Malaria's global impact is expansive and includes the extremes of the healthcare system ranging from international travelers returning to nonendemic regions with tertiary referral medical care to residents in hyperendemic regions without access to medical care. Implementation of prompt and accurate diagnosis is needed to curb the expanding global impact of malaria associated with ever-increasing antimalarial drug resistance. Traditionally, malaria is diagnosed using clinical criteria and/or light microscopy even though both strategies are clearly inadequate in many healthcare settings. Hand held immunochromatographic rapid diagnostic tests (RDTs) have been recognized as an ideal alternative method for diagnosing malaria. Numerous malaria RDTs have been developed and are widely available; however, an assortment of issues related to these products have become apparent. This review provides a summary of RDT including effectiveness and strategies to select the ideal RDT in varying healthcare settings. According to the World Malaria Report released by the World Health Organization (WHO) in 2008, there were 247 million malaria cases among 3.3 billion people at risk in 2006 from 109 countries resulting in an estimated 881,000 deaths. These deaths were primarily in Africa (91%) and in children under 5 years of age (85%) , 3433, 3Malaria RDT employ lateral flow immunochromatographic technology similar to rapid pregnancy tests. In these assays, the clinical sample migrates as a liquid across the surface of a nitrocellulose membrane by capillary action , 33, 34.Plasmodium species or are conserved across all four of the human malaria parasites. Falciparum-specific monoclonals include histidine-rich protein-2 (HRP-2) and P. falciparum lactate dehydrogenase (pfLDH) [The malaria antigens currently used as diagnostic targets are either specific to a  (pfLDH) , 33. Tar (pfLDH) –38. P. falciparum-specific water-soluble protein, localized in the parasite cytoplasm and on the surface membrane of infected erythrocyte. It is present on protrusions, known as knobs, thought to account for sequestration of the trophozoites and schizonts in postcapillary venules. There is increasing concentration of HRP-2 as the parasite advances from ring stage to trophozoite, and it readily diffuses into the plasma [P. falciparum gametocytes. This possibly allows detection at lower parasitemias and at detectable levels 28 days after clinical presentation, well after resolution of symptoms and apparent clearance of parasites from patients [P. falciparum could be detected. HRP-2 is a e plasma , 40. HRPpatients –44. Therpatients , 46. An P. falciparum or P. vivax [P. ovale- or P. malariae-specific antigens have not been widely tested [P. falciparum after the clinical infection is cleared [Another antigen target to detect sexual and asexual stage malaria parasites is Plasmodium lactose dehydrogenase (pLDH), which is the final enzyme in the malaria parasite's glycolytic pathway. Monoclonal antibodies against pLDH can target all human malaria species or can specifically differentiate P. vivax . AldolasP. vivax , 48. Othy tested , 49, 50. cleared .P. falciparum when using RDT. However, data assessing the utility of pLDH and aldolase in nonfalciparum infections is limited.Numerous reviews have highlighted the rapid turnover of commercially available products and varying quality control issues in manufacture and product stability –29, 52. ® Malaria test kit is the only US FDA approved kit. It is based upon the HRP-2 and aldolase antigens . One large trial revealed an overall sensitivity of 82% [P. falciparum was 94% [P. falciparum and 83% for P. vivax. Venipuncture produced similar results to fingerstick for the detection of P. falciparum and P. vivax  and 50% for P. ovale (1 of 2 positive samples), although these numbers were too small to determine reliable sensitivity and specificity. Although the kit is not approved for diagnosing mixed P. falciparum and P. vivax infections, the sensitivity was 94%. No clear data exists for using RDT to detect a recently described species of malaria, P. knowlesi, that has been reported to infect humans [Currently the BinaxNOWy of 82% . The ove was 94% . Again,  was 94% . The finP. vivax . Accordiectively . The sent humans . ® Malaria test kit meets many of the needs required of an ideal malaria diagnostic platform; however, it has also a number of limitations including the FDA indications comment that this kit is only for use in laboratories that have or can acquire blood samples containing P. falciparum for use as a positive control. It is also approved for use in the evaluation of symptomatic patients, with negative results requiring confirmation by thick and thin smears. Therefore individual clinicians or patients themselves might not have rapid results to initiate immediate therapy. Other limitations include the kit's ability to detect viable and nonviable malaria organisms, including gametocytes and sequestered P. falciparum parasites. In some settings, such as pregnancy, this is possibly advantageous; however, this prohibits monitoring the level of parasitemia, which is often used in management decisions. Additionally this prevents the monitoring of therapeutic response as antigens persist after elimination of the parasite. Finally, positive rheumatoid factor has been associated with false positive results. Overall, many of these limitations also plague other RDT [Overall, the BinaxNOWther RDT , 27.P. falciparum malaria is associated with placental sequestration of parasites that can reduce the sensitivity of microscopic diagnosis. In this clinical scenario, the detection of HRP-2 might improve diagnostic capability as this antigen is recovered peripherally [P. falciparum has been made on convalescent serologic (day 14–21 after febrile illness) or post-mortem assessments, all supporting possible diagnosis after initiation of empiric therapy [It has been estimated that 16 million RDTs were delivered in 2006 of which 10.8 million were in Africa and 2.8 million in India . Among ppherally . Howeverpherally –59. The  therapy , 60.Other possible indications for malaria RDT could include malaria prevalence surveys; however, they are insensitive for use in asymptomatic screening and high throughput detection cannot be achieved with individually packaged RDT , 61–64. P. falciparum. It is unclear if the risk of not treating false-negative tests outweighs the benefits of empiric therapy. Selection of specific malaria RDT is based upon region specific criteria including the expected health benefit, implementation plans, monitoring process, and cost with a focus on expected species of infection, level of parasitemia, and treatment paradigms. Parasitological confirmation of the diagnosis of malaria is recommended in all cases except for children under 5 years of age residing in areas of high prevalence of P. falciparum only or where nonfalciparum species occur as coinfections with P. falciparum. The HRP-2-based kits are probably best in this region because of overall improved antigen detection for P. falciparum. Zone 2 occurs in endemic areas of Asia, the Americas, and in isolated areas in Africa specifically the Ethiopian highlands, where falciparum and nonfalciparum malaria typically cocirculate. RDT in these regions will need to distinguish between falciparum and nonfalciparum infections. Zone 3 contains areas with nonfalciparum malaria only; including the P. vivax areas of East Asia and Central America. Here, RDT should focus on P. vivax-specific or pan-Plasmodium specific antigen detection without a need to detect or differentiate falciparum. Even in P. falciparum predominate regions, it is possible for 1–10% of patients to be coinfected including cases requiring anti-relapse therapy with primaquine [The WHO has typically outlined 3 broad zones for selecting devices. Zone 1 occurs primarily in Sub-Saharan Africa and in lowland Papua New Guinea where infections occur with imaquine . www.unwto.org/index.php, accessed 24 January 2009). These travelers might require management of their malaria infection while abroad or upon returning home; however, laboratory personnel in nonendemic regions often lack experience or expertise in microscopic diagnosis of malaria [Although the major burden of malaria is in endemic countries, malarious regions are frequent destinations of the roughly 900 million yearly international travelers ( malaria . Based u malaria , 67. HowPlasmodium, and infections at low concentrations of parasites, along with an inability to monitor response to therapy. In addition, in the case of a negative result, microscopy is still recommended. Therefore RDTs do not eliminate the need to obtain thick and thin smears, and maintaining expertise in microscopy is still a global priority until a new gold-standard is developed. However, malaria RDTs are ushering in a new era of diagnosis to improve the overall global healthcare system. Malaria is a life-threatening infection impacting the most developed countries of the world along with regions of the world lacking basic healthcare infrastructure. Increasing burden of disease, emerging antimalarial drug resistance, and broad implementation of ACT are placing greater emphasis on rapid and accurate diagnosis of patients infected with malaria. Given the difficulty performing microscopy, especially in endemic areas, alternative diagnostic strategies are needed. A highly effective RDT could avert over 100.000 malaria related deaths and about 400 million unnecessary treatments . In addiThe opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the US Department of the Army, the US Department of Defense, or the US government. The authors are employees of the US government and this work was performed as part of their official duties. As such, there is no copyright to be transferred.


