in antibody levels. Seroprevalence data were fitted to reversible catalytic model to estimate sero-conversion rates.Total IgG responses to both antigens in an age stratified cohort  were measured from South-eastern Ghana. 295 randomly selected sera were analyzed from archived samples belonging to a cohort study that were followed at 3 consecutive survey months (n = 885); February, May and August 2009. Temporal variations in seroprevalence of both antigens as well as differences between the age-stratified cohorts were determined by 19 were high  and 57.6%, 52.3% and 43.6% in respective order from Feb. to August. Repeated measures ANOVA showed differences in median antibody levels across surveys with specific significant differences between February and May but not August by post hoc Dunn’s multiple comparison tests for gSG6-P1. For MSP119, no differences were observed in antibody levels between February and May but a significant decline was observed from May to August. Seroconversion rates for gSG6-P1 increased by 1.5 folds from February to August and 3 folds for MSP119.Whereas parasite prevalence was generally low 2.4%, 2.7% and 2.4% with no apparent trends with season, seroprevalence to both gSG6-P1 and MSP1Data suggests exposure to infectious bites may be declining whereas mosquito bites remains high. Sustained malaria control efforts and surveillance are needed to drive malaria further down and to prevent catastrophic rebound. Operational factors for scaling up have been discussed. Malaria elimination has again been accepted as the goal of malaria control efforts with thirty-two out of the remaining ninety-nine malaria endemic countries currently implementing malaria elimination strategies . As inteMa) and the sporozoite rate , is the gold standard for measuring malaria transmission intensity. It is the most direct way of detecting human exposure to infectious bites and mosquito population monitoring. However under conditions of very low malaria transmission the EIR suffers from well recognized limitations  where Pt is the proportion of individuals aged t that is seropositive, Lambda λ is the annual rate of seroconversion and ρ is the annual rate of reversion to seronegative. This was done to investigate the relationship between force of vector and parasite exposure with age. All data were analysed and graphed using GraphPad Prism software .Seroprevalence was defined as the number of positive responders out of the total number of participants tested. The temporal variations in seroprevalence of both antigens as well as differences between the age-stratified cohorts were determined by A total of 295 subjects had data available for all the 3 surveys  and thus all analysis were based on the 295 selected samples. 27% out of the subjects were less than 5 yrs whilst the majority of the study subjects were between the ages of 5 and 14. Only 12% of the participants in the selected cohort were in the in the age bracket of 15–29  were high throughout the three survey months, although a 10% decline in seroprevalence was observed between 1st and 2nd survey months (February and May), this was just at the threshold of statistical significance . There was a significant but gradual decline in exposures to the blood stage P. falciparum merozoite surface proteins (MSP119.), MSP119 specific seroprevalence observed in 1st survey declined by 5% in the 2nd survey and a further 10% in August , May  and August . High exposures to the salivary gland peptide corresponded to strong association with age observed in the first and second survey months . gSG6-P1 responses had very similar trends compared with that observed in MSP119 which also had a strong correlation in the first survey with a gradual decline in the second and third surveys  and second (May 2009) survey months, detecting temporal variations in vector exposure among the cohorts at different time points. The Friedman’s test (Repeated measures ANOVA and non parametric) revealed significant differences in antibody levels to gSG6-P1 between the first, second and third survey months at the population level.  as well as post hoc Dunn’s multiple comparison tests between Feb and May: (p < 0.0001), Feb and August (p < 0.0001), but not May and August (p > 0.5) . However, post hoc analysis revealed no significant differences between first and second (February and May) survey months (p >0.05) but significant decline were observed in the antibody levels between the second and third (May-August) survey months (p < 0.05)  Figure .Generally, median antibody levels to all antigens differed among the age groups throughout the surveys  Figure . Howeverp = 0.0002), May  and August  with increasingly higher antibody levels. However, the Dunn’s multiple comparison tests (post hoc test) between specific pairs of age-stratified cohorts showed intriguing differences: consistently, there were significant differences between < 4 and the 5–14 age groups across all survey months, Feb (p < 0.001) May (p < 0.01) and Aug (p < 0.01) with higher antibody levels observed in the 