In this position paper, the European Society for Clinical Microbiology and Infectious Diseases, Study Group on Clinical Parasitology, summarizes main issues regarding the management of imported malaria cases. Malaria is a rare diagnosis in Europe, but it is a medical emergency. A travel history is the key to suspecting malaria and is mandatory in patients with fever. There are no specific clinical signs or symptoms of malaria although fever is seen in almost all non-immune patients. Migrants from malaria endemic areas may have few symptoms.Plasmodium falciparum malaria is either with oral artemisinin combination therapy (ACT) or with the combination atovaquone/proguanil. Two forms of ACT are available in Europe: artemether/lumefantrine and dihydroartemisinin/piperaquine. ACT is also effective against Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi, but these species can be treated with chloroquine. Treatment of persistent liver forms in P. vivax and P. ovale with primaquine is indicated after excluding glucose 6 phosphate dehydrogenase deficiency. There are modified schedules and drug options for the treatment of malaria in special patient groups, such as children and pregnant women. The potential for drug interactions and the role of food in the absorption of anti-malarials are important considerations in the choice of treatment.Malaria diagnostics should be performed immediately on suspicion of malaria and the gold- standard is microscopy of Giemsa-stained thick and thin blood films. A Rapid Diagnostic Test (RDT) may be used as an initial screening tool, but does not replace urgent microscopy which should be done in parallel. Delays in microscopy, however, should not lead to delayed initiation of appropriate treatment. Patients diagnosed with malaria should usually be hospitalized. If outpatient management is preferred, as is the practice in some European centres, patients must usually be followed closely (at least daily) until clinical and parasitological cure. Treatment of uncomplicated Complicated malaria is treated with intravenous artesunate resulting in a much more rapid decrease in parasite density compared to quinine. Patients treated with intravenous artesunate should be closely monitored for haemolysis for four weeks after treatment. There is a concern in some countries about the lack of artesunate produced according to Good Manufacturing Practice (GMP). Malaria continues to pose challenges in diagnosis and management and remains an infrequently encountered infection for many physicians in non-endemic areas. Malaria symptoms are non-specific and cannot easily be distinguished from a wide range of other febrile conditions on clinical grounds alone. Thus, aPlasmodium falciparum or Plasmodium knowlesi, the parasitaemia (expressed as number of parasites per microlitre of blood or as a percentage of red cells infected) is an essential parameter for determining whether the patient has complicated malaria or not, and to monitor treatment effect.Malaria cannot be confirmed clinically. The gold standard for malaria diagnosis is light microscopy of Giemsa-stained thin and thick blood films, which requires a high level of expertise. This is not always available around the clock outside specialized centres and rapid diagnostic tests (RDTs) are, therefore, increasingly used. However, RDTs have limitations and once a malaria diagnosis has been made, the infecting species present must be determined. Furthermore, in the case of The management of malaria in non-endemic areas may vary between centres. For example, in a retrospective analysis of over 500 patients from five European countries treated between 2003 and 2009, 18 different combination regimens were used[Malaria is one of the major global public health challenges with an estimated 225 million clinical cases and more than 655.000 deaths in 2011, mainly in children aged less than five years old from sub-Saharan Africa. However, recent studies have found that the mortality may be grossly underestimated-5. In EuPlasmodium falciparum malaria cases varies from 0.4% in a large cohort from France up to 5% in a recent cluster of cases imported from The Gambia[Malaria imported to Europe is seen in travellers returning from or migrants coming from endemic areas and migrants living in Europe returning from visiting friends and relatives (VFR). VFR children are particularly at risk. In 2010, WHO reports that 6,244 cases of malaria were imported in Europe down from 14,703 cases in 2000 show ince Gambia.The malaria programme in the WHO European Region, reported an eight-fold increase in the number of imported malaria cases between 1972 and 1988 , followed by a more gradual rise in 2000  with France, the UK, Germany and Italy accounting for more than 70% of all cases.More than five million African migrants may currently be living in Europe, one third of them originating from sub-Saharan Africa. The proMalaria infection among migrants, for example, asymptomatic individuals with sub-microscopic parasitaemia, could increase the risk of transmission leading to re-introduction of malaria in certain areas that have competent vectors and climatic conditions as seen in Greece in 2011. MoreoveP. ovale infections and mixed infections is very similar to the incidence found in West Africans[Migrants commonly believe they are immune to malaria, but theAfricans-29.Anopheles mosquitoes carrying malaria parasites are transported by aeroplane to a non-endemic area and take a blood meal from someone living close to the destination airport[Rare modes of transmission mean that patients with fever and without a travel history to endemic areas might need to be tested for malaria. These include so called “airport malaria” where  airport,31, and  airport. Malaria airport. Transmi airport and febr airport.The pathophysiology of malaria involves the cytoadherence of infected erythrocytes (discussed below) and consequent microvascular obstruction, as well as destruction of infected erythrocytes and the host’s response to the released substances. Chills and fever are associated with high levels of tumour necrosis factor released from monocytes stimulated by antigens released by bursting schizonts. Thrombocytopenia may be immune-mediated or due to activation of the coagulation cascade. AnaemiaP. falciparum-infected erythrocytes adhere to microvascular endothelium in a process known as sequestration, which allows them to avoid being removed in the spleen. This is favoured by other processes, such as a reduction of red cell deformability, rosetting of non-infected erythrocytes around the infected erythrocytes, platelet-mediated aggregation of infected erythrocytes[hrocytes-41. Sequhrocytes,43. Sequhrocytes. A studyhrocytes, that voP. falciparum become symptomatic within 30 days after return from the malaria endemic area, but longer incubation periods are seen with the other species and are prolonged by incomplete malaria chemoprophylaxis which may suppress parasitaemia without achieving full protection. A recent study from Portugal including 284 patients (46% non-immunes and 54% semi-immunes) found that the diagnosis was made between the day of return from the malarious area and up to 47 days later; a single non-immune patient was first diagnosed on the 120th day after leaving Angola[Most infections due to P. vivax and P. ovale, every 72 hours (quartan) for P. malariae and 24 hours (quotidian) for P. knowlesi. In P. falciparum malaria the fever usually lacks a regular pattern.Prodromal symptoms, which may precede the fever for up to two days are fatigue, loss of appetite, headache and body pains. In non-immune patients, malaria usually starts suddenly with a severe feeling of sickness and fever - often reaching 39°C and higher. Not allCommon symptoms are headache and myalgia. Other symptoms may include nausea, vomiting, dry cough, icterus, confusion and respiratory distress. Compromised circulation leads to renal failure and impaired tissue perfusion resulting in acidosis. Gastrointestinal complaints unrelated to treatment, including vomiting and diarrhoea are less frequent. Patients with significant fever paroxysms may initially have a normal temperature between the fevers and feel relatively well.Clinical examination is non-specific or even completely unremarkable. It often takes some days before anaemia or hepatosplenomegaly develop. Coryza, swelling of lymph nodes and eosinophilia are not seen in malaria.Malaria in adult migrants is characterized by a milder clinical presentation, lower levels of parasitaemia, shorter parasite clearance time after treatment and shorter fever duration compared to malaria in travellers due to previously acquired semi-immunity,47-49. CP. falciparum infection (detected by PCR) has been found among asymptomatic sub-Saharan African migrants after their migration to Europe[A high proportion of migrants have few symptoms and present long after arrival in the host country, with peo Europe,55,56.Diagnostic tests for malaria should be performed in any ill patient who has a history of exposure, i.e. patients with a history of travel to malaria-endemic areas, whether or not they are febrile at presentation.Pregnant women visiting endemic areas or arriving from areas of malaria transmission are at greater risk of clinical malaria during pregnancy than nonMost parameters are non-specific. Normochromic normocytic anaemia is observed in the majority of infected patients, but is rare in the first few days of illness. The most consistent finding is thrombocytopaenia and a study from the UK found that children with malaria and a platelet count of < 50,000 per ml had an odds ratio of 8.3 for admission to intensive care units. As the Although commonly unaffected except in case of a secondary infection , the leucocyte count may be raised or greatly diminished in very severe malaria cases and there may be slight monocytosis and lymphocytopenia. The C-reactive protein, procalcitonin, fibrinogen, orosomucoid and cytokine levels are raised in acute malaria. Thrombocytopaenia with a lower concentration of fibrinogen and elevated fibrin degradation products strongly suggest disseminated intravascular coagulation (DIC). Moderate hyponatraemia can be seen, but plasma potassium level is normal. BicarboBlood cultures should be obtained on admission as malaria infection can be complicated by septicaemia,65. All Microscopic examination of Giemsa-stained thin and thick blood films remains the gold standard because it is rapid, easy to perform and sensitive with a sEarly diagnosis is important to prevent uncomplicated malaria progressing to complicated disease. Patients with malaria should be managed in centres with the ability to quantify the parasitaemia . After the start of treatment, there is a lag-phase before the parasite density begins to decline and therFor falciparum malaria, many RDTs show 100% parasite detection score down to a parasite density level of 200 parasites per microlitre, equivalent to a parasitaemia of approximately 0.004%. PolymerP. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi based on the detection of pan-malarial antigens aldolase and LDH antigen[Plasmodium knowlesi infections will be detected by rapid tests which include the pan plasmodial aldolase or LDH antigens[RDTs are increasingly used in medical centres with limited access to experienced microscopists, however, a rapid test cannot determine the parasite density. False negative RDTs in patients with very high parasite densities have been described, probably due to the so-called “pro-zone” phenomenon,72. This antigen. Plasmodantigens,77. The antigens. CliniciP. falciparum infections acquired in Cambodia and the border regions of Thailand with Myanmar, where multi-drug resistant P. falciparum strains are highly prevalent.Treatment should provide rapid clinical and parasitological cure within three days. Oral ACT is the standard treatment of uncomplicated malaria as recommended by WHO,79 Tabl. CurrentArtemether/lumefantrine has to be administered with fatty food to obtain optimal plasma drug concentrations. DihydroAtovaquone-proguanil can be used as first-line treatment for uncomplicated malaria and needs to be administered with fatty food to increase bioavailability Table. AtovaquSecond-line anti-malarial treatments used when first-line anti-malarials are not available or excluded due to other reasons such as intolerance include mefloquine monotherapy for infections originating from regions without established mefloquine resistance . The use of mefloquine at treatment doses has been associated with a significantly higher incidence of serious neuropsychiatric adverse events. CombinaQuinine drug combinations have excellent efficacy, but tolerability is generally poor due to prolonged treatment courses and the occurrence of characteristic adverse effects (cinchonism). The WHOP. falciparum malaria because of widespread resistance. However, chloroquine remains effective in Haiti, Dominican Republic, Middle East and Central America north of the Panama Canal, and may be considered as an alternative treatment if ACT cannot be used.The use of chloroquine is not recommended for the treatment of severe malaria in non-immunes and 5% or more in patients from endemic areas[In the case of failed anti-malarial chemoprophylaxis, an anti-malarial drug different from the chemoprophylactic drug taken should be used for treatment. Finally, it is important to note that despite the fact that oral anti-malarials are recommended for the treatment of uncomplicated malaria, it is sometimes necessary for the responsible physician to use intravenous treatment with artesunate or quinine as recommended for the treatment of severe malaria. This decision may be taken based on evidence of important co-morbidities, intractable vomiting, or on clinical concern of the physician. The clinical criteria for severe malaria are shown in Tableic areas.P. falciparum malaria should, in general, be admitted to hospital, since monitoring of prognostic parameters including parasitaemia, treatment adherence and if needed, transfer to intensive care units, would normally be instantly available. Repeated monitoring of blood pressure, urinary output and oxygen saturation may be indicated. However, management as outpatients may be considered in uncomplicated cases in some healthcare systems where daily follow up until clearance of parasitaemia and fever and monitoring of treatment adherence can be undertaken. Persons migrating from malaria endemic regions may fall into this category.Patients suffering from P. falciparum, especially P. knowlesi. Plasmodium vivax can be severe in non-immunes[The clinical criteria for severe malaria are shown in Table-immunes. The cri-immunes.P. falciparum malaria is an emergency which may become rapidly fatal[Severe imported ly fatal. Intravely fatal and a rely fatal. IntraveIVA is superior to intravenous quinine (IVQ) in overall survival and safer and simpler to administer-89. IVA IVA produced under European Good Manufacturing Practice (GMP) standards is not yet available. Nevertheless, in some European countries (particularly in France), IVA manufactured in China  has been approved by the National Drug Agency and is now available with a specific temporary authorization of use (see section: «unlicensed drugs » below).IVQ is the drug of choice if IVA is not immediately available. An ECG should be obtained before starting IVQ. Use of a loading dose is recommended to rapidly obtain a therapeutic serum quinine level. The loaA full seven day course of IVQ is rarely completed. Quinine treatment can be changed either to an ACT or to an oral quinine-antibiotic combination as soon as the parasite density decreases and the patient tolerates oral treatment. The same principles apply for intravenous artesunate, where treatment is switched to oral ACT as soon as the parasite density has fallen adequately. In adults, doxycycline (or clindamycin during pregnancy) should be used in association with quinine if the quinine course cannot be followed by a course of ACT. Mefloquine should be avoided in patients with cerebral malaria even in the recovery phase. IVQ should be completed with a full course of ACT, atovaquone/proguanil or mefloquine.P. falciparum malaria there was no observed improvement in patient outcome or acid–base status with fluid loading. Neither CVP  nor PAoP (Pulmonary Artery occlusion Pressure) correlated with markers of end-organ perfusion or respiratory status[Amongst the various scoring systems for adult malaria patients requiring ICU treatment outside endemic areas, SAPS II and the WHO score appear to be most reliable,84,94. Fy status. In the y status.Acute renal impairment and failure is frequent and indications for acute dialysis do not differ from acute renal failure in other conditions. Hyponatraemia is often seen in falciparum malaria, caused by reduced kidney function and consecutive dilutional hyponatraemia or by an Anti Diuretic Hormone Syndrome in the case of euvolemia. In both cases, treatment is by fluid restriction. Precise monitoring of the fluid balance is essential.not be given, as they lead to prolongation of coma time and worsen the prognosis[In the case of cerebral malaria the usual supportive measures practiced in neurological intensive care medicine are recommended. However, corticosteroids as well as mannitol should rognosis. HypoglyThere is no consensus on the indications, benefits and dangers involved in exchange blood transfusion, so it should not be used. AutomatWHO guidelines recommend IVA in preference to quinine for the treatment of severe malaria in adults. At presSince 2011, the French National Health Agency (AFSSAPS), now named (ANSM) has temporarily authorized the import and use of IVA  via ACR-Pharmaceuticals, the Netherlands, granting it a temporary authorization of use. HoweverPaediatric dosages are provided in TableP. falciparum malaria in paediatric patients in Europe  are appropriate second line drugs. Tetracyclines are contra-indicated in children below 8 years of age (below 13 years in some countries).Quinine-clindamycin or mefloquine mono-therapy  is an important concern in young children. The use of paediatric drug formulations has been shown to improve the tolerability of antimalarials. To date, artemether/lumefantrine and dihydroartemisinin/piperaquine are useful first line paediatric anti-malarials. Artmether/lumefantrine is available as a dispersible tablet formulation (registered in Switzerland only). Atovaquone/proguanil is also available as paediatric tablets. Anti-malarial treatment of severe malaria in children follows similar algorithms as for adult patients and is based on prompt administration of intravenous artesunate (or quinine if artesunate is not available). Children presenting with malaria are likely to have high fever which increases the risk of vomiting and seizures. Fever should be reduced by tepid sponging and the use of an anti-pyretic such as paracetamol. which can be administered rectally, though paracetamol prolongs parasite clearance times in children.Bacterial infections including sepsis and meningitis are more common in paediatric patients compared to adults in malaria endemic regions, and blood culture should be obtained on admission. Invasive non-typhoidal salmonella infections are among the most common invasive bacterial pathogens in some regions of Africa where malaria is highly endemic.Congenital transmission of malaria to the newborn is a rare event and is estimated to occur in only 1% of newborns delivered by mothers with malaria. Clinical disease in the infant usually develops 2–8 weeks postpartum and includes non-specific symptoms such as fever, vomiting, diarrhoea, and poor feeding. To date, little information is available for the appropriate treatment of cases of congenital malaria and in patients of less than 5 kg bodyweight. Therefore, weight adjusted treatment with artemether/lumefantrine, dihydroartemisinin/piperaquine and atovaquone/proguanil may be considered for this indication.Pregnant