Over diagnosis of malaria contributes to improper treatment, wastage of drugs and resistance to the few available drugs. This paper attempts to estimate the rates of over diagnosis of malaria among children attending dispensaries in rural Tanzania and examines the potential cost implications of improving the quality of diagnosis.The magnitude of over diagnosis of malaria was estimated by comparing the proportion of outpatient attendees of all ages clinically diagnosed as malaria to the proportion of attendees having a positive malaria rapid diagnostic test over a two month period. Pattern of causes of illness observed in a <2 year old cohort of children over one year was compared to the pattern of causes of illness in <5 year old children recorded in the routine health care system during the same period. Drug and diagnostic costs were modelled using local and international prices. Over diagnosis of malaria by the routine outpatient care system compared to RDT confirmed cases of malaria was highest among <5 year old children in the low transmission site  followed by the ≥5 year age group in the lower transmission site (RR 14.0 95%CI 8.2–24.2). In the low transmission site the proportion of morbidity attributable to malaria was substantially lower in <2 year old cohort compared to children seen at routine care system. . A higher proportion of children were diagnosed with ARI in the <2 year old cohort compared to children seen at the routine care system . Using a RDT reduced overall drug and diagnostic costs by 10% in the high transmission site and by 15% in the low transmission site compared to total diagnostic and drug costs of treatment based on clinical judgment in routine health care system.The introduction of RDTs is likely to lead to financial savings. However, improving diagnosis to one disease may lead to over diagnosis of another illness. Quality improvement is complex but introducing RDTs for the diagnosis of malaria is a good start. Over diagnosis of malaria has been reported widely in both outpatient st line treatment for malaria. Microscopy or rapid diagnostic tests (RDTs) are used to confirm cases in health facilities that have these resources In Tanzania artemisinin-based combination therapy (ACT) is the 1There is a growing debate about the cost effectiveness of introducing RDTs in view of the adoption of expensive ACTs The study was conducted in a low and a moderate malaria transmission area in Tanzania. The low transmission site, Same, located in Kilimanjaro region  from January to December 2007 were collected from the routine Health Management Information System (HMIS) registers kept at each health facility.f, Span Diagnostics, Surat, India,). The RDT results were reported to the health facility's clinical staff for making treatment decision.This component of the study was done in July to August 2007 in the 8 study health facilities. The study objectives were explained to all outpatient attendees  requested to take part in the study. All patients attending for any illness and giving written consent were checked for malaria using an RDT (ParaHIT- Data on causes of morbidity in 2 months to 2 year-old children who were enrolled in the IPTi study (reported elsewhere The cost of treatment of morbidity pattern in <5 year old children based on the HMIS data of the year of 2007(data source (i) outlined above) was compared to that of the morbidity pattern observed in the IPTi study cohort (data source (iii) outlined above). The costing was undertaken from the perspective of the government health system and focused on the costs of diagnostic tools and drugs. Wider treatment costs to caretakers and providers associated with inpatient and outpatient care were not included in the analysis. Categories of cause of morbidity were based on those listed in the HMIS data and these were: malaria, acute respiratory infection, pneumonia, eye infection, ear infection, skin infections, urinary tract infection and ‘other’ . Treatment regimens and their associated costs were based on recommended first and second line treatment for a one year old infant weighing approximately 10kg, and the proportion of children likely to receive the various drugs was extrapolated from the prescribing practices of the study clinicians from the IPTi study cohort. For example, to determine the average unit cost of treating ARI, a weighted average was calculated to reflect the type and frequency of all the drugs given to all children identified and treated for ARI in the IPTi cohort. It was assumed that extrapolating the unit costs of treating illnesses based on the prescribing practices of the IPTi study clinicians would closely mirror the prescribing practice advocated by national treatment guidelines. Drug and diagnostic costs are modelled using local The base case costing scenario assumed that in the IPTi study cohort all suspected cases of malaria received an RDT at a unit cost of US$0.75 Ethical permission was granted by the Ethical Review Boards of the National Institute of Medical Research of Tanzania and the London School of Hygiene and Tropical Medicine.The percentage of outpatient attendees who were RDT positive and the percentage of attendees diagnosed as malaria in the HMIS data over the 2 month study period (July and August 2007) were compared in <5 and ≥5 year old age groups. The distribution of causes of morbidity in <5 year old children reported in the Routine Health care system data was compared to the distribution of causes of morbidity in the placebo group of the IPTi study cohort.Potential cost savings by improving diagnosis were estimated by comparing the cost of treatment of causes of morbidity in <5 year old children reported by the Routine Health care system to the cost of treatment of morbidity observed in the placebo group of the IPTi study cohort. Results are presented as total cost to the public health care system (per 100 infants) and as ratio between IPTi cohort costs versus Routine health care system costs. Univariate sensitivity analysis was conducted on the main variables that were uncertain, prone to change over time and cost drivers. Specifically, the cost of the RDT was varied from US$1 to US$0.50, ACT costs from US$1 to US$0.50 and the rate of routine microscopy use from 100% to 0%.The comparison of the diagnosis of malaria confirmed by RDT and diagnosed clinically at the study health facilities are shown in In the low transmission site, the variation in the proportion of patients having a RDT confirmed malaria did not vary substantially between the health facilities; it ranged from 0 to 2% in <5 year old children and 1–2% in ≥5 year old age group. However, the clinical diagnosis of malaria varied dramatically from 0–44% in <5 year old children and 16–51% in ≥5 year age group.In the moderate transmission site, the variability in RDT confirmed and clinically diagnosed malaria between the health facilities was relatively low in both age groups except at Majengo dispensary which serves an urban population. At this urban health facility, the disparity between the RDT confirmed and clinically diagnoses malaria was high in <5 year old children (RR 5.6 95%CI 2.6–12.2) and in ≥5 years age group (RR 19.6 95%CI 8.9–43.2). The results from this urban dispensary are similar to those from the low transmission site.A comparison of the distribution of causes of morbidity in the <2 year old children enrolled in the IPTi study cohort diagnosed by study clinicians The same pattern was observed in the high transmission site. A higher proportion of children were diagnosed with malaria in the children seen in the routine care system compared to that in the IPTi cohort  and the proportion of children with a diagnosis of ARI was lower in the routine care compared to the IPTi cohort The unit costs of treating each causes of morbidity are shown in The costs of treating each category of morbidity per 100 children based on the proportional morbidity in the IPTi cohort and children treated at the routine care system is presented in In the moderate transmission site, there were 7% savings to 15% additional costs if the cost of ACT varied from US$1 to US$ 0.50 per child treated respectively. The use of RDTs to improve the accuracy of malaria diagnosis was only cost saving if ACTs cost more than US$ 0.63. In the low transmission site, if the cost of ACT varied from US$1 to US$ 0.50 per child treated, there was a 17% savings to 7% additional costs respectively. RDTs were only cost saving if ACTs were more than US$0.50.The frequency of use of microscopy in routine care system had a limited impact on cost savings. More importantly, however, even if rates of microscopy use varied from 100% to 0%, the use of RDTs in the IPTi cohort was always cost savings in both transmission settings when compared to the routine care system. In the moderate transmission setting, if microscopes were used 100% of the time to diagnose malaria in routine care then the RDT group showed 15% savings, if microscopes were never used, the IPTi cohort showed 4% cost savings compared to routine care system. In the low transmission settings the savings were between 19% (100% microscopy use in routine care system ) and 11% (no microscopy use).Over diagnosis of malaria occurs at high rates in both low and moderate transmission settings. It is interesting to note that although malaria is less common in the low transmission setting and the risk ratio (RR) of over diagnosis is high, the actual numbers of those wrongly diagnosed as having malaria is greater in the moderate transmission setting as malaria is more frequently diagnosed. This over diagnosis of malaria occurs largely at the expense of under diagnosing ARI and pneumonia. This study shows that in both study districts malaria is significantly over-diagnosed at the primary health care level and that this over-diagnosis is massive in low transmission areas as well as in small urban settings (townships) such as district capitals in moderate transmission areas. The high burden of malaria was observed mostly in health facilities which were located in rural areas, and this important difference has not been detected by the routine care system. Rural communities bear the brunt of malaria burden. However, due to growing townships in rural areas this population at risk of malaria is decreasing. The population in small townships is now living with the trappings of modern urban life of improved housing, access to health care, access to education, higher population density and decreased malaria transmission Over diagnosis is costly in physical and financial terms. The physical cost is in people who are ill due to a cause other than malaria and not getting the appropriate treatment leading to severe disease and mortality Our cost analysis suggests that there is a thresholds where the price of an infant dose of ACT no longer makes RDT use cost saving. In the low transmission setting this is US$ 0.50 and in the moderate transmission setting this is US$ 0.63. If, however, the cost of ACT was to remain at US$0.95, even if the cost of the RDT were to increase to US$1 