5–14 year cohorts. However, there were no differences seen in the antibody levels of anti-gSG6P1 in the 5–14 and the >14 age group in the first and second survey months (P > 0.05), a decline in between the third survey with the > 15 year cohort having lower gSG6-P1 levels but this was not statistically significant  increased significantly between <5 and 5–14 age groups in the first and second survey months but not the third  May  August . Similar to gSG6-P1, there were no differences in the antibody levels observed between the 5–14 and the adult age-groups throughout the three survey months.Median antibody levels to the merozoite surface protein , also recorded a somewhat corresponding increase  tested 9 Figure , MSP119 9 Figure  . Generat Figure . MSP119 Metrics of malaria transmission change on different temporal scales, mirroring among other things the changes in vector exposure, parasite infections in humans, as well as the dynamics of changing human immunity. The key component in deciding the appropriateness of malaria transmission metrics as end points for measuring changes in transmission is determined not only by costs, precision, and accuracy, but also by the intrinsic variability of the metric across space and time . Using a19 was 40% and above. Similar findings have been reported elsewhere. In Somalia, when no parasites were detected both in wet and dry seasons after screening more than one-thousand people, 17.9% and 19.3% of MSP119 seroprevalence was found respectively [19 reached peaks above 50% [The use of serological markers of vector and parasite exposure to track changes in malaria transmission over time has several competitive advantages over other metrics. Chiefly, serological markers are more sensitive and robust ,16,14. Fectively . Similarbove 50% .et al. studied human immune response to gSG6-P1 salivary peptide in five communities in northern Senegal where malaria transmission has been described as low, with the An. gambiae s. l. as the principal vector. They observed a significant increase in IgG levels to gSG6-P1 during the peak exposure to Anopheles bites, and a corresponding decrease right after the end of the exposure season [Anopheles exposure between villages [Serological markers, in particular gSG6-P1, are robust in tracking temporal changes in vector exposure and risk of pathogen transmission in the younger population under intense transmission intensity ,31,32,40villages ,31.Anopheles gambiae, a principal malaria vector in the district, requires relatively shallow and transient breeding aquatic habitat and therefore its abundance does not peak immediately after heavy rainfall but must tarry for the flooding waters to recede.In the current study it was observed that Total IgG anti-gSG6-P1 levels showed significant differences between February and May but showed sustained high levels in August. Asutuare is a sugar-cane growing area; a cash crop that thrives in water logged areas, thus the area has several irrigation schemes that virtually flood the area all year round exposing inhabitants to high vector exposure. High vector exposure as evidenced from the anti-gSG6-P1 responses was observed in the month of February, a relatively dry month, this may be due to lagged effect of high rainfall recorded two-three months earlier in 2008 . Meteoro19 Age-specific seroprevalence has been used to estimate seroconversion rates (SCR) as a measure of malaria transmission intensity. Earlier studies in Tanzania have shown that these estimates are tightly correlated with EIR measurement [19 seroprevalence has been described as an equivalent of the force of infection and correlated with the entomological inoculation rate. This measures the rate of parasite exposure in the population with age (or time) [19 SCR increased about 3 fold from February to August. Although seroprevalence decreased approximately 8% (gSG6-P1) and 14% (MSP119) at the population level from the February to August  to 2.4% (August), age stratifications revealed that parasite prevalence increased with age in the paediatric population which happens to be 88% of the total study population  ,16. In tst Table , the SCRAnopheles population density increased. It has been suggested that the gSG6-P1 is a reliable marker for measuring human–vector contact [ma measured in EIR estimation, this is because human immune response to gSG6-P1 increases and decreases sharply following high and low vector exposure seasons, and transient exposure in naïve individuals, this has been observed by other scientist elsewhere [Anopheles breeding sites [The force of exposure to vector bites as seen in seroprevalence and antibody levels in study participants increased 1.5 fold from February to August. This implies that the aggressiveness of vector bites increased or the  contact . Possibllsewhere ,42. In Llsewhere ,46. A silsewhere ,20,47. Ilsewhere . It has Parasite prevalence (PR) in the current study was generally low, with no exposure in the adult population >15 years, this has been reported by other scientists working in the