women are at increased risk for malaria related morbidity and mortality. This increased risk extends to the post-partum period. PregnanPregnant women presenting with malaria in the first trimester should be treated with quinine and clindamycin for 7 days. Artesunate and clindamycin for seven days can be used if there is treatment failure with the quinine/clindamycin combination Table66. CurrenPlasmodium ovale and P. malariae generally remain sensitive to chloroquine in all endemic areas, despite reports of delayed parasite clearance time[Plasmodium vivax sensitivity to chloroquine has declined steadily in Indonesia, Peru and Oceania[P. vivax[P. falciparum with this parasite or with P. ovale or P. malariae[P.vivax with a treatment success of 100%[P. vivax[P. vivax, but it is not an ideal treatment because of low tolerability and it may lead to early relapses[nce time. Plasmod Oceania and a pa[P. vivax and it m malariae. Mefloqu of 100%. Monothe[P. vivax. Monotherelapses. First-lPlasmodium vivax and P. ovale infections, but not P. malariae, require treatment with primaquine (PQ) for 14 days to eradicate liver hypnozoites and thus prevent relapses. Plasmodium vivax strains with reduced susceptibility to primaquine are found in southern regions of Oceania and South-East Asia and require a higher dose of primaquine  for 14 days to prevent relapses[P. vivax and P. ovale after G6PD testing according to the CDC[P. vivax. Primaquine should be administered concomitantly with the blood schizonticide or as soon as possible after treatment.relapses. The CDCrelapses, and thi the CDC. Other cPrimaquine is contraindicated in patients with deficiency of the enzyme glucose-6-phosphate dehydrogenase. In patiP. knowlesi, a well-know primate malaria species that has recently recognized as causing a significant number of human malaria cases in South-East Asia and especially Malaysian Borneo, is not standardized, and WHO has not yet provided recommendations[P.falciparum, P. knowlesi may cause severe cases, with fatality rates as high as 27%, especially in older or female patients. Uncomplicated P. knowlesi cases can be treated with ACT, chloroquine, quinine, or atovaquone/proguanil[P. knowlesi, and they have not been described in animal models[P. knowlesi cases, the use of IVA was associated with a lower case-fatality rate (17% vs 31%) and lower median parasite clearance time (2 days vs 4 days) than IVQ[P. knowlesi should be treated with chloroquine or an ACT drug and complicated P. knowlesi with IVA.Treatment of ndations. Evidencroguanil. Mefloqul models,123. A rthan IVQ. Thus unThe effect of food on the absorption and pharmacokinetics of anti-malarials is important Table. In geneThe most prominent drug/drug interaction is the effect on QTc-prolongation with quinine, artemether/lumefantrine as well as with mefloquine. The effect of the individual drugs can be enhanced if they are used together, consecutively, or at the same time as other cardioactive drugs, such as ketoconazole or phenothiazines. Quinine and atovaquone/proguanil can enhance the effect of anticoagulants. Concomitant treatment with rifampicin, metoclopramide or tetracycline can result in a lower plasma concentration of atovaquone/proguanil.Treatment of HIV/malaria co-infected patients has potential for drug interactions Table. A recenAll hospitals in Europe should have plans for diagnosing and managing patients with malaria. Obtaining a travel history is mandatory for all patients with fever. If RDTs indicate the diagnosis of malaria but microscopy cannot be performed adequate treatment should be started straight away and the patient promptly transferred to a health care facility where microscopy can be done. In the case of a negative RDT but high suspicion of malaria, patients should also be transferred to a centre with expertise in the microscopic diagnosis of malaria. ACT should be available for treatment of uncomplicated malaria and intravenous artesunate available for severe or complicated malaria. Intravenous quinine should be available if it is not possible to maintain a stock of intravenous artesunate.A standing committee is proposed to harmonize guidelines as far as possible, whilst recognizing the need in some areas for individual/country-specific decision-making. The main recommendations for non-specialist physicians are summarized on TableGerd Burchard has received speakers' honoraria from GlaxoSmithKline, Novartis Pharma and Sigma-Tau. Peter L. Chiodini has received speakers' honoraria from GlaxoSmithKline. Patricia Schlagenhauf has received speakers' honoraria and research funds from GlaxoSmithKline, speakers honoraria,research funds and consultancy fees from F. Hoffmann-La Roche and she is a member of the sigma-tau advisory board. Eskild Petersen has received speakers a honoraria from GlaxoSmithKline.EP coordinated the project and manuscript, all authors contributed sections to the manuscript and all authors read and approved the final manuscript.


Haemoglobinuria is one of the manifestations of severe malaria and results from severe intravascular haemolysis. Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been implicated in its aetiology. Haemoglobinuria may be associated with severe anaemia and, less frequently, acute renal failure.A prospective case-control study was carried out to determine the incidence of haemoglobinuria as confirmed by dipstick urinalysis, microscopy and spectrophotometric measurement, among children with severe malaria. A total of 251 children presenting at the Children’s Emergency Ward with severe malaria were recruited over a period of 21 months. The G6PD status and the outcomes of severe malaria in children with and without haemoglobinuria was studied with respect to renal failure, the recurrence of haemoglobinuria and blood pressure changes over a three-month follow-up period.It was found that the incidence of haemoglobinuria among children with severe malaria is 19.1%. Children <5 years constituted 76.8% of all the study patients. Patients with haemoglobinuria had median age of 52.5 months, which was significantly higher than 35 months in patients without haemoglobinuria (p=0.001). Although, haemaglobinuria was commoner among boys (54.2%) than girls (45.8%), the difference was not statistically significant. There were no significant differences between children with and without haemoglobinuria regarding their nutritional status or parasite densities. Among the clinical features of the study patients, only jaundice was significantly associated with haemoglobinuria (p=0.0001). Renal failure occurred in three out of 48 children with haemoglobinuria and in none of the 203 without. There was not recurrence of haemoglobinuria in the follow-up period. At discharge, blood pressure was elevated in six children (one previously haemoglobinuric), but all returned to normal within the follow-up period.Haemoglobinuria was a prominent feature of severe malaria and it was significantly associated with jaundice at presentation. Haemoglobinuria was commoner in older children than younger children but not related to sex. G6PD deficiency was not an independent predictor of the occurrence or outcome of haemoglobinuria. Blood pressure was not affected by haemoglobinuria on admission nor during follow-up. Plasmodium falciparum accounts for 96-98% of malaria infections, while other infections are due to Plasmodium malariae[It is estimated that 300 million clinical cases of malaria occur each year in malaria-endemic regions. Most of the deaths from malaria are due to the severe forms of the disease, especially cerebral malaria and severe malarial anaemia. Recent  malariae.falciparum malaria may manifest with wide variety of features as described by WHO haemoglobinuria being one of them[Malarial illnesses may take a variety of clinical forms, differing in pattern and severity, from uncomplicated to severe and complicated malaria. Severe  of them. HaemoglRetrospective studies of malaria have reported haemoglobinuria incidence in adults ranging from 15% to 63%,10. SomeP. falciparum-endemic areas who took quinine irregularly or glucose-6-phosphate dehydrogenase (G6PD) deficiency have been implicated in the aetiology of haemoglobinuria. Haemoglobinuria can also occur in patients with normal erythrocyte G6PD levels who receive quinine for severe malaria[Several factors such as immune hypersensitivity in long-term residents of  malaria,20. Howe malaria.The clinical features peculiar to malaria-induced haemoglobinuria are mainly features of intravascular haemolysis and these features may be associated with anaemia. They generally include fever, abdominal pain, back pain or pain in the limbs, dyspnoea, tachycardia, a non-specific complaint of generalized malaise or dizziness,22. ShocHaemoglobinuria was previously thought to be innocuous and self-limiting, usually resolving within three days of onset. However, studies have shown that haemogobinuria may result in acute renal failure. Krishna and Karnad in a proThe prevalence of haemoglobinuria, its clinical profile, the associated frequency of G6PD deficiency and its effects on the outcome of severe malaria among Nigerian children, are unknown. In the present study, haemoglobinuria among children with severe malaria was prospectively investigated using a quantitative spectrophotometric method as distiAll study participants were recruited under the auspices of the Childhood Malaria Research Group (CMRG) at the 600-bed tertiary hospital, University College Hospital (UCH) in the city of Ibadan, Nigeria in west sub-Saharan Africa. Ibadan is the capital city of Oyo State in the South-West of Nigeria. It has a population of 2,550,293 people and annual growth rate of 2.83% (2006 census). The popThis study was carried out at the Otunba Tunwase Children Emergency Ward (OTCHEW) of the Department of Paediatrics of the University College Hospital, Ibadan. The OTCHEW is one of the six wards in the Department of Paediatrics and it has thirty-five bed-spaces for admission. The number of