RDTs would still be cost saving. Regardless of the level of microscopy use in routine practice, RDT use would always be cost saving in both transmission settings. This is likely to be due to the low quality of routine microscopy We have looked at the cost implications of using RDTs to better identify malaria episodes and in addition the costs associated with treating a range of non malaria illness episodes. The paper does not undertake a rigorous cost effectiveness analysis In southern Mozambique the use of RDTs in all suspected cases of malaria was shown to be cost-saving when parasite prevalence among clinically diagnosed malaria cases was low to moderate. However, the study also suggested that targeting RDTs at the group older than 5 years and treating children less than 5 years on the basis of clinical diagnosis was even more cost-saving The paper uses data from different sources and although we make direct comparisons, readers should consider the extrapolations that we have made. There are certain limitations when making comparisons between Routine Health care system and RDT survey data, as the Routine Health care system data contains the same people who are sampled in the RDT survey and the Routine Health care system data is known to be incomplete as not all attendees would have been registered. However, the Routine Health care system data is used for government statistics and its weaknesses have to be acknowledged and accepted The costing part has its limitations. Most notably, we do not consider the effect of variability in the specificity and sensitivity of RDTs on the cost savings. The sensitivity and specificity of RDTs is reported to be 90% and 95% respectively compared with expert slide reading In both the RDT survey and the IPTi study cohort There has been a decrease in malaria across the region which national statistics and clinical diagnoses have not reflected. In this paper we show that improving accuracy of diagnosis by using RDTs and with the benefit of continuous supervision by a research team, similar to continuous medical education (CME) widely adopted in developed countries, over diagnosis of malaria can be detected and appropriate treatments can be given to patients. RDT testing should be implemented nationally and a record of RDT positive diagnoses fed back to the National Malaria Control Program, where control activities could focus on the places where malaria is truly a problem. Health education about malaria and the alternative diagnoses needs to be directed at clinicians and in a more general way to the general population. The extent to which RDTs are cost saving will be associated with the frequency of their use. If the practice of over diagnosis of malaria is widespread, an initial over use of RDTs can be expected, which will carry additional costs. Overtime however, the frequency of true negative readings should go a long way to reassure health care providers and the general population that the prominence given to malaria is unfounded, and therefore RDT use in general should fall. Currently supervisory visits focus on preventive services especially EPI and more recently IMCI. We suggest that these visits should be broader and include supervision on curative services for all age groups and focus on improving diagnosis. This study sheds some light on what diagnoses health services should expect and suggests that respiratory tract disease and diarrhoea are still major causes of morbidity in Africa even in areas of high transmission of malaria.Malaria transmission differs enormously throughout Tanzania


Malaria remains a significant burden in sub-Saharan Africa. However, data on burden of congenital and neonatal malaria is scarce and contradictory, with some recent studies reporting a high burden. Using prospectively collected data on neonatal admissions to a rural district hospital in a region of stable malaria endemicity in Kenya, the prevalence of congenital and neonatal malaria was described.st January 2002 to 31st December 2009, admission and discharge information on all neonates admitted to Kilifi District Hospital was collected. At admission, blood was also drawn for routine investigations, which included a full blood count, blood culture and blood slide for malaria parasites.From 1Plasmodium falciparum malaria parasitaemia, of whom 11 were admitted within the first week of life and thus classified as congenital parasitaemia, were identified. 7/18 (39%) had fever. Parasite densities were low, ≤50 per μl in 14 cases. The presence of parasitaemia was associated with low haemoglobin (Hb) of <10 g/dl (χ2 10.9 P = 0.001). The case fatality rate of those with and without parasitaemia was similar. Plasmodium falciparum parasitaemia was identified as the cause of symptoms in four neonates.Of the 5,114 neonates admitted during the eight-year surveillance period, blood slide for malaria parasites was performed in 4,790 (93.7%). 18 (0.35%) neonates with Congenital and neonatal malaria are rare in this malaria endemic region. Performing a blood slide for malaria parasites among sick neonates in malaria endemic regions is advisable. This study does not support routine treatment with anti-malarial drugs among admitted neonates with or without fever even in a malaria endemic region. Malaria remains one of the most significant disease burdens in resource poor countries of sub-Saharan Africa especially among children under the age of five years and pregnant women -4. HowevMalaria contributes significantly to perinatal disease burden in terms of pregnancy losses, prematurity due to preterm labor and intra-uterine growth retardation -12. Despst January 2002 to 31st December 2009.Given the limitations of the published data, this study describes the prevalence and outcome of congenital and neonatal malaria in all neonates admitted to a rural district hospital in a malaria endemic region in Kenya over an eight-year period from 1Plasmodium falciparum parasite prevalence in children aged 2-10 years ranges from 1.3-25.9 [2 is continuously monitored and vital events updated every four to six months. The entire area was mapped using global positioning system (GPS) and both mapping and population data are linked on-line to the hospital admission data.The study was conducted at the Kenya Medical Research Institute (KEMRI) centre located at Kilifi District Hospital on the coast of Kenya, 60 kilometers north of Mombasa. More than seventy percent of neonates admitted to the hospital are born at home in a rural community where the entomological inoculation rate is between 0 and 120 infected bites per year, and the 1.3-25.9 ,22. This1.3-25.9 ,24. In aGovernment-employed clinicians in the outpatient department, working independently of the inpatient research team, refer children for admission. In addition, sick neonates are admitted directly from the maternity department. At admission and discharge/death, standardized clinical and laboratory data are prospectively collected by research clinicians and directly entered into a computer database. Consent for use of the data is obtained from the guardian of every child at the point of admission. The Kenya National Scientific and Ethical Review Board approved this study (SCC protocol 1592).Plasmodium species in the first seven days of life, and neonatal malaria as intra-erythrocytic asexual forms of Plasmodium species in the first 28 days of life in a sick neonate was used [A neonate was defined as any child admitted aged 28 days or younger . The diswas used . Any neowas used . Meningiwas used . This apAt admission, the research clinician performed emergency procedures such as correction of airway, breathing, circulation, hypothermia or hypoglycaemia before taking a formal admission history and physical examination. A venous blood sample was routinely drawn (according to weight but not exceeding 3 mls) for a full blood count, blood slide for malaria parasite examination, blood glucose, electrolytes and microbiological culture. As per World Health Organization (WHO) guidelines, meningitis was considered in all neonates presenting with any one of: drowsiness, lethargy, reduced feeding, high-pitched cry, irritability, apnoeic episodes, a bulging fontanel, unconsciousness or seizures and where not contraindicated, a lumbar puncture was performed . Researc® 11 .Admitting clinicians entered individual patient clinical data at point of contact using a FileMaker Pro database . Likewise, laboratory data was double entered and verified using FoxPro for windows . Pearson's chi square test (or Fisher's exact test as appropriate) was used to compare proportions. Analysis was performed using STATAP. falciparum malaria and no neonates with parasitaemia due to other plasmodial species were identified. Eleven of the eighteen were admitted within the first week of life and were thus classified as congenital parasitaemia. Parasite densities were low and in 14 of the cases were fewer than 50 per μl. Three neonates had parasite densities of 2,480, 11,600 and 22,560 per μl respectively and one neonate had 38/500(7.6%) red blood cells parasitized . All the cases were admitted between the years 2002 and 2005.During the study period, a total of 5,114  neonates were admitted to Kilifi District Hospital. Most (67%) of the neonates admitted were less than one week old. Blood film results for malaria parasites were available for 4,790 (93.7%) of all neonates: 3,187(92.6%) in the first week of life and 1,606 (96%) thereafter. 