same area . The pre19) provide a useful sero-surveillance tool for tracking malaria transmission intensity. In order to scale.Seroprevalence and seroconversion rates of vector (gSG6-P1) and parasite proteins  together with parasite prevalence, we have identified low parasite prevalence, high vector exposure and small changes in malaria transmission intensity from February through May and then to August 2009. Seroconversion rates for gSG6-P1 increased by 1.5 folds from February to August and 3 folds for MSP119. Possibly, exposure to infectious bites may be declining whereas mosquito bites remains high. Malaria transmission in Asutuare as well as many other areas in Africa where endemicity is low needs careful monitoring with sensitive and robust tools to ascertain progress and to forestall a potential catastrophic rebound.Using antibody levels to gSG6-P1 and MSP1


Plasmodium falciparum-induced malaria can cause placental lesions and intrauterine growth restriction (IUGR). There are few published studies on Plasmodium vivax-induced malaria in pregnancy. Ultrasound is an efficient method for evaluating foetal biometry and placenta. The present study aimed to investigate the occurrence of increased placental thickness, foetal biometry and the amniotic fluid via ultrasound in a cohort of pregnant women with vivax malaria in Manaus, Amazonas, Brazil.During pregnancy, A cohort study was conducted among 118 pregnant women with vivax malaria and 191 pregnant women without malaria. Foetal biometry, placental thicknesses and the amniotic fluid were evaluated via ultrasound. Biometric data were distributed by the trimester in which the infection occurred and converted to Z scores. The results were compared between the groups.vs 0%; p <0.001). The Z scores of biometric parameters were not statistically significant when comparing the groups or according to the time of infection. In ultrasound results of the 118 pregnant women with malaria, seven (6%) showed low foetal weight, two (1.7%) showed oligohydramnios and one (0.85%) showed foetal malformation. There was no significant difference when these variables were compared to those of the control group.Among pregnant women from the cohort, increased placental thickness was observed in ten women with malaria  contains supplementary material, which is available to authorized users. Plasmodium vivax [Malaria is among the most important public health issues worldwide . In Latium vivax ,5,6.Plasmodium sp. infections [Plasmodium falciparum-induced malaria can cause placental lesions and changes in foetal oxygenation that lead to neonatal impairments, such as intrauterine growth restriction (IUGR), decreased amniotic fluid, prematurity and brain injuries caused by hypoxia and intrauterine death [P. vivax-infected pregnant women are more often anaemic and have newborns with lower birth weights [Pregnant women are more susceptible than the general population to fections , and malfections -11. Durine death -14. Stud weights ,15-17.Ultrasound is a useful tool for detecting IUGR and determining the gestational age by foetal biometry . PlacentIn cases of placental insufficiency and chronic hypoxia, the foetus adapts to the new situation by protecting its important organs. This mechanism leads to decreased diuresis and secretion of pulmonary fluids and subsequently leads to oligohydramnios, which is usually detected via ultrasound There is a lack of studies on ultrasound evaluations of foetal growth, amniotic fluid and placentas in pregnant women with vivax malaria. The present study aimed to investigate the occurrence of changes in placenta, foetal growth and amniotic fluid volumes by ultrasound in a cohort of pregnant women with vivax malaria in Manaus, Amazonas, Brazil.A prospective cohort study of pregnant women with and without vivax malaria, all of whom had gold standard first trimester ultrasound dating scans (CRL ≤14 wks), were enrolled from January 2006 to July 2007 in the city of Manaus at the Dr Heitor Vieira Dourado Tropical Medicine Foundation . This institution is also a primary care centre, diagnosing and treating patients with malaria even if they have no clinical complications. The unexposed group was selected from another close health unit devoted to antenatal care, attending patients with the same epidemiological profile in the same period, matched by maternal age and gestational age at inclusion.The unexposed group was recruited in another clinic because the FMT-HVD is a reference hospital for infectious disease and infections in pregnancy were an excluding factor in these study (they can lead to IUGR and other diseases). The study design planned to include one unexposed woman for each exposed pregnant woman. Most of the population from Manaus is of mixed races, therefore, ethnic group was not specified. Routine blood examinations were performed in all of them. The same physician performed the ultrasound in both groups.The microscopic diagnosis was performed by thick blood smear in all women. Maternal parasite load was estimated using a