admissions per year is approximately 2,000. About twice that number are treated there without needing admission.P. falciparum parasitaemia with normal cerebrospinal fluid. A Blantyre coma score less or equal to 2 was used to define coma status. Severe malarial anemia (SMA) cases were defined as conscious children with packed cell volume (PCV) less than 16% in the presence of P. falciparum parasitaemia. Other forms of severe malaria (OFSM) were defined according to WHO criteria[This was a prospective case control study. After obtaining informed consent, children were recruited between the ages of six months and twelve years using WHO severe malaria criteria. Cerebracriteria and requcriteria.The cases were defined as children with severe malaria presenting with haemoglobinuria. Age- and sex-matched severe malaria patients without haemoglbinuria served as controls. Age matching criteria were +/− three months for six to 24 months and +/− six months for older children. Children were excluded from the study if they had been transfused within three months before presentation, or there was history of severe trauma or burns within the preceding month or they had a haemoglobinopathy or they presented with haematuria.Peripheral blood films, thick and thin, for microscopic examination for malaria parasites were carried out on admission. About 5ml of venous blood was withdrawn from each study participant into EDTA tubes for haemoglobin electrophoresis and G6PD screening. The blood pressure of each patient presenting with severe malaria was measured on admission.Dipstick urinalysis was carried out in all enrolled children. Urine samples that tested positive for blood were divided into two aliquots. Urine microscopy was carried out on one aliquot to exclude haematuria while the second was preserved at −20°C to measure haemoblogin concentration. Urine racks containing aliquots from each time the child passed urine, were kept for all patients with dark coloured urine to monitor the clearing of the urine.nd, 4th, 8th and 12th weeks after recruitment. At each follow-up clinic visit, children were screened for malaria parasites and their clinical status, including blood pressure measurements, was documented. Urinalysis was repeated for both the cases and the controls. Those who tested positive for blood had urine microscopy repeated to exclude haematuria.Children with haemoglobinuria and their matched controls were followed up during the 2About 10ml of urine was collected and subjected to urinalysis immediately. Otherwise, it was stored in the fridge at 4°C and the urinalysis done within 24 h. Multistix strip (DIALAB Ref G04010C) was compEach child’s caregiver was interviewed using a structured project questionnaire. History was obtained from the caregiver and, if old enough, the child. A complete physical examination was carried out on each patient. All findings from physical examination and other relevant information including the results of G6PD test, PCV, parasite counts, haemoglobin electrophoresis were recorded into the structured project questionnaire.PCV was determined using the capillary method and spinning in a Hawksley micro-haematocrit centrifuge at 11,000g for 5 min at room temperature. The packed cell volume was then read with a haematocrit reader.Thick and thin films were made on the same slide for each patient. The thin film was fixed with methanol immediately and the slide was allowed to dry, then flooded with Giemsa already diluted 1 in 10 with phosphate buffer, pH 7.2. The stain was washed off with distilled water, after allowing it to stand for about 10 min, and then air-dried. The stained films were then examined microscopically for malaria parasites using a x100 objective (oil immersion) and x10 eye piece lenses i.e. total magnification of x1,000. The parasites were counted against 200 white blood cells (WBC) and parasite density was calculated for each patient based on an assumed total WBC of 8000/μL of blood.G6PD status of recruited children was determined using the fluorescent spot test method of Beutler and Mitchell.t-test. Data not normally distributed were compared using the Mann-Whitney U-test. Continuous variable estimates were expressed as mean (±SD) or median (inter-quartile range) while categorical variables were in proportions, ratios and percentages. Statistical significance level was set at p<0.05.Data was entered into a microcomputer and analyzed using the Epi-Info Version 6 and SPSS 11.0 for windows. Categorical variables were compared using either the uncorrected chi-square test or Fisher’s exact test while continuous variables were analyzed using the Student’s A total of 265 children with severe malaria were screened over a period of 21 months (January 2006 to September 2007) and 251 eligible children were recruited into this study. These comprise 102 (40.6%) females and 149 (59.4%) males giving a female to male ratio of 1:1.5 . These consisted of 6 12.5%) of the 48 patients with haemoglobinuria compared with 14 (6.9%) of the 203 patients without haemoglobinuria . Three samples were lost during storage.Out of the 42 patients with haemoglobinuria who survived 19 (45%), 29 (69%), 13 (31%) and 27 (64%) were followed up at two weeks, four weeks, eight weeks and 12 weeks respectively. For those without haemoglobinuria 24 (12.6%), 39 (20.6%), nine (4.7%) and 44 (23.2%) were followed up at 2, 4, 8 and 12 weeks respectively. The follow-up at three months involved 15 males (55.6%) and 12 (44.4%) females with haemoglobinuria and 30 (68.2%) males and 14 (31.8%) without haemoglobinuria. Two of the patients with haemoglobinuria had recurrent haemoglobinuria at one month while haemoglobinuria never occurred in the group of patients without initial haemoglobinuria.The blood pressures of the study patients on admission and during follow-up are shown in Table The present study has shown that the incidence of haemoglobinuria among children with severe malaria is 19.1%. This value is higher than the previously reported range of 2% to 17%,17,18 buThere was a significant difference in the age-distribution of patients with or without haemoglobinuria. The proportion of children above five years with haemoglobinuria was higher than those without haemoglobinuria. This finding suggests that haemoglobinuria may be commoner in children above five years. A previous prospective study reported a higher incidence of haemoglobinuria among those more than four years old. HoweverThe proportion of boys in our study was higher than girls, a finding that is consistent with previous studies from the same centre,29. ThouThere was no significant difference in the nutritional status of patients with or without haemoglobinuria. The weight-for-age z-score was consistent with previous findings that severe malaria generally occurring in children whose nutritional is within normal limits,30.Almost half of the study subjects were SMA patients, similar to what has been reported in studies from Yemen, Sudan, and UgaJaundice was significantly associated with haemoglobinuria, which is in accordance with previous studies on blackwater fever that have reported jaundice and pallor as major manifestations,20,33,34Occurrence of haemoglobinuria has been previously linked with parasitaemia. While some researchers have associated blackwater fever with scanty or no parasitaemia,20, otheThe frequency of G6PD deficiency among severe malaria patients in the present study was 9.2%. This was close to the frequency of 6% reported from the same centre in the 1960’s. The frePrevious studies have implicated the repeated exposure to quinine in the aetiology of Blackwater fever,20,34. WThe overall mortality rate of severe malaria from this study was 8% with CM accounting for majority of the cases; this finding is consistent with previous studies. HaemoglP. falciparum is known to exhibit cytoadherrence in deep vascular tissues, it is tempting to speculate that the ischaemia consequent upon cytoadherence in the renal vasculature stimulates the rennin-angiotensin mechanism, which raises the blood pressure. The rapid resolution of the rise in blood pressure is consistent with the idea of a rapid reversal of the renal vascular cytoadherence and ischaemia following the clearance of the parasitaemia.The urine of all the patients with haemoglobinuria became visually clear over a period of three days. Comparison of the blood pressure of patients with or without haemoglobinuria showed no significant difference on admission and during follow-up. Even the three patients who had renal failure had normal blood pressure on admission. Of these, the two survivors had normal blood pressure throughout the follow-up period. In all six children (one in the group with haemoglobinuria) who had mild elevation of blood pressure in the acute phase, the blood pressure returned to normal within the follow-up period. This indicates that blood pressure elevation in severe falciparum malaria is transient. However, our period of observation (follow-up) was limited to three months. The possibility of subsequent blood pressure elevation during longer follow-up cannot be excluded. The mechanism of this blood pressure elevation is not clear. The proportions involved are small. But if anything, it is slightly lower in the haemoglobinuric group (1/48 ie 2.1%) than the group without haemoglobinuria (5/203 ie 2.5%). This suggests that direct toxicity of free haemoglobin on renal tissues is not likely to be a major factor. Since The authors declare that they have no competing interests.DF-R, OS and WAA conceived, designed, deployed and directed the case-controlled study at the Childhood Malaria Research Group, Ibadan, Nigeria. WAA, BJB, AEO, NKA, SO, KO, FOA, WAS, OS and DF-R carried out patient recruitment and follow-up, sample collection, storage and transport. WAA, FAO, JIA carried out haemoglobinuria tests. WAA, BJB, AEO, OS and DF-R analyzed data. DF-R, OS and WAA produced figures and tables and wrote the manuscript. All authors read and approved the final manuscript.