18 (0.35%) neonates with a positive blood slide for 2 0.049 P = 0.824). Overall the presence of parasitaemia was associated with an Hb of <10 g/dl (χ2 10.9 P = 0.001). Only one case presented with convulsions at admission. The prevalence of hypoglycaemia (blood glucose <2.6 mmols/l) at admission did not differ between the cases with parasitaemia and those without (χ2 0.26 p = 0.61)The summary of the clinical and laboratory characteristics for the 18 neonates with parasitaemia is as shown  drugs in addition to antimicrobial drugs for suspected sepsis. Of the remaining, there were seven diagnosed as neonatal sepsis, four cases of prematurity with severe respiratory distress, two cases of neonatal jaundice and possibly sepsis too, and one case of meningitis  of the neonates with parasitaemia died compared to 1,006 (19.7%) of those without parasitaemia (Fisher's exact P = 0.38). Four of the neonates with parasitaemia died within 24 hours of admissions, two possibly from overwhelming sepsis, one from a combination of severe jaundice and possible sepsis and the last from complications of prematurity with respiratory distress. The fifth died on the 10Information on the burden of malaria in early infancy, especially the neonatal period is scarce, inconclusive and contradictory, with a wide range of results reported ,15,16. IThe result of this study therefore suggests that the burden of neonatal malaria is much less than previously reported. Several single centre studies in Nigeria have reported a parasitaemia prevalence in excess of 20% among sick neonates admitted to different hospitals in the country, and in a sizeable proportion of them, symptomatology attributed to the parasitaemia ,18,20,31The marked differences in prevalence of congenital and neonatal parasitaemia are difficult to explain. Plausible explanations include real epidemiological differences and operational factors. In several recent studies, malaria slides read in routine service have been shown to have very poor positive predictive value when compared to expert microscopy ,35. In oIt is worth noting that all the cases of neonatal and congenital malaria were identified in the earlier years 2002-2005). It is plausible that this could partly be explained by the increased drive to combat malaria in the region through a combination of strategies such as increased coverage of insecticide treated mosquito nets, improving antenatal care and uptake of Intermittent preventive Treatment in pregnancy (IPTP), and finally change to a more effective anti-malarial drug that occurred during the decade . It is p. This fiA major limitation of this study is that not all neonates with low parasitaemia were treated with antimalaria drugs, although all survived except the ones who died from complications of prematurity or sepsis. It is however worth noting that studies have documented that most cases of such low parasitaemia clear spontaneously without treatment ,18,31. IThe results of this study suggest that it is worthwhile to perform blood films on hospitalized neonates with fever in malaria endemic regions since though it is rare, congenital and neonatal malaria does occur. Even where resources are extremely limited, investigation for malaria especially among febrile neonates not responding to standard antibiotics treatment should be undertaken. However, in many instances a positive slide may not explain the cause of illness. Neonates with a positive slide should therefore still receive antimicrobial drugs for sepsis as well as antimalarial drugs. The results however do not support routine treatment with anti-malarial drugs for admitted neonates with or without fever, in this malaria endemic region.The authors declare that they have no competing interests.MKM participated in study design, analyzed the data and drafted the manuscript. AT helped in data analysis and participated in manuscript writing. NM & JB & KM supervised clinical care and participated in manuscript writing. PK assisted in study design and manuscript preparation. CN conceived the study, helped in study design and manuscript writing. All authors read and approved the final manuscript.Table showing a summary of key studies describing malaria in the neonatal period.Click here for fileTable Summary of the cases with neonatal parasitaemia admitted to Kilifi District Hospital.Click here for file


Plasmodium falciparum malaria is a complex disease in which genetic and environmental factors influence susceptibility. IgG isotypes are in part genetically controlled, and GM/KM allotypes are believed to be involved in this control.In this study, 216 individuals from Daraweesh, an area of seasonal malaria transmission in Sudan, were followed for nine years for malaria infection. Total IgG and IgG isotypes against four malaria antigens, MSP2-3D7, MSP2-FC27, AMA1, and Pf332-C231 were measured in plasma obtained from the cohort at the end of the study, during the dry malaria-free period. The GM/KM allotypes of the donors were determined.The GM 1,17 5,13,14,6 phenotype was associated with a higher incidence of malaria compared with the non-1,17 5,13,14,6 phenotypes (P = 0.037). Paradoxically, the carriers of the GM 1,17 5,13,14,6 phenotype had significantly higher baseline levels of total IgG and non-cytophilic IgG isotypes as compared to non-carriers. The KM allotypes influence on IgG isotypes level was limited. Finally, the differences in the baseline concentrations of total IgG and IgG isotypes between the different GK/KM phenotype carriers were antigen-dependent.The results show that GM but not KM allotypes appeared to influence host susceptibility to uncomplicated malaria as well as the antibody profile of the donors, and the carriers of the GM 1,17 5,13,14,6 phenotype were the most susceptibleP. falciparum malaria and antigen-dependent influence on total IgG and IgG subclasses.The GM allotypes have significant influence on susceptibility to uncomplicated  Selection for resistance to malaria among inhabitants of malaria endemic regions may have influenced polymorphisms in genes encoding a variety of proteins involved in immunity . For exaThe association of GM/KM allotypes with susceptibility to several different diseases has been reported  and theiIn the current study, a hypothesis suggesting that the GM/KM make-up of individual immunoglobulin affects IgG isotype levels, depending on target malaria antigen, was examined. Consequently, the GM/KM allotypes might influence the host susceptibility to malaria. Therefore, ten GM  and 2 KM  allotypes were investigated and combined with nine years of longitudinal malaria incidence data collection. In addition, baseline antibody response to four leading asexual blood-stage malaria vaccine-candidate antigens, comprising the apical membrane antigen-1 (AMA-1), merozoite surface protein-2 , and Pf332-C231, was analysed to test this hypothesis. Results revealed that, development of protective immunity is not only attributed to repeated exposure with increasing age [The study was carried out in Daraweesh village, eastern Sudan, where malaria is hypoendemic with occasional quite severe 'malaria seasons'. The malaria transmission in the region is strictly seasonal but markedly unstable; in 'wet years' it peaks in October/November, after the summer rain, although a few sporadic cases also occur between February and August. In Daraweesh, malaria affects all age groups, although the incidence decreases after twenty years of age. A detailed geographical, demographic and social description of the area has previously been reported ,15.The inhabitants of Daraweesh are descendents of a founder population of the West African Fulani-speaking group, originally from Burkina Faso. The village founders migrated to the Sudan more than hundred years ago although the ethnic identity (and Fulani language) has been maintained by frequent inter-marriage between descendents of the small founding group and some marriage with other Sudanese Fulani immigrants. In year 2000, the total population was approximately 500 inhabitants and during the 13-year study period, it ranged between, 400 to 600 individuals. The plasma samples used in this study were collected in the dry season in May 2005. All donors were malaria-free, no malaria parasite was detected by microscopy or PCR in any of the blood samples.From 1991 to 2004, the inhabitants of Daraweesh were under regular clinical and parasitological malaria surveillance ,15; excePeripheral blood samples (3 ml) were collected from each donor into a vacuum EDTA tube. After centrifugation the plasma was collected into cryotubes and stored at -20°C until used. The study had received ethical clearance from the ethical committee at the Faculty of Medicine, University of Khartoum, and the Sudanese Federal Ministry of Health. Consent was obtained from the village members regarding the whole project and from the individual donors, when blood samples were collected.Escherichia coli and refolded in vitro. Both the 3D7 and FC27 forms of MSP 2 were expressed in E. coli with C-terminal hexa-His tags. The expression and purification of these proteins will be described elsewhere (manuscript in preparation). The recombinant Pf332-C231 corresponds to a 231-amino acid fragment in the C-terminal part of Pf332. The plates were incubated overnight at 4°C, and then blocked for 2 hrs with 0.5% bovine serum albumin (BSA) diluted in carbonate buffer (pH 9.6). Plasma samples diluted in incubation buffer (PBS + 0.5% BSA), 1:1,000 (IgG) and 1:400 (IgG1-4), were added in duplicate and incubated for 1 h at 37°C. The plates were then washed four times, and bound IgG antibodies were detected with goat anti-human IgG-ALP (1:2000) . IgG subclasses were analysed with their respective biotin conjugated mouse anti-human subclass specific monoclonal antibodies: mouse anti-human IgG1 1:1,000 , mouse anti-human IgG2 1:3,000 , mouse anti-human IgG3 1:1,000  and mouse anti-human IgG4 1:2000 . Alkaline phosphatase (ALP) conjugated streptavidin (Mabtech) diluted 1:2,000 was added to detect bound antibodies of IgG2-4, while ALP-conjugated to goat anti-mouse Ig  were used for IgG1 antibodies and the plates were developed with nitrophenyl phosphate . The absorbance was read at 405 nm using a Vmax™ Kinetic microplate reader  and the antibody concentrations were deduced from the log-log correlative coefficient of each IgG subclass standard curve (six dilutions of myeloma proteins of IgG1-4 subclasses), ranging from 0.01 to 3 μg/ml for IgG1, 0.001 to 0.3 μg/ml for IgG2, 0.001 to 0.1 μg/ml for IgG3 and 0.01 to 1 μg/ml for IgG4 according to the manufacturer's recommendation . In plots, the antibody concentrations were transformed into log 10 values without any changes in the P - values obtained from the analysis of the raw values.An indirect enzyme-linked immunosorbent assay, ELISA,  was usedAllotyping of the serum samples from the above subjects was carried out for G1M , G2M (23), G3M , and KM  determinants by a standard haemagglutination-inhibition method . Human eSigma Stat software was used for data analysis. There is almost absolute linkage disequilibrium between individual GM alleles ; therefoThe individual GM allelic type  5 and 17 were detected in all (100%) donors involved in this study. The vast majority of the donors had the GM 1 (99.5%) and GM 14 (98.6%) alleles. The GM 13 82.4%) allotype was the next most frequent, while half of the population carried the GM 6 allotype (49.5%). The other allotypes; 3, 21 and 23, were rare , followed by 1,17 5,14,6 (1.5 ± 1.6) and 1,17 5,13,14 (1.5 ± 1.3) phenotype carriers, , showed that carriers of 1,17 5,13,14,6 phenotype as compared to the non-carriers, had experienced statistically discernibly increased number of malaria episodes over the years of the follow up (P = 0.037). The carriers of the other three major GM phenotypes as compared with the non-carriers of the corresponding phenotypes were equally susceptible to malaria  instead of GM allotypes. Results showed that there was less influence of the KM allotype on the total IgG and IgG isotypes concentration, regardless of the target antigen. The only significant differences were the higher concentration of total IgG to MSP2-FC27 and to Pf332-C231 antigens in the plasma obtained from the KM 3 compared to that obtained from the KM 1,3 phenotype carriers  and the less susceptible , worth further investigation. In this study, the GM 1,17 5,14 carriers, compared with the carriers of the other phenotypes or the non-carriers of 1,17 5,14 phenotype, had experienced the least number of malaria episodes although the differences were not significant. The significantly raised antibodies in the plasma of the carriers of the GM 1,17 5,13,14,6 phenotype, were mainly