semi-quantitative method as recommended by the Brazilian Ministry of Health: 200–300 parasites per cu mm; 301–500 parasites/cu mm; 501–10,000 parasites/cu mm; 10,001-100,000 parasites/cu mm and 100,000 or more parasites/cu mm.Parasite densities with differential counting of asexual stages were estimated by experienced microscopists, by counting the number of parasites in 200 leukocytes in high magnification fields, and the number of leukocytes/cu mm.The thick blood smear was performed at the moment of recruitment and monthly when the patient returned to antenatal evaluation.The patients recruited for the study were interviewed and responded to a questionnaire regarding clinical and demographic data and were subsequently referred to ultrasound evaluation. The ultrasound examinations were performed at the time of the malaria diagnosis and at 30-day intervals until the delivery date. The examinations were performed with the GE Voluson Expert and GE P6 devices  by a single examiner who was certified in ultrasound examination by the Brazilian College of Radiology (Colégio Brasileiro de Radiologia) and the Brazilian Federation of Gynecology and Obstetrics (Federação Brasileira de Ginecologia e Obstetrícia) and in Foetal Medicine by the Brazilian Federation of Gynecology and Obstetrics.et al. [The patients were examined while in the supine position for bi-dimensional evaluations. They were instructed to relax the abdominal muscles and not to position the arms under the head or cross the legs in order to prevent muscle contracture. The gestational age was estimated from the crown-rump length (CRL) during first trimester ultrasounds, according to the technique reported by Grisolia et al. . During et al. ; the AC et al. ; and, thet al. . From 18et al. . Three mIn an attempt to eliminate problems caused by variations in the measurements according to the gestational age, the Z score was used to compare the ultrasound results between the two groups. When the score was two standard deviations or more above or below the mean, it was considered altered.The Z scores were evaluated for all parameters according to the following formula: Z = (X-M(GA))/SD(GA), where X is the measured value , M(GA) is the mean of the approximate value for the evaluated gestational age according to the normality, and SD(GA) is the standard deviation of the mean for the gestational age according to the standard curve.et al. [The amniotic fluid was evaluated according to the amniotic fluid index (AFI) proposed by Phelan et al. . The plaet al. . The resIn the group of pregnant women with malaria, the treatment comprised the use of chloroquine , according to the recommendation of the Brazilian Ministry of Health . In patiIn both groups, obstetric complications were treated at the local maternity hospitals in the region, according to proximity to the patient’s house; the births also occurred at these hospitals. Approximately one month after delivery, the patients came back to another medical evaluation, and newborn data such as body weight, Apgar scores and the presence of complications were collected from the newborn cards that were filled out at the maternity hospitals.P. vivax on the microscopic thick film examination;Vivax malaria: presence of Maternal anaemia: haemoglobin lower than 11 mg/dl;Maternal weight: measured upon enrolment and at each antenatal consultation until childbirth. Mother’s weight prior to pregnancy was based on antenatal dates. Weight gain was defined as the difference in maternal weight between the first and last weight measured at antenatal consultation;Low ultrasound weight: weight less than the tenth percentile for the gestational age;Low weight at birth: weight at birth less than the tenth percentile for the gestational age.  ;Pre-term babies: newborns with gestational age less than 37 weeks according to the first trimester ultrasound;IUGR: foetuses at term with low weight , were excluded from the study. Patients whose gestational age could not be evaluated correctly because they did not undergo a first trimester ultrasound and patients who did not attend the follow-up examinations were also excluded from the analysis.The results were tabulated with Excel  and analysed with Epi-Info for Windows, version 3.5.3  The dataThe patients were informed about their participation in the study, and their consent was obtained through informed consent terms that were signed by the patients or their legal guardians. The present study was approved by the Research Ethics Committee of the Fundação de Medicina Tropical Dr Heitor Vieira Dourado (FMT-HVD) under protocol number 0836–05.During the study period, 214 pregnant women with malaria and 289 without malaria were referred for ultrasound examination. Among the group of pregnant women with malaria, three were excluded because of multiple pregnancy, 50 because of concomitant diseases , 20 because of falciparum malaria and 23 due to loss to follow-up . Among the group of pregnant women without malaria, six were excluded because of multiple pregnancy, 53 because of