The polyomavirus family is rapidly expanding with twelve new human viruses identified since 2007. A significant number of the new human polyomaviruses (HPyV) has been found on the skin. Whether these viruses share biological properties and should be grouped together is unknown. Here we investigated the serological behavior of cutaneous HPyVs in a general population. 799 sera from immunocompetent Australian individuals aged between 0–87 were analyzed with a Luminex xMAP technology-based immunoassay for the presence of VP1-directed IgG antibodies against MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, and BKPyV as a control. Except for HPyV9, overall seropositivity was high for the cutanous polyomaviruses (66–81% in adults), and gradually increased with age. Children below 6 months displayed seropositivity rates comparable to the adults, indicative of maternal antibodies. TSPyV seroreactivity levels strongly increased after age 2 and waned later in life comparable to BKPyV, whereas MCPyV, HPyV6 and HPyV7 seroreactivity remained rather stable throughout. Based on the identified serologic profiles, MCPyV seems to cluster with HPyV6 and HPyV7, and TSPyV and HPyV9 by themselves. These profiles indicate heterogeneity among cutaneous polyomaviruses and probably reflect differences in exposure and pathogenic behavior of these viruses. Polyomaviridae constitute a family of small DNA viruses that infect a variety of hosts. In recent years twelve new human polyomaviruses (HPyVs) were identified, among which the KI and WU polyomaviruses Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses The A number of the new HPyVs has been found on the skin , with frequencies of 40–80% for MCPyV Two of the cutaneous HPyVs, MCPyV and TSPyV, are considered to be pathogenic, and have stong evidence of causality in Merkel cell carcinoma (MCC) and trichodysplasia spinulosa (TS), respectively. MCPyV is clonally integrated in 80% of MCC, a rare but aggressive neuroendocrine skin tumor most commonly found in elderly and immunosuppressed persons Seroepidemiologic studies of the BK (BKPyV) and JC polyomaviruses (JCPyV) have indicated that these viruses are ubiquitous and infect the general population early in life with an overall seroprevalence of 60–80%, respectively In this study we systematically determined and compared seropositivity and seroreactivity rates of the pathogenic cutaneous polyomaviruses MCPyV and TSPyV with those of HPyV6, HPyV7 and HPyV9 so far without attributable disease. To that purpose, we extended our previously described Luminex xMAP technology-based TSPyV VP1 polyomavirus seroassay For the purposes of this study, sera from a group of 799 immunocompetent Australian individuals were assessed. These samples were collected during October 2008 – June 2009 as part of a larger study investigating the seroprevalence of Q Fever in Queensland, Australia Additional sera (n = 110) collected for routine diagnostic testing were obtained from the public Toowoomba Hospital, which services the regional city of Toowoomba and the surrounding rural population. Sera from both hospitals were originally collected for a wide range of routine serological and biochemical testing. Clinical records were used, when available, to exclude immunocompromized patient sera from the candidate sample pool. A third sample group (n = 186) was obtained from children's sera submitted for routine allergy screening at a Brisbane-based private pathology clinic.Table S1). Appropriate approval for this research was obtained from the Queensland Children's Health Services Ethics Committee. Individual patient consent was not sought due to the retrospective nature of the study. The data were analyzed anonymously.The age range of the total study population was 0–87 years , with an overrepresentation of ages from 0–10 yrs. For sub-group analyses to determine seroprevalence by age, the total population was divided in 14 different age groups: 0–0.5 years (n = 31), 0.6–1.9 (n = 63), 2–3 (n = 62), 4–5 (n = 58), 6–7 (n = 58), 8–9 (n = 70), 10–14 (n = 55), 15–19 (n = 59), 20–29 (n = 59), 30–39 (n = 64), 40–49 (n = 54), 50–59 (n = 58), 60–69 (n = 54), and >70 years of age (n = 54)  5′- GGATCCGGATCCCCCTGTCAAAGAAAAGGAAATGG-3′ and HPyV7 anti sense 5′-GTCGACGTCGACAATTCCTTCTTGTTGTCTGTG-3′. For PCR and cloning strategy details see van der Meijden et al. Escherichia coli strain as described In the polyomavirus multiplex serology methodology, Glutathione-S-Transferase (GST)-VP1.tag fusion proteins were used as antigen For the measurement of antibodies against the HPyVs described above, a multiplex antibody binding assay was performed (Luminex based xMAP technology). This assay is based on fluorescent beads chemically cross-linked to glutathione-casein (GC) to which, through affinity purification of 1 mg/ml crude bacterial GST-VP1.tag fusion protein lysate, the GST-VP1.tag fusion proteins could bind ). Cut-off values were calculated by the mean seroresponse for each virus of the serononresponders plus three times the standard deviation. While doing so, the following cut-off values were obtained: MCPyV, 321 MFI; HPyV6, 129 MFI; HPyV7, 604 MFI; TSPyV, 250 MFI; HPyV9, 317 MFI; BKPyV, 428 MFI.Immunoassay cut-off values were based on the group of immunocompetent children aged 7–24 months (n = 63) among the study population. A frequency distribution analysis with a bin width of 250 MFI was performed on the seroresponses of the cut-off population for each HPyV tested, to determine the seronegative population, which was defined by samples falling within bins with a frequency percentage above 10% (2) were calculated to determine the association between seroresponses against different polyomaviruses, as shown in Pearson correlation coefficients . Seroresponses were common for TSPyV, HPyV6 and HPyV7, intermediate for MCPyV, and low for HPyV9. The highest seroresponses were measured for BKPyV.To measure seroresponses against MCPyV, HPyV6, HPyV7, TSPyV, HPyV9 and BKPyV, the VP1 major capsid protein of each polyomavirus was expressed and coupled to specifically colored Luminex beads. Seroresponses against the VP1-coated antigenic beads were measured for each individual within the study population using the Luminex xMAP technology. The obtained serological measurements are shown separately for each polyomavirus in 2 = 0.181) . In Table S2).To determine the seroprevalences in our study population for each of the viruses, seropositivity was assessed by calculating the proportion of sera that displayed seroreactivity above the established cut-off value for each virus  seropositivity rates increased to 68% for MCPyV, 76% for HPyV6, 66% for HPyV7 and 81% for TSPyV. Adult seropositivity for HPyV9 remained low (24%). Only 4 adult samples (1.2%) were seronegative for any of the cutaneous HPyVs. Seropositivity for all of the tested polyomaviruses was observed in 56 individuals (7%) of which 43 (77%) were adults. and Table S2). This phenomenon was also observed for BKPyV, where a seropositivity of 90% was measured in the very young, in line with very high seropositivity rates in the higher age groups.Except for HPyV9, in the age group below 6 months of age, seropositivity was significantly higher than in the next age group and comparable to the levels measured in the adults (Table S3). This pattern mirrored to what was observed for BKPyV in the same analysis . Lower immunogenicity of the used MCPyV strain 344 might explain this observation, although it contains aspartic acid (D) and arginine (R) at positions 288 and 316, respectively, important for proper folding of the MCPyV VP1 protein and epitope recognition 2-value of 0.181 in our study, and a Spearman correlation coefficient of 0.433 in the French Minor cross-serorecognition was observed between HPyV6 and HPyV7 but this correlation was very poor, both in the overall comparison and in the competition experiments. Another recent study using VP1 VLP antigen also had some indication of cross-serorecognition of these viruses We cannot rule out the possibility of crossreactivity between the six HPyVs analyzed in this study and other polyomaviruses, especially ones with high VP1 homology The observed seropositivity rates were within the range of previously published studies The observed seropositivity patterns of MCPyV, HPyV6, HPyV7 and TSPyV indicate that primary infections commonly occur in children and young adults. The increase of seropositivity with age for most of the viruses tested is compatible with the model of continuing primary infections throughout adult life. High seropositivity rates were detected in the first 6 months of life for all viruses except HPyV9. The rapid decline to the lowest measured seropositivity rates for each virus in the consecutive (0.6–1.9 yrs) age group, strongly suggests the presence of maternal antibodies during infancy. Maternal antibodies against KIPyV and WUPyV in young infants have been previously reported by Nguyen et al. Differences were observed with respect to the intensity of virus-specific seroreactivities among seropositive individuals. Whereas MCPyV, HPyV6 and HPyV7 seroreactivity moderately increased with age, the seroreactivity for TSPyV rapidly increased, analogous to BKPyV seroreactivity, as reported also by others Table S1). No information was available on ethnicity, and specifically Australian Indigenous identity, so associations in this regard could not be investigated. Given the general demographics of the areas that were serviced by the three health