the total IgG and IgG2, but not the IgG3 isotype. The levels of IgG4 isotype to all antigens were generally very low, not associated with age and apparently were not increased after acute infection (unpublished data). The allotype-specific variation in the levels of IgG subclasses was antigen-dependent. This finding is supporting and adding to a previous report suggesting inherent ability of individual malaria antigens in antibody class switching .The GM 1,17 5,13,14,6 phenotype was the second most prevalent phenotype in the area. This same phenotype was found to be more dominant in the Massalit, a tribe presumed to be more susceptible to malaria than Fulani . The aboThe influence of GM/KM allotypes on antibody response is assumed to be due to alteration of antibody specificity through variable regions  or by moRecently, it has also been found that, there is a higher prevalence than previously expected, of primary partial immune deficiencies including IgG isotypes and GM allotypes, in apparently healthy individuals . FinallyThis study shows that, the GM allotypes are involved in susceptibility to uncomplicated malaria. Furthermore, there are indications for involvement of GM and to a lesser extent of KM allotypes in control of total IgG and IgG isotype concentrations, depending on target malaria antigen. The GM 1,17 5,13,14,6 phenotype was associated with higher baseline levels of total IgG and non-cytophilic IgG2. While GM 1,17 5,14 phenotype was associated with least susceptibility and highest baseline IgG3 response. Finally, the allotype GM 6, which is believed to be absent in Europeans was detected in 50% of the study population (Fulani).The authors declare that they have no competing interests.HG, DA and TT planed, established, maintained and supported the Daraweesh nine-years clinical and parasitological data; HG, AN and GE supervised and collected the field samples; AR, NI, MTB, KB, GH and JP were responsible for the laboratory data, HG, DA, TT, MTB, KB, JP and GH, were responsible for the study design, data analysis, results interpretation and paper drafting, all authors contributed to manuscript writing


Past studies in India included only symptomatic pregnant women and thus may have overestimated the proportion of women with malaria. Given the large population at risk, a cross sectional study was conducted in order to better define the burden of malaria in pregnancy in Jharkhand, a malaria-endemic state in central-east India.Cross-sectional surveys at antenatal clinics and delivery units were performed over a 12-month period at two district hospitals in urban and semi-urban areas, and a rural mission hospital. Malaria was diagnosed by Giemsa-stained blood smear and/or rapid diagnostic test using peripheral or placental blood.Plasmodium falciparum, 37.2% Plasmodium vivax, and 9.3% mixed infections). Peripheral parasitaemia was more common in pregnant women attending antenatal clinics in rural sites  and in those who were younger than 20 years . Among delivery unit participants, 1.7% (12/717) had peripheral parasitaemia and 2.4% (17/712) had placental parasitaemia. Women attending delivery units were more likely to be parasitaemic if they were in their first or second pregnancy , had fever in the last week , or had rural residence . Malaria control measures including indoor residual spraying (IRS) and untreated bed nets were common, whereas insecticide-treated bed nets (ITN) and malaria chemoprophylaxis were rarely used.2,386 pregnant women were enrolled at the antenatal clinics and 718 at the delivery units. 1.8% (43/2382) of the antenatal clinic cohort had a positive diagnostic test for malaria (53.5% The prevalence of malaria among pregnant women was relatively low. However, given the large at-risk population in this malaria-endemic region of India, there is a need to enhance ITN availability and use for prevention of malaria in pregnancy, and to improve case management of symptomatic pregnant women. Plasmodium falciparum have increased in India in recent years [Malaria is a disease of global importance that results in 300-660 million cases annually and an estimated 2.2 billion people at risk of infection . Approxint years .Malaria in pregnancy (MIP) poses substantial risk to the mother, foetus and neonate. In settings with either stable or unstable transmission, MIP has serious public health consequences. In areas of stable malaria transmission, clinically symptomatic infections are rare and the main consequence is an increased risk of maternal anaemia ,7 low biP. falciparum and P. vivax are associated with adverse pregnancy outcomes, these studies primarily focused on symptomatic pregnant women [P. falciparum and P. vivax during pregnancy and at delivery. The study was conducted in the state of Jharkhand in east India, with the ultimate goal of enhancing the development of evidence-based policies to reduce the burden of disease due to MIP in this region of India.Past MIP studies in India have demonstrated the important contribution of malaria to maternal and neonatal morbidity and mortality ,12. Althnt women . Relativnt women ,14. GiveP. falciparum accounting for 44% of the cases [This study consisted of a series of cross-sectional surveys conducted in three hospitals  in two districts in Jharkhand, India Figure . Jharkhahe cases . The Jhahe cases ,17. MostThe two rural study sites were selected in the district of Gumla , which at the time of the study, was considered to be a malaria-endemic area of the state of Jharkhand. In contrast, the third site, Ranchi District , was selected to represent an urban district with low transmission of malaria. Thus, the three sites were meant to provide a reasonable representation of typical conditions that would be found in Jharkhand. The District Level Household and Facility Survey conducted between December 2007 and April 2008 revealed that 56% of women had at least one antenatal clinic (ANC) visit and 18% overall had institutional deliveries including 59% in urban areas but only 13% in rural settings[The SPR in Ranchi District was 7.2% in 2005 . IRS in The study had three components with recruitment targeted to women presenting to ANC, delivery units (DU), or the inpatient antepartum ward. For the ANC component, pregnant women aged ≥15 years who reported to the study site for routine care were screened and enrolled. Those who had previously participated in this component of the study or were unwilling to provide written informed consent were excluded. For the DU component, women aged ≥15 years who presented for delivery and were willing to provide written informed consent were enrolled. For inpatients, pregnant women with an admission diagnosis of malaria, anaemia, or a febrile illness of unknown origin were screened for study participation. Those whose malaria-related diagnosis was confirmed or who were treated for malaria were enrolled after obtaining informed consent. Case report forms and study procedures were evaluated in a pilot study and modified accordingly.Trained study personnel interviewed the enrolled women and collected information on socio-demographic characteristics ; reproductive history including gravidity; history of fever and anti-malarial drug use; and use of anti-malarial prevention measures. A complete physical examination including the determination of gestational age from the height of fundus, measurement of axillary temperature with digital thermometer, and other vital signs was also performed. Blood was obtained by finger-stick for malaria blood film preparation, rapid diagnostic test (RDT), and haemoglobin determination. Women with positive RDT results or who were anemic were referred immediately to the hospital physician for treatment. The clinic staff was informed of additional parasitaemic individuals identified through blood smears so that they could be appropriately treated.Pregnant women enrolled at the DUs were interviewed, with data collection focused on socio-demographic characteristics, obstetric complications, history of fever and anti-malarial use during pregnancy, and the use of anti-malarial prevention measures. Blood was obtained by finger-stick soon after delivery for haemoglobin determination, RDT, and malaria blood film preparation. Placental blood by aspiration and an impression smear from the maternal side of the placenta were obtained to determine placental parasitaemia by thick smears and RDT. A drop of cord blood was also taken to prepare a blood smear. All neonates were weighed with an electronic digital scale to the nearest 10 grams and the gestational ages of all live births were estimated within 24 hours of delivery by means of a modified Ballard examination . Women wEnrolled subjects were interviewed and information on socio-economic status, reproductive history including obstetric history, history of fever and anti-malarial drug use, and the use of anti-malarial prevention measures was collected. Data including recorded clinical signs, results of laboratory investigations, treatments administered, admission and discharge diagnosis and outcome of admission were extracted from the subject's hospital record.Plasmodium species. All smears were re-checked by a member of the parasitology laboratory at the National Institute of Malaria Research Field Station in Jabalpur. The First Response Malaria Pf/Pv test , a parasite lactic dehydrogenase antigen test, was used to perform the RDTs. This test has a sensitivity of 93% and specificity of 85% for detection of malaria in non-pregnant individuals [P. falciparum and 69% and 99.4% for P. vivax, respectively [Thick and thin smears prepared from peripheral blood of ANC and DU subjects, placental blood and placental impression from DU subjects, and cord blood were Giemsa-stained and examined under high power. Asexual parasite forms were counted until a minimum of 200 white cells had been counted. Slides were considered negative only if no parasites were seen after identifying 500 leukocytes. Parasite densities were estimated using an assumed total white blood cell count of 8,000 leukocytes/μL of blood [ividuals . The Firectively . A portaThe following definitions were used:P. falciparum or P. vivax parasitic forms on blood smears or positive RDT.Peripheral parasitaemia: presence of asexual Placental malaria: presence of malaria parasites on impression smear of maternal side of placenta or by RDT.P. falciparum or P. vivax on thick blood smear or a positive RDT.Symptomatic malaria infection: history of fever within the last week or temperature ≥ 37.5°C associated with the presence of asexual forms of Severe malaria: a malaria attack associated with any of the following: cerebral malaria, severe anaemia, renal failure, pulmonary oedema, hypoglycaemia, shock, spontaneous bleeding, or repeated convulsionsAnaemia: haemoglobin <110 g/L.Severe anaemia: haemoglobin <70 g/L.Low birth weight: birth weight < 2,500 g.Prematurity: gestational age <37 weeks as assessed by Ballard examination .Stillbirth: death of foetus prior to delivery.The study was approved by the Institutional Review Boards of Boston University