concomitant diseases , and 39 due to loss to follow-up .There were no significant differences between the groups regarding to the mean age, education, parity, haemoglobin levels, and maternal weight gain during pregnancy Table . The meaOf the ultrasounds from the 118 pregnant women with malaria, seven (6%) showed low foetal weights, two (1.7%) showed oligohydramnios, and one (0.85%) showed foetal malformation. There was no significant difference when these variables were compared to those of the control group. However, increased placental thickness  was only observed in ten pregnant women with malaria with anterior and posterior placenta (8.5%)  levels above 11 mg/dl and above the malaria group Hb mean (10.9 mg/dl). This group had no cases of IUGR or low birth weight and only one patient had pre-term labour with 34 weeks. None of those patients with increased placental thickness had had oligohydramnios, lateral or fundal placenta. Data from increased placental thickness group are presented in Additional file Comparing increased placental thickness group with placental normal thickness group, there were no statistical differences between maternal age, parity, mean gestational age, Hb levels at presentation, maternal weight gain, BPD, CC, AC Z-score, birth weight and gestational age at delivery Table .Table 3Placental analysis revealed a statistically significant increase of thickness in patients with malaria than in the without malaria group p <0,01)  in the pregnant women with and without malaria were compared to the fifth, 50k et al. . In bothThe Z scores were compared according to the trimester in which the patients contracted malaria. The first ultrasonography after malaria diagnosis and the last ultrasonography before the birth were compared. When comparing the groups, no significant differences were found in the mean Z scores of the ultrasounds with regard to the trimester of infection p >0.05 in all parameters  or the inclusion in the study  with respect to impairment in the foetal weight, BPD, HC, FL, or AC. No significant impairments were found between the groups and the exposure time Figure . An analTable Among the 118 pregnant women with vivax malaria, 109 (92.4%) initiated treatment on the day of symptom onset. The other nine initiated treatment up to five days after symptom onset. All initiated treatment on the same day of diagnosis of vivax malaria by the thick blood film. Comparing the mean Hb levels with increased placental thickness, low birth weight or pre-term births there were no significant difference between the groups of pregnant women with lower Hb levels and increased placental thickness Table .Table 5The time to the first ultrasound examination ranged from 0–15 days after the diagnosis of the symptoms, with a mean of five days. Sixteen patients evolved with relapse throughout the follow-up period, however these patients did not present any ultrasound differences as compared to those that did not relapse.The present study evaluates foetal and placental ultrasound changes in pregnant women with vivax malaria in Latin America. Significant differences in the placental thicknesses between the malaria and non-malaria groups were revealed via placental ultrasound. Ultrasound studies of placental thickness have reported that placentas with increased thicknesses are more likely to be associated with hydrops, molar pregnancy, other infections during pregnancy, triploidy, maternal diabetes mellitus, anaemia and aneuploidy ,38.Increased placental thickness does not comprise a specific diagnosis of any disease. However, it can act as an alert to identify foetuses at high risk of disease resulting in inflammation, oedema or compensatory hypertrophy .et al. [Lee et al. -23 studyet al. [et al. [Godfrey et al.  in theiret al.  studying [et al.  studying [et al. . There wet al. [P. vivax causes inflammation in the intervillous spaces [When using three-dimensional ultrasound to study placental volumes in pregnant women with vivax and falciparum malaria in Thailand, Rijken et al.  found noet al. . No studs spaces ,44.P. falciparum and P. vivax infections on placental histopathology in low malaria transmission areas, McGready et al. [P. falciparum-infected patients, especially those who were infected soon before delivery. The P. vivax infections in this study by McGready et al. [In a study of the effects of placental y et al.  found hiy et al.  were assy et al. . StudiesThe foetal biometry measurements, including BPD, FL, AC, and foetal weight, are important tools with which to evaluate gestational age, IUGR and detecting obstetrics problems. It is fundamental to know the correct gestational age measured by a first trimester ultrasonography and compare the foetal biometry and the weight expected for the gestational age, as was done in this study -48.th percentile for the 38- to 40-week age range, according to a study by Pedreira et al. on gestational age-based foetal weight parameters in Brazil [In the present study, most of the pregnant women developed malaria during the second and