centres, we expect the large majority of the study population to be Caucasian. Furthermore, we compared available clinical data of patients with either upper or lower quartile seroresponsiveness for each HPyV tested for conditions including upper or lower respiratory disease, gastroenteritis, organ dysfunction or reported cutaneous abnormalities, but no specific patterns or associations with disease were found.Further analysis of our serological data revealed no associations between seropositivity and sex (data not shown). The association between HPyV9 seroprevalence and men described by Nicol et al. Based on our data, three different serological profiles seem apparent for the cutaneous polyomaviruses. Profile 1 was exhibited by MCPyV, HPyV6 and HPyV7. It is characterized by a significant increase in seropositivity with age, while the intensity of the seroresponses remain rather stable. Profile 2 was observed for TSPyV. Comparable to Profile 1, Profile 2 is characterized by a significant increase in seropositivity with age, in this case combined with a steep seroreactivity increase during childhood that seems to wane later in life. Profile 3 observed for HPyV9 displayed low seropositivity throughout all ages, with occasionally high seroresponses.per se. The low prevalence of TSPyV on the skin of healthy individuals (<5%) The biological basis behind each polyomavirus' serological profile is unknown so far. In part they may reflect differences in virus exposure, porte d'entree and/or antigen presentation. Furthermore, the extent of infection within the host and immunogenicity of the virus possibly play a role. The close resemblance of Profile 2 (TSPyV) with that of BKPyV, for instance, could suggest that TSPyV causes a generalized primary infection with viremia. Consequently, this virus should not be considered a cutaneous pathogen Conversely, it is tempting to speculate that serological Profile 1  indicates infection without viremia, for example limited to the skin. In light of the many reports that revealed the presence of DNA from these viruses in several different  clinical samples, however, it is too early to draw any conclusions in this regard. The same is true for HPyV9 (Profile 3) that has been found both on skin and in blood To summarize, on the basis of observed seropositivity and seroreactivity rates three different serologic profiles were discerned for the polyomaviruses found on the skin. These profiles probably reflect differences in exposure and/or in host and pathogenic behavior of these polyomaviruses, and further study in this regard is needed. Until that information is available, prudence is called to group these new viruses together as cutaneotropic.Figure S1Frequency distribution of seroresponses. Presented are seroresponses of children aged 0.6–1.9 years (n = 63) for MCPyV, HPyV6, HPyV7, TSPyV, HPyV9 and BKPyV. Bins with a width of 250 MFI were used and samples falling within bins with a frequency percentage > 10% were included for the cut-off value calculation (red bars).(TIF)Click here for additional data file.Table S1Distribution of the serum samples over the three study centres of sample collection.(TIF)Click here for additional data file.Table S2Associations (odds ratios) between seropositivity and age.(TIF)Click here for additional data file.Table S3Median and mean seroreactivity among seropositive patients per age group.(TIF)Click here for additional data file.Dataset S1Dataset S1.(XLS)Click here for additional data file.


Intermittent preventive treatment (IPT) has recently been accepted as an important component of the malaria control strategy. Intermittent preventive treatment for children (IPTc) combined with timely treatment of malaria related febrile illness at home to reduce parasite prevalence and malaria morbidity in children aged between six and 60 months in a coastal community in Ghana. This paper reports persistence of reduced parasitaemia two years into the intervention. The baseline and year-one-evaluation findings were published earlier.The main objective in the second year was to demonstrate whether the two interventions would further reduce parasite prevalence and malaria-related febrile illness in the study population.This was an intervention study designed to compare baseline and evaluation findings without a control group. The study combined home-based delivery of intermittent preventive treatment for children (IPTc) aged 6 - 60 months and home treatment of suspected febrile malaria-related illness within 24 hours. All children aged 6 - 60 months received home-based delivery of intermittent preventive treatment using amodiaquine + artesunate, delivered at home by community assistants every four months (6 times in 24 months). Malaria parasite prevalence surveys were conducted before the first and after the third and sixth IPTc to the children. The evaluation surveys were done four months after the third and sixth IPTc was given.Parasite prevalence which reduced from 25% to 3.0% at year-one evaluation had reduced further from 3% to 1% at year-two-evaluation. At baseline, 13.8% of the children were febrile (axilary temperature of ≥37.5°C) compared to 2.2% at year-one-evaluation while 2.1% were febrile at year-two-evaluation.The year-two-evaluation result indicates that IPTc given three times in a year (every four months) combined with timely treatment of febrile malaria illness, is effective to reduce malaria parasite prevalence in children aged 6 to 60 months in the study community. This must give hope to malaria control programme managers in sub-Saharan Africa where the burden of the disease is most debilitating. Malaria is estimated to cause between 300 and 500 million clinical cases with about 700,000 to 1.6 million deaths every year. About 94% of these deaths are believed to occur in sub-Saharan Africa . In 2006Like elsewhere in Africa, malaria control in Ghana relies on early diagnosis and prompt treatment of suspected cases (fevers) and the home is where early recognition and, in most cases, prompt treatment is initiated -8. IPTc Malaria control through prompt and effective treatment remains a challenge, especially in areas where access to early effective treatment is a problem. It is in these areas where IPT, especially IPTc could become an important component of the malaria control strategy, since it has been demonstrated that IPTi given with childhood vaccinations reduced incidence of first episode of malaria and also reduced severe anaemia by more than 50% during the first year of life -13. It wThe Ghana Health Service had aimed at treating 55% of under-five febrile malaria cases within 24 hours of symptom onset by 2007, as opposed to the Abuja target of 60% by 2005, but three clear years after the expiration date of 2007, this target is yet to be achieved. This study was, therefore, designed to assess the potential of a strategy of IPTc delivered at home and timely treatment of febrile illness to reduce parasite prevalence and malaria morbidity in the children-under 5 years as well as contribute to achieving the target of getting 55% of children under five into treatment within 24 hours. The baseline and year-one-evaluation results were published in 2009 . This paThis paper is to further demonstrate the feasibility of controlling febrile malaria illness in the home through combined interventions of intermittent preventive treatment and timely febrile malaria illness management in children aged 6 - 60 months in southern Ghana .The study site was discussed in detail in the earlier publication . In brieThis was an intervention study designed to compare baseline and evaluation findings without a control group.IPTc was delivered every four months beginning in July 2007  and was evaluated in July, four months after the last round of IPTc and just before the fourth round of IPTc [Any child with febrile illness suspected to have malaria by the caretaker was reported to the community assistants who then evaluated and treated those who met the protocol criteria for suspected febrile malaria illness, which was a child with axillary temperature of ≥ 37.5°C; whose caretaker reported suspected febrile malaria together with one or more of the following; headache, hot body, weakness, loss of appetite, among others, with no other overtly observed or reported condition was treated with the full three-day course of artesunate and amodiaquine .During IPTc rounds and prevalence surveys, a simple questionnaire was administered to caretakers to record febrile illnesses in the seven days prior to those events. That is, caretakers whose children received IPTc at any round and those who participated in the prevalence surveys responded to the questions. Questions asked included: Has your child been well in the past seven days, including now? If no, what is/was wrong with him/her? What was done for the child when you found out that he/she was not well? During the prevalence surveys, however, additional questions were asked about bed net usage.This was an implementation research which aimed at enrolling every child aged six to 60 months living in the study area into the study with the consent of their caretakers, mostly parents. However, for the prevalence survey, a sample size of 174 was needed to determine malaria parasite prevalence estimated at 20% in the study population . At the ® photometer  at the field site [A diagnosis of malaria infection was made with light microscopy of thick and thin blood smears. The thick and thin blood