and the Centers for Disease Control and Prevention, the Ethics Committee of the National Institute of Malaria Research (NIMR) in India, the Scientific Advisory Committee of the NIMR and the Health Ministry Screening Committee of the Indian Council of Medical Research.P. falciparum or P. vivax parasitaemia were evaluated by univariate analysis and then adjusted for significant predictors in multivariate analysis.All case report forms were checked for completeness and inappropriate or illogical responses. The forms were double-entered using CS-Pro, with range, consistency, and edit checks built into the data entry programme for quality control. The two databases were validated and all inconsistencies and differences were resolved. Statistical analyses were performed using SAS software version 9.1 . Categorical data are presented as frequency counts (percent) and compared using the chi-square or Fisher's exact statistic as appropriate. Continuous data are presented as means (± standard deviation) and compared using the t-test or analysis of variance as appropriate. Since most participants did not know their exact date of birth, we have presented participants' ages in ranges based on their estimations. Risk factors for either Recruitment and enrollment took place from December 2006 to December 2007. Of 2499 pregnant women screened during their ANC visits, 60 were not eligible because they had previously participated in the study, 53 refused to provide informed consent, and 2,386 were enrolled. In the DU, 739 pregnant women were screened and all of them were eligible, although 21 refused to provide consent and therefore 718 were enrolled.Most pregnant women attending ANC were in the 20 to 34 year old age range and had some level of formal education Table . The vasP. falciparum was identified in 53.5% of parasitemic individuals while P. vivax was found in 37.2% and 9.3% of infections were mixed. Peripheral parasitaemia was over three times more likely among women living in rural areas when compared with those from urban or semi-urban sites , and among primigravidae and secundigravidae relative to multigravidae . Primigravidae had higher parasite densities than women with one or more past pregnancies but this difference was not statistically significant . Parasitaemia was more commonly encountered in pregnant women who had a history of fever within the week prior to enrollment or were febrile at the time of the study visit . Overall 51.2% (22/43) of the pregnant women with a positive malaria diagnostic test at the time of ANC visit were symptomatic. The majority of positive malaria tests occurred from April to December with the greatest number in July, corresponding to the monsoon season.A positive diagnostic test for malaria was obtained in 1.8% 43/2382) of the total cohort , P. vivax in 17% (2/12), and mixed infection in 8% (1/12). The mean density of parasitaemia in the women with positive blood smears was 14,272 asexual forms/μL . The peripheral parasitaemia density was significantly higher for primigravid women than in those who had one or more prior pregnancies . A greater proportion of the women presenting to the semi-urban and rural sites were parasitaemic but this difference was not significant  .8) Table . PrimigrPlacental impression yielded one more case  than placental blood smears . The mean density of placental parasitaemia in the 10 women with positive smears was 13,200 asexual forms/μL . An additional seven cases of placental parasitaemia were identified by RDT yielding an overall prevalence of placental parasitaemia of 2.4% with non-significantly greater proportions seen at the non-urban sites and in primigravidae and secundigravidae. Symptomatic malaria infections were present in 42% of women with peripheral parasitaemia (5/12) and 41% of those with placental parasitaemia (7/17). Pregnant women with peripheral parasitaemia were more likely to have either a self-reported fever or fever measured at enrollment than those who were aparasitaemic . Similarly, more women with placental parasitaemia had fever (reported or measured) compared with women whose placentas did not harbour parasites (41.2% vs. 11.0% p = 0.002). As observed in the ANC participants, most episodes of parasitaemia occurred in July and August during the monsoon season.More than half of the DU participants were anemic but only 4.5% had severe anaemia Table . For DU Excluding the 6 sets of twins delivered, 20.9% (141/675) of the babies delivered by the DU participants had LBW. There were slightly more LBW infants among mothers with placental malaria, but this difference was not significant (p = 0.53)  of the inpatients were between 20 and 34 years old (82%). Six (22%) were primigravidae, 10 (37%) secundigravidae, and 11 (41%) multigravidae. The majority was from scheduled tribes (59%) with the others belonging to general caste (19%), scheduled caste (11%) and other backward castes (11%). Similar to the ANC and DU participants, 78% reported having a bed net in the household and 63% reported sleeping under it most nights although only 7.4% were insecticide-treated. Sixty-seven percent of their homes had been sprayed with insecticide and only one woman had been taking chemoprophylaxis during pregnancy.P. falciparum was the cause of malaria for 84% (21/25) of the pregnant women, while two (8%) had P. vivax and two (8%) had mixed infection with P. falciparum and P. vivax. Eight (29.6%) of the admitted pregnant women had severe malaria  based on WHO criteria [P. falciparum was responsible for severe malaria in seven of eight women, while the last had a mixed infection. All admitted pregnant women were treated with parenteral or oral quinine, arteether or artesunate with excellent responses; none died.Malaria was confirmed by microscopy or RDT in 92.6% (25/27) of the inpatients. criteria . Of the P. falciparum, is responsible for a substantial portion of serious illness requiring hospital admission for pregnant women in this region.There was a relatively low prevalence of malaria among pregnant women attending ANCs and delivering in the study site hospitals in Jharkhand. Previous studies of MIP in India found similar to higher prevalence rates, ranging from 1.4% to 20% ,24,25 HoMalaria occurred more commonly in women in rural areas and those who were in their first or second pregnancy, as has been seen in studies of MIP in sub-Saharan Africa . The higOverall, there was a substantial burden of anaemia among pregnant women. While the proportion of pregnant women suffering from severe anaemia was relatively small (~4% in the ANC cohort), there was a significant association with malaria. Among women in the DU cohort, there was no association between parasitaemia and severe anaemia. The lack of an association with severe anaemia can be potentially attributed to the smaller sample size in the DU study cohort. Although none of the associations was significant, adverse maternal and birth outcomes including LBW, prematurity, stillbirth, and gestational hypertension all occurred more commonly in pregnant women with parasitaemia. Despite the low frequency of MIP, the large population of Jharkhand, nearly 22 million people , means tThe EMCP was active in both Ranchi and Gumla Districts until 2005 when the program ended. The low prevalence of malaria among pregnant women in the current study might have resulted from enhanced detection and treatment of symptomatic individuals in the community through personnel trained by this program. However, the very low rate of ownership of ITNs suggests that this component of the EMCP has not effectively reached this vulnerable population although it was encouraging to find that many households had bed nets and that they were used on a regular basis. The enhanced provision of ITNs and their regular retreatment are cornerstones of the EMCP . Their nDespite its existence as an official guideline at the time, chloroquine was almost never used for prevention of MIP. Although chloroquine resistance has been rising in India ,5, this One major limitation of this study is that the cross-sectional surveys were facility-based. Since a great part of the malaria burden is thought to occur in marginalized, remote tribal populations, the findings in this study may not be generalizable to all areas and populations within the state of Jharkhand. In addition, because this was a cross-sectional rather than a longitudinal study, the actual burden of MIP in this region may have been underestimated. Thus, the study design did not allow for an understanding of the natural history of parasitaemia in pregnant women or the frequency of malaria attacks during the course of gestation. The use of placental smear in this study instead of histopathology might have underestimated the burden of malaria since histopathology is more sensitive in identifying placental malaria ,21. FinaGiven the large at-risk population in this malaria-endemic region of India, there is a need to enhance ITN use for the prevention of MIP. Fortunately, there is already a culture of bed net use. If ITNs become more widely available, they would likely be acceptable to pregnant women barring any problems with their perception of risk associated with the chemicals used to treat the nets. Since the retreatment of ITNs presents many logistical challenges, future efforts should focus on the provision of long-lasting ITNs. An additional challenge will be to increase their availability and to make them affordable for Indians living below the poverty line. There should be a focus on improving case management of symptomatic pregnant women, and evaluating the efficacy and effectiveness of the intermittent screening and treatment strategy. IPTp does not appear to be a rational choice for reducing the burden of MIP, and chloroquine prophylaxis may not be effective due to rising resistance and, in any case, is not being implemented in Jharkhand. This requires enhancing the availability and use of diagnostic tests including RDTs and ensuring the availability of safe and effective drugs for the treatment of pregnant women with malaria.Davidson Hamer has one competing interest to declare, specifically equity ownership in Inverness Medical Innovations, a company that produces a malaria rapid diagnostic test. All other authors declare that they have no competing interests.DHH, BJW, KYA, MD, VU, WBM, and NS contributed to the conception and design of the study. MPH, JT, PG, MIB, MS, KA, APD, and NS all contributed to study implementation and data collection. BJW, MPS, and WBM performed data analyses and DHH, KYA, MD, VU, MIB, JT, and NS assisted with interpretation of data. DHH, BJW, KYA, and NS drafted the manuscript. All authors contributed to and approved the final manuscript.The United States Agency for International Development (USAID)/India mission provided funding for this study to the Child and Family Applied Research project at Boston University, Boston, by means of the USAID cooperative agreement (GHS-A-00-03-00020-00). The opinions expressed herein are those of the authors and do not necessarily reflect the views of USAID.