third trimesters of gestation, and an asymmetric growth restriction with an abdominal circumference reduction was expected, but not observed. In both groups, the weights were similar to those considered adequate in the 50n Brazil .Some studies showed that recently infected women supposedly have a tendency towards more favourable perinatal outcomes because of the absence of immunological memory ,50,51. Iet al. concluded that earlier treatment yielded fewer or even no effects throughout the gestation [In a study of foetal biometry via umbilical artery and uterine artery Doppler in women with vivax malaria in Msambweni, Dent estation . Rikjen,estation . Schmiegestation . Thus, aThe antenatal supplementation with daily iron is effective in reducing the risk of low birth weight ,56. ThisIt is worth noting that no changes in the demographic data were observed among the groups of pregnant women, suggesting that the groups were homogeneous and that epidemiological factors were not confounding factors in this study.P. vivax chloroquine-resistance is supposed to be around 5% [In the present study, the changes in the placental thickness, although significant, did not cause foetal repercussions at birth. There were no morphological or foetal changes that could be considered characteristic of foetal involvement in vivax malaria. The fact that the ultrasound examinations agreed with those from a cohort group of unexposed pregnant women was most likely due to the area being of low endemicity, and therefore, all pregnant women in the present study participated in follow-ups in an active detection system that allowed early diagnosis and treatment of new malarial episodes. The treatment and control of infections and relapses ensured that the behaviour of the infected group was similar to that of the group of pregnant women without malaria. In these study, malaria was an acute and non-chronic event with acute consequences that, in most cases, did not have any consequences and retreated spontaneously after treatment, thus demonstrating the benefits of adequate treatment and close follow-up of infected cases. All women were treated with chloroquine, and recent data showed that round 5% .P. vivax infection; only thick blood smears were performed; PCR to diagnose low parasitaemia could not be performed; patients performed weekly prophylaxis with chloroquine without supervision; some infections such as syphilis, toxoplasmosis and rubella were excluded because are routinely tested in antenatal care, but other infections, although rare, were not excluded and could have lead to increased placental thickness; the examinations were performed by an ultrasound examiner who was not blinded to the presence or absence of infection, leading to bias. Furthermore, variables derived from ultrasound biometry followed the Hadlock reference table from a North American population, impairing a precise estimation in the Amazonian population. However, using a control group this limitation was surpassed.Limitations of the present study were low power of the study in order to estimate the frequency of low birth weight in The placental thickness changes were significant but caused no foetal repercussions at birth. The ultrasound findings except placental thickness were similar in both groups, possibly because this is a low-endemic area and the pregnant women in the study were followed up in an active detection system that allowed early diagnosis and treatment of new malaria episodes.From the public health perspective, the finding of increased placental thickness could be a sentinel marker of malarial infection and this information could be useful for those who perform ultrasound examinations in remote areas, alerting the need of thick blood smear systematically. However, further studies in other areas are needed to confirm this finding.


Malaria is the most common disease transmitted by the bite by an infected female anopheles mosquito and caused by the plasmodium parasite. It is mostly prevalent in subtropical regions receiving abundant rain and supporting copious mosquito breeding. This disease is generally detected by the microscopic examination of blood films or antigen based rapid diagnostic test. Only occasionally the parasite DNA is detected using polymerase chain reaction in certain advanced, expensive laboratories.An innovative spectral detection method based on the fluorescence spectra of a set of blood plasma biomolecules  and red blood cell (RBC)-associated porphyrin is being evolved by our group.The research so far has exhibited sensitivity and specificity values exceeding 90% based on the spectral features of blood components of 14 malaria patients and 20 numbers of age adjusted normal controls. The fluorescent biomolecules go out of proportion when the malarial parasite breaks down the hemoglobin of blood.This technique has the potential to be used as an alternative diagnostic procedure for malaria since the instrumentation involved is portable and inexpensive.http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_182The