smears were stained with 3% Giemsa for 30 minutes. Parasite density was determined by counting the number of asexual parasites per 200 white blood cells, and calculated per μL assuming a white blood cell count of 8,000 cells per μL. Sexual parasite count was done per 1,000 white blood cells. A smear was declared negative if examination of 100 thick-film fields did not reveal the presence of asexual parasites. Quality control was done by allowing a second microscopist to read a random sample of 10% of all slides to confirm the absence or presence of parasites. Haemoglobin concentration was determined from finger pricks using a portable automated HemocueThe Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana and the Ethical Review Committee of WHO reviewed and approved the study. Each mother/caretaker was informed of the objectives, methods, anticipated benefits and potential hazards of the study. Caretakers were also informed that they were at liberty to withdraw their children from the study at any time without penalty. They were assured that all information collected during this study would be kept confidential and that in any resulting publication it would not be possible to link the data to individuals in the study .This paper presents the two-year-evaluation of combined IPTc and timely treatment febrile illnesses . Most of the children received IPTc six times in 24 months .Plasmodium falciparum, the dominant species in Ghana. Other clinical and parasitological findings are presented in Table Malaria parasite prevalence at year-one evaluation was 3.0% (10 out of 357), which further dropped significantly to about 1.0% (3 out of 337) at year-two-evaluation, a significant reduction of over 60.0%, p < 0.001, Fisher exact test). This showed a point parasite prevalence reduction of over 90.0% in 24 months . There was a significant relationship between febrile status and parasitaemia at year-one-evaluation  but this was not the case at year-two evaluation. Two out of the three parasite positive cases receive IPTc four times and the remaining one received it twice. All the positive infections were In this study, anaemia was defined as haemoglobin <10 g/dl and trend analysis showed a significant reduction from baseline through to year-two evaluation (p = 0.003). Comparing the baseline level of 27.6% to year-two evaluation level of 15.7% showed about 43.1% reduction in anaemia cases in 24 months  a number of febrile illnesses were reported for the children within seven days prior to data collection Table . A trendIPTc was delivered to children aged 6 to 60 months in the study community six times . There have been marginal increases in the number of children at each subsequent treatment round until the fourth, where it dropped marginally from 433 (IPTc3) to 415 (IPTc4), and then increased to 419 (IPTc5) and 421 (IPTc6). The decrease was largely due to some children attaining the age of more than 60 months and were dropped out of the study. At the time of year-two-evaluation, majority of the children have received all six IPTc doses. A few were lost at various IPTc rounds due mainly to either the movement of these children out of the study community at one point or the other, or the entry points of participants, since enrolment was prospective and any child who attained the age of six month at any given time was enrolled. However, the number of dropouts at each round was too small for any meaningful comparative analysis to be done on them. During each IPTc round, a number of febrile illnesses were reported by caretakers within the past seven days prior to treatment and trend analysis showed significant reductions in reported fever as the number of IPTc rounds increases  and headache (0.021) reported as the IPTc rounds increased but not for chills and rigors, and vomiting Table .There was virtually no serious adverse event reported after drug administrations. However, some caretakers reported that their children were weak for about two to four hours after taking the medicines and these were; five (1.21%) IPTc1, four (0.95%) IPTc2, seven (1.65%) IPTc3, four (0.93%) IPTc4, five (1.13%) IPTc5 and 5 (1.11%) IPTc6. None of these cases warranted any medical intervention. A few caretakers also reported that their children did eat very well after taking the drugs but this could not be independently verified.As this was at year-one evaluation, no caretaker had lost a child in 12 months preceding the year-two evaluation, indicating that, no caretaker had lost a child in the twenty-four months of project implementation, except two infants who died at birth and this was attributed to the absence of a midwife at the clinic at the time of their birth.As expected, over 98% of the caretakers were mothers of the children. Caretakers  reported that they and their families slept under bed nets the previous night. Comparatively, insecticide-treated net usage has increased marginally from 60.0% at year-one evaluation to 63.0% at year-two evaluation. However, compared to baseline (38.5%), there was a significant increase of 63.6% in the use of treated bed net in 24 months.This paper presents the two-year-evaluation of intermittent preventive treatment for children (IPTc) combined with timely treatment at home for malaria control, targeting children aged 6 - 60 months old. The main finding at year-two-evaluation was a further reduction of about 66.6%  in malaria prevalence in the study population compared to about 88.0% reduction reported between baseline and year-one-evaluation  -12,15-18Anaemia in the children, defined as haemoglobin <10g/dl, further improved by 27.4% . Compared to baseline, this was over 65.0% reduction in all cause anaemia prevalence in the study population. This compares well with a recent randomized trial in Tanzania, which showed that IPT given to infants at the time of childhood immunization reduced the incidence of the first episode of malaria and anaemia by more than 50.0% during the first year of life ,12. HoweMalaria-related morbidity in the study population, expressed by the presence of fever and other malaria-related signs and symptoms reported, has reduced as captured either during prevalence surveys or IPTc administration. Timely treatment of febrile malaria cases in the community did not follow any pattern. However, it could be argued that the community assistants who were on hand to deliver effective treatment in a timely fashion have contributed to the marked reduction of parastaemia seen during evaluation surveys. A community randomized trial showed a slightly higher coverage in the community-based delivery arm compared to facility-based arm . This shet al [et al [Findings reported here present a challenge to the existing practice, especially in most sub-Saharan African countries where malaria diagnosis is mostly based on presumption without confirmation. As community interventions or access to treatment increases, this may lead to fewer malaria infections, which may pose the danger of over diagnosis and treatment with expensive drugs for people who do not need them . Controlet al , this mal [et al  also makThe use of IPTc combined with timely home treatment to control malaria was found to reduce malaria prevalence in children aged six to 60 months . AlthougAt this point, it may be recommended to reduce the frequency of treatment from every four months to every six months or providing treatment at the beginning of each peak season (two peak seasons in southern Ghana). Either way, the timing must be related to the major peak season in the study area. At certain point it should be necessary to treat the general population at least once in a year to reduce the circulating parasites in the population such that mosquitoes will have very little or no parasite to transmit. Findings reported in this paper indicates that, with consistent efforts, malaria could be put under control such that it may no longer be a major public health problem, especially in sub-Sahara Africa where it is a threat to millions of people. Also, the study shows that it is possible to train community assistants to deliver IPTc together with home management of malaria on timely basis with very little supervision. However, additional training must be provided to enable community assistance to use RDT in the community so as to move away from presumptive treatment as malaria incidence continues to go down in communities.One major limitation of the study was that rapid diagnostic test (RDT) was not used to confirm the presence of parasite before timely treatment was provided as a result of the ethical consideration not to allow field assistants to take blood from the children. Another weakness of the study is that there was no control group to compare the result so it was not possible to attribute all the reduction in parasite prevalence to the interventions. Also, the contribution of each of the intervention to the reduction of parasite prevalence in the study population was not measured. All these must be taken into consideration when designing s similar study in future.The authors declare that they have no competing interests.CKA was involved in the conception, design and implementation, data analysis and writing of this paper. KAK was involved in the design and writing of this paper. AS-K was involved in the implementation and writing of this paper. MGW was involved in the conception, design and writing of this paper. All the authors have read and approved the final version of the manuscript.