In malaria-endemic areas, reliably establishing parasitaemia for diagnosis of malaria can be difficult. A retinopathy with some features unique to severe malaria with a predictive value on prognosis, has been described. Detection of this retinopathy could be a useful diagnostic tool. This study was designed to determine the diagnostic usefulness of retinopathy on ophthalmoscopy in severe malaria syndromes: Cerebral malaria (CM) and non-cerebral severe malaria (non-CM), i.e. malaria with respiratory distress (RD) and malaria with severe anaemia (SA), in Ghanaian children. Secondly, to determine any association between retinopathy and the occurrence of convulsions in patients with CM.A cross-sectional study of consecutive patients on admission with severe malaria who were assessed for retinal signs, at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, from July to August 2002 was done. All children had dilated-fundus examination by direct and indirect ophthalmoscopy.Fifty-eight children aged between six months and nine years were recruited. Twenty six(45%) had CM, 22 with convulsion; 26(45%) had SA and six(10%) had RD.Any retinopathy was seen in: CM 19(73%), SA 14(54%), RD 3(50.0%), CM with convulsion 15(68%) and CM without convulsion 4(100%). Comparison between CM versus non-CM groups showed a significant risk relationship between retinal whitening and CM. There was no significant association with papilloedema, macular whitening, macular haemorrhage, retinal haemorrhage, vessel abnormality and cotton wool spots.Tortuous and engorged retinal veins, not previously described as a feature of CM, was the most common vascular abnormality(15/58 = 26%) and was detected even in the absence of papilloedema.Retinal whitening, a sign suggestive of retinal ischaemia, was significantly more common in CM than in non-CM syndromes. However, the high prevalence of any retinopathy in the latter suggests that the brain and the retina may be suffering from ischaemia in both CM and non-CM. Malaria is an important cause of morbidity and mortality especially in sub-Saharan Africa , where cIn malaria-endemic areas, reliably establishing parasitaemia for diagnosis and differentiating between malaria and other causes of febrile disease can be difficult , often cA combination of retinal abnormalities have been described to be associated with severe malaria. Retinal changes were described more than 50 years ago in cerebral malaria . Later mCharacteristic features of these retinal changes include whitening of the peripheral retina and macula , retinal vessel abnormalities, papilloedema, and multiple retinal haemorrhages often with pale centres ,16,22-24The detection of malarial retinopathy  could therefore be a useful diagnostic tool for the clinician to confirm the diagnosis of severe malaria, particularly in a comatose parasitaemic child. The severity of retinopathy  and papilloedema may also be useful in predicting the likelihood of death in cerebral malaria ,25. SpecIn Ghana, malaria is endemic and a leading cause of death in children ,27. ImprA cross-sectional study of consecutive patients on admission with severe malaria who were assessed for retinal signs, at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, from July to August 2002 was carried out. Ethical approval for the study was obtained from the University of Ghana Medical School Ethics and Protocol Review Committee, and participation was dependent upon signed informed consent from the parents or guardian of the patients.Children aged six months to nine years, presenting with signs and symptoms suggestive of malaria, axillary temperature ≥37.5°C and no other obvious cause of fever were consecutively screened for inclusion by a project physician.P. falciparum parasitaemia who fell into one of the following categories of severe malaria: SA: haemoglobin (Hb) < 5 g/dL, fully conscious with no episodes of severe bleeding, and no reported or observed convulsions; CM: Blantyre coma score ≤ 3 and duration of coma > 60 minutes despite correction of any hypoglycaemia, any Hb value and no record of recent severe head trauma, no other cause of coma or neurological diseases; RD: rapid breathing plus one or more of the following: alar flare, chest recessions, use of accessory muscles for respiration, or abnormally deep breathing. Patients with evidence of other infectious disease, abnormal cerebrospinal fluid and positive sickling status were excluded. Clinical parameters were documented on standard case record forms.Inclusion criteria were: Patients with All children were examined once by an ophthalmologist within 12 hours of admission, by direct and indirect ophthalmoscopy after pupil dilatation with tropicamide 1% and phenylephrine 2.5%. The retinal findings were recorded on predesigned standardized charts specifically including the previously reported findings associated with CM. Ophthalmoscopy was repeated for all patients at one week, and those who had persistent abnormalities were re-examined after one month. The eye findings used in the analysis for a child were the changes documented during admission.Plasmodium parasites using Giemsa-stained thick and thin blood films. Cerebrospinal fluid examination was done in patients with altered consciousness as soon as it was considered clinically safe in order to rule out meningitis.Haemoglobin was measured using an 18-parameter, automatic haematology analyzer ; screening for sickling using the sodium metabisulphite method; microscopic detection and identification of As per institutional practice at the time, patients with CM and those with other forms of severe malaria who could not retain oral medication were treated with intramuscular quinine sulphate . IM quinine was changed to syrup quinine at the same dosage when patients regained full consciousness or after 72 hours (whichever was earlier), via nasogastric tube to complete a 7-day course. Patients with other forms of severe malaria who were able to take medications orally were treated with amodiaquine 10 mg/kg body weight per day, as single daily doses for three days. Patients with SA or RD were given humidified oxygen and children with SA were transfused with blood. Hypoglycaemia was corrected with intravenous 10% dextrose solution. Paracetamol and anticonvulsant therapy were administered as clinically indicated at standard dosage.Data was analysed using SPSS Version 16.0. Continuous numerical variables were summarised as mean and standard deviation (SD) and categorical variables as percentages with binomial confidence intervals. Group comparisons were done using Chi-square test (with Yate's correction for 2 × 2 comparisons) or Fischer's exact test as appropriate. Binary logistic regression was used for analysis of associations between retinal signs and level of haemoglobin. P-values < 0.05 were considered statistically significant.Fifty-eight children aged between six months and nine years were recruited. Twenty-nine (50%) were males. The types of severe malaria encountered were: CM 26 (45%), SA 26 (45%) and RD 6 10%). Twenty-two (85%) of the patients with CM had at least one convulsion either observed during admission or reported by parents or guardians or both. Nineteen (73%) patients with CM, 14 (54%) with SA and three (50.0%) with RD i.e. a total of 36 out of the 58 patients (62%), showed one or more retinal abnormalities  13 (22%); macular whitening 19 (33%); macular haemorrhages 5 (9%) and retinal haemorrhages  20 (35%); papilloedema 12 (21%); cotton wool spots 3 (5%), and vessel abnormalities 16 (28%). The vessel abnormalities observed included whitening of the vessels especially at the periphery of the retina and white tram lining of vessels. However, orange discoloration of vessels was not encountered.The commonest vascular abnormality was engorgement and tortuosity of veins (15/58 = 26%). These vessels extended from the periphery to the optic disc and 8 out of 58 (14%) were not associated with papilloedema.In order to assess the clinical usefulness of retinopathy on ophthalmoscopy in differentiating between CM and non-cerebral severe malaria, i.e. the pooled group of SA and RD patients, the frequency of occurrence of retinopathy in the two groups was compared Table . There wThere was a significant risk relationship between retinal whitening and CM as compared with the non-CM . It should be noted, however, that less than 50% of CM patients displayed these particular finding. A logistic binary regression analysis done to examine the effect of the level of haemoglobin and anaemia on the presence or absence of retinal whitening in the CM group failed to show any significant influence  (Data not shown). There were no significant risk relationships seen between CM and the rest of the retinal signs studied.Fifteen (68%) of the CM patients with convulsions and 4100%) of the CM patients without convulsions showed one or more retinal signs  who had some retinopathy initially, i.e. the changes were reversible and none of the patients had developed new retinopathies. In particular, the engorged and tortuous vessels had disappeared by one week in 13 out of the 15 children and in the remaining 2 children this finding had disappeared by the one-month follow-up.This study demonstrated retinopathies in all types of severe malaria corroborating the findings from the Gambia, Kenya, Malawi and Mali ,16,22,23Retinal whitening was the only retinopathy that showed significant association with CM in this study. Retinal whitening, which has been suggested as an indicator of retinal ischaemia due to capillary non-perfusion  and its Of interest, however, is that this current study detected similar proportions of the various retinopathies as found in older studies from Malawi ,32. ThisNo deaths were encountered in this study and so the associations between retinal changes and deaths could not be studied. Other studies have demonstrated increased mortality and a positive predictive value of some malarial retinopathies  for fatality and severe malaria especially CM ,16,22,25It was not possible to determine if there was any association between convulsion and retinopathy in CM because of the small number of patients who had no convulsion.The two patients with neurological sequelae   had retiThe limitations of this study are that: since all the patients studied survived, it was impossible to determine the prognostic significance of the retinopathies observed for mortality. The small number of patients analysed for each type of severe malaria and the lack of comparison with non-cerebral malaria comas or uncomplicated malaria also contributed further to the limitations. Finally, this study used bedside ophthalmoscopy to test the clinical relevance of retinopathy in patients with severe malaria without fundus photography. Thus detailed measurement and evaluation of the calibre of the blood vessels encountered could not be done reliably.This study demonstrated a high occurrence (50-75%) of retinal changes in the patients with severe malaria . Whereas detection of malarial retinopathy through ophthalmoscopy may potentially be very useful from a diagnostic, prognostic, and scientific point of view, the high prevalence of any retinopathy in the non-cerebral severe malaria group limits the use of malarial retinopathy in differentiating CM from other severe malaria syndromes.Retinal whitening, a sign suggestive of retinal ischaemia, was found in both CM and non-CM syndromes, suggesting that the brain suffers from ischaemia in CM just as the retina, both forming part of the central nervous system.Finally, once skill is acquired with the indirect ophthalmoscope the features of retinopathy are clearly evident and their diagnostic and prognostic value can then be utilised. It is the technique and equipment of indirect ophthalmoscopy which remain the barriers to wider uptake and usefulness.The authors declare that they have no competing interests.EV participated in the design of the study, performed the ocular examination and write up of the manuscript. NCT conceived and participated in the design of the study, performed the ocular examination and helped to draft the manuscript. AB was responsible for coordination of study. AGO and GBQ were responsible for patient selection and helped to prepare manuscript. NTA performed the statistical analysis and interpreted the data. KJAL conceived and participated in design of the study and helped to draft the manuscript.All authors read and approved the final manuscript.