virtual slide(s) for this article can be found here:  Malaria is considered as one of the major health problems in subtropical countries like India and Bangladesh. It is a mosquito borne infectious disease, but caused by the parasite of the genus Plasmodium. The disease is transmitted through the bite of mosquito infected by the parasite. The mosquito bite introduces the malarial parasite from its saliva into the blood of the human host. The parasite travels to the liver to reproduce and mature up . The comWhen the sporozoites of the malaria parasites enter into the bloodstream through a mosquito bite, they migrate to the liver. The sporozoites infect the liver hepatocytes and get multiplied into merozoites asexually inside the hepatocytes (8–30 days). After maturing, they break the cell walls of hepatocytes and enter into the blood stream. Like the sporozoites, which multiplied inside the hepatocytes, the merozoites infect RBCs, get multiplied further within, finally breaking out of RBCs to invade fresh RBCs .In humans, malaria is caused by five species of Plasmodium including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. The three major human malaria species in India are Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae .Plasmodium falciparum is the most dangerous form of malaria with the highest rate of mortality. The treatment for malaria will be provided on the basis of the species identified upon standard diagnosis. In the states where Plasmodium falciparum and Plasmodium vivax co-exist, fluctuating proportions of the two species complicate the diagnosis and treatment process .The diversity of malaria in India is also attributed through the increased diversity of malaria vector species. In India, of the 60 species of mosquito available, six were found to be the vector for malaria. Anopheles culicifacies with its indistinguishable sibling species: A, B, C, D, and, E is the major vector of malaria in India which accounts for 60 - 65% of the malaria burden. It also acts as a model organism to study the host-vector-parasite interactions . RSpectral diagnosis is a newly evolving technique for detecting diseases, particularly cancer -16, thalThe methods and materials for spectral diagnosis of cancer and hemoglobinopathies have been presented in a number of papers earlier -19. The Controls: Exactly 5 ml of venous blood from each of the 20 healthy volunteers (age range: 10 to 20 years) was collected in a violet sterile vial that contained the anticoagulant EDTA. The vial was gently rocked five times to adequately mix the EDTA and whole blood, and the samples was centrifuged at 3000 rotational speed (RPM) for 15 minutes. Clear, pale, greenish-yellow plasma supernatant was obtained by such centrifugation. A total of 1.5 ml of supernatant was removed from the top layer for spectrofluorimetric analysis, leaving the buffy coat and the formed elements, as undisturbed sediment. The blood plasma sample was subjected to synchronous fluorescence excitation spectral analyses without any other treatment.Next, the buffy coat, which contained mostly white blood cells (WBCs) , was removed and discarded, and exactly 1 ml of the thick formed elements from the bottom layer, which contained mostly RBCs, was removed to a sterile vial and mixed with 2 ml of analytical grade acetone. Proper care was taken to ensure that the formed elements did not develop lumps. After thorough mixing to enable the acetone to extract fluorophores within and around the cells, the sample was centrifuged again (3000 rpm for 15 minutes). The resulting supernatant was subjected to fluorescence emission spectra analysis at an excitation wavelength of 400 nm.Patients: The same protocol was used to process blood samples from confirmed patients suffering from malaria caused by the parasite plasmodium falciparum as diagnosed by professional pathologists of General hospital, Chennai. Samples were obtained before any treatment. The patients were informed about the investigation, and proper consents were obtained. The samples were collected as per the ethical committee approval (GACN/KP/CNBT/8428) The malaria subjects consisted of 14 patients , and had different levels of clinical severity.The instrument used was a spectrofluorometer  capable of collecting excitation, emission, and synchronous spectra in the 200–800 nm range. An excitation and emission slit width of 10 nm and scan speeds of 1000 nm/min were used. The samples were placed in quartz cuvettes and illuminated by a specified wavelength of light with a 10-nm spectral width and a spot size of 3 × 2 mm. The power at the point of illumination was approximately 20 μW, which was too low to induce photo bleaching. This finding was confirmed by repeating the experiment three times for each sample and observing no inter-replicate spectral differences.Three types of spectra are measured in the field of fluorescence spectroscopy. In fluorescence emission spectra (FES), one particular wavelength is selected for the excitation of a molecule, and the fluorescence emission