Anopheles gambiae are an additional cause for concern. In this study, the effect of the short residual life of bendiocarb insecticide and of children spending time outdoors at night, on malaria infection prevalence was examined.Malaria is endemic on Bioko Island, Equatorial Guinea, with year-round transmission. In 2004 an intensive malaria control strategy primarily based on indoor residual spraying (IRS) was launched. The limited residual life of IRS poses particular challenges in a setting with year-round transmission, such as Bioko. Recent reports of outdoor biting by Data from the 2011 annual malaria indicator survey and from standard WHO cone bioassays were used to examine the relationship between time since IRS, mosquito mortality and prevalence of infection in children. How often children spend time outside at night and the association of this behaviour with malaria infection were also examined.Prevalence of malaria infection in two to 14 year-olds in 2011 was 18.4%, 21.0% and 28.1% in communities with median time since IRS of three, four and five months respectively. After adjusting for confounders, each extra month since IRS corresponded to an odds ratio (OR) of 1.44 (95% CI 1.15–1.81) for infection prevalence in two to 14 year-olds. Mosquito mortality was 100%, 96%, 81% and 78%, at month 2, 3, 4 and 5 respectively after spraying. Only 4.1% of children spent time outside the night before the survey between the hours of 22.00 and 06.00 and those who did were not at a higher risk of infection . Sleeping under a mosquito net provided additive protection .The results demonstrate the epidemiological impact of reduced mosquito mortality with time since IRS. The study underscores that in settings of year-round transmission there is a compelling need for longer-lasting IRS insecticides, but that in the interim, high coverage of long-lasting insecticidal nets (LLINs) may ameliorate the loss of effect of current shorter-lasting IRS insecticides. Moreover, continued use of IRS and LLINs for indoor-oriented vector control is warranted given that there is no evidence that spending time outdoors at night increases infection prevalence in children. Unprecedented scaling up of indoor residual spraying (IRS) and long-lasting insecticidal nets (LLIN) has led to major successes in the fight against malaria in recent years, with some countries recording more than 50% reductions in the annual number of malaria cases over the past decade . DespiteWorld Health Organization (WHO) position statements of 2006 and 2007  supporteIRS implementation is based on the premise that the residual active ingredient of the insecticide remains at sufficient concentration on treated interior walls to kill mosquitoes throughout the malaria season, or until the following spray round. The relatively short residual lives of WHOPES-approved products  present Anopheles mosquitoes. Recent reports about outdoor biting behaviour observed in Anopheles gambiae raise questions about the future effectiveness of these indoor-oriented vector control methods [Anopheles gambiae s.s. using human landing collections. In view of this finding the potential impact of being outdoors at night on the effectiveness of indoor vector control strategies as measured by infection prevalence was examined in this study.The effectiveness of both IRS and LLINs is based on the assumption that most people are indoors or under an LLIN during the night-time, peak-biting hours of  methods ,8. A recAn. gambiae and Anopheles funestus respectively were recorded [kdr) gene in An. gambiae s.s. [On Bioko Island, Equatorial Guinea  malaria is endemic with continuous year-round transmission. Before 2004, annual entomological inoculation rates (EIR) of over 250 and 750 infectious bites per person per year by recorded . In 2004iae s.s.  led to aiae s.s. .In 2005, intermittent preventative treatment for pregnant women (IPTp), case management using artemisinin-based combination therapy (ACT), improvement in diagnosis through training in microscopy and rapid diagnostic tests (RDT) together with the training of health facility staff were introduced as additional measures.In 2007, 110,000 PermaNet 2.0  LLINs were distributed through a mass distribution campaign, assisting householders in hanging one net per sleeping area at an average of three nets per household. Initially very high levels of LLIN ownership and usage were achieved , but thiMalaria prevalence in children ages 2–15 years declined from 45% (95% CI 40%–51%) in 2004, before the introduction of intensive malaria control to 20% (95% CI 15%–26%) in 2011. Moderate to severe anaemia (Hg < 8 g/dL) dropped from 15% to 2% over the same period, and all cause under-five mortality fell from 152 per 1,000 births to 55 per 1,000 in the first four years post intervention . The 200et al.[Plasmodium falciparum using RDT . Children testing positive for parasitaemia, with haemoglobin <11 g/dL, or who were febrile, were referred to a local field clinic for appropriate treatment .Annual household malaria indicator surveys have been conducted on Bioko since 2004. The data used in this paper are from the survey conducted in 2011 and, for comparison, the 2004 baseline survey. Eighteen sentinel sites were designated on the island and houses were randomly sampled from each site, using lists constructed at each site by enumerating all households, using personal digital assistants (PDAs) equipped with global positioning systems (GPSs). The survey instrument was based on the malaria indicator survey developed by the Roll Back Malaria Monitoring and Evaluation Reference Group . In the et al. found thet al.,14-16. SData on insecticide residual efficacy were obtained from WHO standard cone bioassays  performeSome of the sentinel sites were large and contained several distinct settlements. Since prevalence of malaria infection and delivery of the interventions could vary between communities in the same sentinel site, the latitude and longitude of each household were used to group houses into separate clusters such that the distance between clusters was at least 1 km. These clusters were used for calculating the following cluster level variables: prevalence of infection, median reported time since last spray, and the proportion of children both sleeping under a net the previous night and living in a sprayed house. Clusters containing fewer than five houses were excluded from analysis. Clusters which were part of the city of Malabo were classified as urban, the others as rural. Household socio-economic status (SES) was calculated using the first principal component score based on variables related to household size, asset ownership, livestock ownership and household utilities. The SES scores were converted to quintiles for analysis.P. falciparum Apical Membrane Antigen-1 (AMA-1) by enzyme linked immunosorbent assay (ELISA), for calculating sero-positivity prevalence by age and for estimating the serological conversion rate (SCR) for each sentinel site [In the annual malaria indicator survey in 2008, filter paper blood samples were collected from 7,387 individuals of all age groups, for detection of antibodies to nel site . SCR hasnel site ,20. In tLogistic regression was used to estimate unadjusted odds ratios for malarial infection of children aged two to 14 years in relation to the following variables: the median reported time since spray for the cluster, whether the child spent time outside the house the previous night, whether the area was urban or rural, the quartile of SCR of the sentinel site, the quintile of SES of the household, the age of the child grouped as two to four, five to seven, eight to eleven and twelve to fourteen years old, and whether the child slept under a net the previous night.a priori as potential confounders. The child spending time outside the previous night and whether the area was urban or rural were not included in the model because they showed no evidence of association with malaria infection and did not confound the association between malaria infection and the exposures of interest.A second logistic regression was used to estimate adjusted odds ratios. Median reported time since spray for the cluster and whether the child slept under a net the previous night were included in the model as the exposures of interest; the SCR quartile of the sentinel site, the SES quintile of the household and the age of the child grouped as above were included Standard errors in both analyses were adjusted to account for the survey design by taking into account the between site variation in prevalence using the svy set of commands in Stata. The primary sampling unit (PSU) was set to be the cluster .To investigate the potential effect of combined vector control on infection prevalence, a linear regression was performed with infection prevalence of the clusters as the dependent variable and the percentage of children who both slept under a net the previous night and live in a sprayed house as an explanatory variable of interest and the SCR of the sentinel site as a second explanatory variable to control for the underlying site-specific transmission intensity. Clusters with fewer than 10 children were excluded from this analysis.All analysis was done using Stata version 12 .Ethics approval for the study was granted by the Equatorial Guinea Ministry of Health and Social Welfare and the London School of Hygiene and Tropical Medicine . Informed written consent was given by each participant or, in the case of children, a responsible adult. In the case of participants being unable to read the text was read, and explained to them and consent was confirmed by an independent witness identified on the consent form.P. falciparum malaria infection in the 2011 survey. Of these records, 1,428 were excluded from the analysis because the house did not have its co-ordinates recorded and could therefore not be assigned to a cluster; a further 55 records were excluded because they fell in clusters with data on fewer than five houses. The analysis was carried out using the data of the remaining 3,909 children from 1,703 households. Overall prevalence of infection in this sample was 21.0% . TableA total of 5,422 children aged two to 14 years of age from 2,337 households were tested for TableThere was strong evidence from the 2011 survey that older children were more likely to be infected .Further evidence for the benefit of use of LLINS in addition to IRS on Bioko is given by a more substantial reduction in prevalence of infection relative to the transmission potential in sites where combined use of both IRS and LLINs is high, compared to places with lower coverage of both interventions combined Figure. This beAdding a third round of spraying to bridge the gap between spray rounds would be prohibitively costly, excessively demanding on the spray programme, and could lead to non-compliance with householders refusing to have their houses sprayed. On the other hand, ensuring the supply of LLINs in communities that receive IRS can remedy the shortcoming associated with short-lasting IRS insecticides, at least until longer-lasting insecticides of the appropriate insecticide class become readily available. Therefore, despite the high persistent coverage of IRS in Bioko, the national programme should give high priority to scaling up LLIN ownership through a new mass distribution campaign. In addition, the national programme should reinforce health promotion messages on the use of nets even when houses have been sprayed.Anopheles mosquitoes seeking bloodmeals indoors from human hosts during night hours, there may be behavioural selection for increased outdoor biting which could compromise conventional vector control efforts. Outdoor biting has been shown to take place in Bioko in human landing catch observations [In Bioko, as elsewhere, there is concern that with the intensification of vector control interventions that kill rvations . The resThe study shows that prevalence of infection is similar across socio-economic groups apart from the wealthiest quintile, who have substantially lower risks of infection. This indicates that the provision of malaria prevention measures are equitable, but that the highest socio-economic quintile is at lower risk, possibly due to better housing, better access to medical care and better health-seeking behaviour.Infection risk in children under 15 years was shown to increase with age in 2011  may mitigate the waning of protective effect conferred by current shorter-lasting IRS insecticides. Moreover, continued use of IRS and LLINs is warranted in the absence of evidence that spending time outdoors at night increases infection prevalence in children.ACT, Artemisinin-based combination therapy; AMA, Apical membrane antigen-1; BIMCP, Bioko island malaria control project; CI, Confidence interval; EIR, Entomological inoculation rate; IPTp, Intermittent preventive treatment in pregnancy; GPS, Global positioning system; IRS, Indoor residual spraying; LLIN, Long-lasting insecticidal net; OR, Odds ratio; PDA, Personal digital assistant; RDP, Rapid diagnostic test; SCR, Serological conversion rate; SES, Socio-economic status; WHO, World Health Organization.The authors declare that they have no competing interests.Conceived and designed the experiments: IK, CS. Performed the experiments: AM, DV. Analysed the data: JB, IK. Wrote the paper: IK, JB, AM, CS, DV, FM, GN. All authors read and approved the final manuscript.