Vascular factors have been implicated in the development of cognitive decline and dementia. The purpose of this study is to determine the association of the Ankle Brachial pressure Index (ABI) and brachial-ankle Pulse Wave Velocity (ba-PWV) to cognitive impairment in a community-dwelling population.The ABI and ba-PWV were measured using the volume-plethymographic apparatus in 388 subjects aged 60 years old and over. The Mini-Mental State Examination was also employed to measure global cognitive status. The effectiveness of the ABI and ba-PWV as putative markers of cognitive impairment were determined by using a multiple logistic regression analysis after adjusting for confounding factors.Subjects with poor cognition were significantly older and less well educated than those with normal cognition. According to the multiple logistic regression analysis, the lowest ABI tertile was found to be a significant independent risk factor  of the cognitive impairment, whereas the highest brachial-ankle PWV tertile was not.A low ABI was an independent risk factor for cognitive impairment in community-dwelling older populations, whereas a high ba-PWV may not be. Further research will be required to analyze ABI and PWV with greater accuracy. The cognitive impairment is an important health issue contributing to disability , morbidiVascular factors have been implicated in the development of cognitive decline  and demeThe ABI is the ratio of the ankle and the brachial systolic blood pressure and is used to assess the severity of arterial occlusion in the leg, and a reduction of ABI suggests the presence of peripheral arterial disease due to atherosclerosis. It is also known that atherosclerosis in the lower legs represent just one manifestation of a similar pathology in other arterial systems ,7. A numAnother assessment for arterial stiffness is PWV, in which the velocity of the pulse wave is measured as it travels a given distance between 2 sites along the arterial system. Since Mizushima et al.  first reThese findings contribute to the growing body of evidence on correlation between arterial stiffness and cognitive impairment. Recently, a measurement device which can simultaneously measure the ABI and brachial-ankle PWV has become available . HoweverIn this study, the ABI and ba-PWV were compared as markers of cognitive impairment in a community-dwelling population, and MMSE was used to assess cognitive status. The ABI and PWV were measured as indicators of arterial structure and function. The present study is considered as the report to obtain a comparative data between the ABI and ba-PWV as markers of cognitive function in a community-dwelling population.The subjects were 388 volunteers  who participated in the Iwaki Health Promotion Project in 2008. The data collection for this study was approved by the Ethics Committee of Hirosaki University School of Medicine and all subjects had provided written informed consent before participating in the project. The demographic data  and life style  were obtained from self-questionnaires and interviews. The height and weight of subjects were measured, and body mass index (BMI) was calculated. Low-density lipoprotein (LDL) cholesterol, triglyceride and HbA1c were also measured by standard analytical techniques.The ankle and brachial pressures were measured using the volume-plethymographic apparatus . In addition to recording the limb lead ECG and mechano-cardiograms were simultaneously recorded by attaching blood pressure cuffs with a tonometic sensor to the upper arm and ankle.The ABI was determined as the ratio of ankle systolic blood pressure to brachial systolic blood pressure. To calculate the ABI, the brachial pressure was measured in the left arm, and the ankle pressure was measured for both the left and right sides with subjects in the supine position and the lowest value of the ABI was taken .The brachial-ankle PWV (ba-PWV) was calculated by time-phase analysis. The time interval between the wave front of the brachial waveform and that of the ankle waveform was defined as the time interval between the brachium and ankle (ΔTba). The distance between sampling points of ba-PWV was calculated automatically according to the height of the subject. The path length from the suprasternal notch to the brachium (Lb) was obtained from superficial measurements and was expressed using the following equation: Lb = 0.2195 × height of the patient (in cm) -2.0734. The path length from the suprasternal notch to the ankle (La) was obtained from superficial measurements and was expressed using the following equation: La = (0.8129 × height of the patient (in cm)+12.328). Finally, the following equation was used to obtain ba-PWV: ba-PWV = (La-Lb)/ΔTba . In thisThe Mini-Mental State Examination (MMSE) was given to all participants to measure their global cognitive status. This test assesses orientation to place and time, short term memory, episodic long-term memory, subtraction, ability to construct a sentence, and oral language ability. The maximum score was set as 30, and poor cognition was defined as a score of less than 24 . SubjectData are presented as mean ± SD. A value of p < 0.05 was considered significant. Student's unpaired t test for continuous variables or chi-square test for categorical variables was used to evaluate the difference in variables between subjects with MMSE≦23 and those with MMSE≧24. The subjects were divided into 3 groups according to their ABI and ba-PWV tertiles, and a multivariate logistic regression analysis was applied to assess the usefulness of the ABI and ba-PWV measurements as markers of cognitive impairment both crude and adjusted conditions for confounding factors  . The datThe subjects were divided into 2 groups according to their MMSE scores . The clinical characteristics of subjects are listed in Table st quartile of ABI  was shown to be an independent risk factors. In crude odds ratio, the 1st quartile of ABI  approached statistical significance. Under both crude and adjusted conditions, amount of education consistently showed lower risk of cognitive impairment. There were no other significant risk factors of cognitive decline.The subjects were grouped according to their ABI tertile  and according to their ba-PWV tertile . Table The ABI and PWV are the markers of atherosclerosis and arterial stiffness respectively. In the present research, their associations with cognitive impairment were examined through cross-sectional assessment among community-dwelling population. The cognitive decline was found to be 3.19 times more prevalent among subjects in the 1st tertile of ABI (0.66 to 1.08). On the other hand, no PWV tertiles showed an association with cognitive impairment in either crude or adjusted conditions. This research is considered as the report suggesting a usefulness of ABI and PWV as markers of cognitive function in a community-dwelling population.The measurement of the ABI provides one of the most practical means to objectively assess the presence of atherosclerosis. A number of previous studies ,8,9 suggOne explanation is that sub-clinical cerebrovascular disease, such as silent cerebral infarctions may mediate the association between atherosclerosis and cognitive impairment. In the present research, brain imaging was not carried out on subjects, thus this was not evaluated. Also atherosclerosis may induce cerebral hypo-perfusion leading to cerebral hypoxia. These conditions may destabilize neurons and synapses and evolve in a neurodegenerative process characterized by formation of senile plaques, neurofibrillary tangles, and amyloid angiography ,23,24. AIn this study, we did not find an association between PWV and risk of cognitive impairment. Some findings obtained from other studies were found to support our results. For example, Dhoat et al  did not In order to assess cognitive status, the MMSE was employed in this study. The MMSE score does not always reflect the cognitive function exactly, as it is sometimes known to be influenced by the education level of the subject. In the present study, the subjects with a normal MMSE score were significantly more educated than those with impaired MMSE scores, and the amount of education was found to have significant association with the MMSE score. This finding was consistent with that of previous reports. In Baltimore longitudinal Study of aging , personsAs all subjects were volunteers who had interests in their health, they may be healthier than those who were not involved in this study. Thus, those who were not would be more likely to do poorly on a community-based cognitive assessment . A "seleIn the present study, the brain imaging was not carried out, thus Alzheimer's disease (AD) was not excluded completely. The aetiology of AD is different from vascular-type dementia. Including subjects with AD could obscure the association between vascular factors and cognitive function. Nevertheless, the result of this study, which showed cognitive impairment in subjects with lower ABI, suggests the predictive value of ABI.In this study, we determined the association between ABI and PWV to cognitive impairment in cross-sectional study. A score in the lowest ABI tertile is an independent risk factor for cognitive impairment, whereas a score in a high ba-PWV tertile was not. However, we are not able to completely rule out an association between increased PWV and risk of cognitive impairment due to a number of factors including the measurement site of PWV, use of the MMSE, having a relatively small sample size and the possibility of a selection bias. Furthermore, a comparison between ABI and PWV was not performed in the longitudinal situation. There is still no cure for dementia, and the risk factors of cognitive decline must be identified to delay its progression. Therefore, further investigation is needed to obtain clear associations between them in the future.The authors declare that they have no competing interests.NS conceived the study, designed the study, conducted the statistical analysis, interpreted the data and wrote the initial draft of the manuscript. SK had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. SK, SN and NYF contributed to study design and assisted in drafting the manuscript. TU and IT completed initial survey construction, recruitment of participants. AK, YS, KD and MM participated in the data collection, and the interpretation of the results. All authors have approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/10/46/prepub