spectrum is obtained by rotating the emission grating over a predetermined range. The reverse is true of fluorescence excitation spectra (FXS), in which the peak emission band of a molecule is selected, and the excitation grating is rotated to scan the excitation spectra. In synchronous excitation spectra (SXS), both gratings are synchronously rotated at offsets of 40 nm or 70 nm to obtain the fluorescence excitation bands for every molecule in the predetermined range. The wavelength offset and scan range are not unique; they are determined empirically by trial and error, for a given set of experimental protocol. After analysing other offsets, including 10 nm and 30 nm, it was determined that the 70 nm offset provided excellent resolution and good contrast between the normal and malaria samples because 70 nm is the Stokes shift  of the mIt is important to draw attention to the fact that concentration quenching and inner filter effect are inherent in such undiluted plasma. Hence the experiments were repeated for both sets of samples, after 5 times and 10 times serial dilution with normal saline. However, discrimination between the samples of patients and controls were best with undiluted samples and hence only this result was presented.Figure 1 = I635/I585, the ratio of intensity of two fluorescence bands cited above, this was about 1.2 ± 0.1 for normal control.It could be seen that there were three important fluorescence bands, 460 nm , 585 , 635 nm . In fluorescence spectroscopy, the accepted convention is to take the ratio of peaks of fluorescence bands, in order to quantity and obtain a measure of endogenous biofluorophores. If we define the ratio R1 = 1.0 ± 0.1. That means, as far as porphyrin is taken as the biomarker, the difference between the malarial sample and normal control sample is only marginal. .Figure 2 = I280/I380, (ratio of peak intensity at 280 nm and 380 nm) was 4 for normal; another ratio parameter R3 = I460/I380 (ratio of peak intensity at 460 nm and 380 nm) was about 1.0 for normal.Figure 2 = 6.9, R3 = 3.2 for the malarial sample. That is, R2 was about 70% and R3 was 300% elevated respectively.Figure 2 and Figure 3 to classify the malaria samples from the control ones. The discrimination between the two sets was quite good.Figure Spectral diagnosis is a newly evolving technique for detecting diseases, particularly cancer -16, thal1 as shown in our earlier papers [1 has decreased only marginally or from the window of R1 the sensitivity and specificity of malaria diagnosis would be very poor.Out of the few diseases diagnosed by spectral features, it was thalassemia closest to the malaria, in terms of spectral manifestation. Thalassemia are inherited diseases due to reduced or defective α or β chain of the hemoglobin (Hb). Because of this, the concentration of RBC is only about 50 to 60% of the normal . For exar papers . In comp2 and R3 as these two parameters area measure of metabolites of hemoglobin [2 and R3 is elevated. When RBC breaks down, a process that is abnormally high in thalassemia patients, the consumption of NADH is high, simultaneously producing equally abnormally high levels of FAD.Another important aspect of thalassemia is the poor average lifetime of RBC, while the average lifetime for normal controls is about 120 days for the thalassemia patients it varied from 80 days to 10 days depending upon the severity. This is because the RBC of thalassemia patients is fragile. This is manifested as the abnormally high value of Rmoglobin -19. For Similar degradation is externally initiated and perpetrated by the parasite which breeds and multiplies inside the RBC of malaria infected patients. In other words, malaria and thalassemia are similar from the point of view of the fragility of RBC the former being initiated by the parasite injection, and the latter being inherited. However, these two are dissimilar from the point of concentration of RBC as the former ones are persons with the same concentration of RBC as those of normal, but the latter ones are subjected with inherited low level of RBC.Most of the symptoms and complications of malaria may be attributed to the rapid and abnormal lysis of the RBC’s and this spectral diagnosis provide the direct evidence for this point of view. The photographs, presented in Figure In this preliminary study, done as a proof of concept, the fluorescence spectral features of blood components of malarial patients have been reported. The study shows that, the malarial patient sample could be distinguished with high sensitivity from the normal control, because plasma of a malaria patient is flooded with the fluorescent decay products of RBC. It is important to mention that this report presents results of only one type of malaria (PF). We need to repeat with other types of malaria to check possible distinction, a task difficult in the conventional technique. In addition, we need to do large-scale, multicenter study to assess the clinical viability of this new technique. We hope to undertake this task in the near future.