Plasmodium falciparum malaria as reflected by lower parasitaemia and fewer clinical symptoms than other sympatric ethnic groups. So far most studies in these groups have been performed on adults, which is why little is known about these responses in children. This study was designed to provide more information on this gap.The Fulani are known to be less susceptible to Circulating inflammatory factors and antibody levels in children from the Fulani and Dogon ethnic groups were measured. The inflammatory cytokines; interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor (TNF) and the chemokines; regulated on activation normal T cell expressed and secreted (RANTES), monokine-induced by IFN-gamma (MIG), monocyte chemotactic protein (MCP)-1 and IFN-gamma-inducible protein (IP)-10 were measured by cytometric bead arrays. The levels of interferon (IFN)-alpha, IFN-gamma and malaria-specific antibodies; immunoglobulin (Ig) G, IgM and IgG subclasses (IgG1-IgG4) were measured by ELISA.The results revealed that the Fulani children had higher levels of all tested cytokines compared to the Dogon, in particular IFN-gamma, a cytokine known to be involved in parasite clearance. Out of all the tested chemokines, only MCP-1 was increased in the Fulani compared to the Dogon. When dividing the children into infected and uninfected individuals, infected Dogon had significantly lower levels of RANTES compared to their uninfected peers, and significantly higher levels of MIG and IP-10 as well as MCP-1, although the latter did not reach statistical significance. In contrast, such patterns were not seen in the infected Fulani children and their chemokine levels remained unchanged upon infection compared to uninfected counterparts. Furthermore, the Fulani also had higher titres of malaria-specific IgG and IgM as well as IgG1-3 subclasses compared to the Dogon.P. falciparum parasites compared to the Dogon and that these differences are evident already at an early age. The inflammatory responses in the Fulani were not influenced by an active infection which could explain why less clinical symptoms are seen in this group.Taken together, this study demonstrates, in accordance with previous work, that Fulani children mount a stronger inflammatory and antibody response against  Plasmodium falciparum remains one of the most common infectious diseases worldwide and is still a major cause of morbidity and mortality in tropical regions, especially in children below the age of five. Immunity to malaria is dependent on both the innate and the adaptive arms (both cell- and antibody mediated) of the immune system, which are required for adequate protection [Infection with otection .P. falciparum but also seem to contribute to immunopathology and the severity of the disease if not controlled properly. Available data are consistent with a requirement for an early production of in particular interferon (IFN)-γ to mount resistance against infection [Inflammatory cytokines play an important role in human immune responses to infection with nfection , and pronfection . Howevernfection . They alnfection . Other cnfection ,6.P. falciparum infections vary with disease severity [Chemokines are chemotactic cytokines that play important roles in bridging the innate and the adaptive immune system . They orseverity  and someseverity -11.P. falciparum infections, the importance of antibody-mediated response against P. falciparum parasites was shown 50 years ago in passive transfer studies, in which immunoglobulin (Ig) G antibodies from immune African adults reduced the level of parasites in Gambian children [P. falciparum infection, whereas the role for IgG2 and IgG4 in terms of protection are less clear and has been suggested to interfere with the protective mechanisms seen by the cytophilic antibodies [Even though the innate immune system is essential during children . Howevertibodies -16. ThusP. falciparum infection between two ethnic groups; the Fulani and the Dogon [P. falciparum infection than the other ethnic groups as reflected by fewer clinical symptoms of malaria and less parasite clones and numbers found in their blood [P. falciparum infection in terms of antigen-presenting cell (APC) subset activation and toll-like receptor (TLR) stimulations [P. falciparum infection in Fulani as compared to other sympatric ethnic groups is not fully understood. Earlier studies have mostly focused on asymptomatic adults, and little is known about this in children. The focus of this study has, therefore, been to investigate inflammatory- and antibody responses in infected and uninfected children from Dogon and Fulani ethnic groups living in Mali, by measuring selected cytokines, chemokines and IgG subclasses in plasma obtained from these children.In Mali, previous studies have shown different susceptibility to he Dogon . Similarhe Dogon . These phe Dogon ,18. Despir blood ,18. In air blood ,16,19 anir blood  antibodiir blood . This hiir blood . The conir blood . Establiir blood , suggestir blood  comparedulations . NeverthP. falciparum parasites. Malaria infection was defined as having a positive thick blood smear with or without any malaria symptoms. Axillary temperature was measured in all children and symptomatic malaria was defined as fever ≥ 37.5°C, or history of fever, plus the presence of any density of parasites in the blood. Among the Dogon, 20 children were undergoing a malaria infection and 20 children were healthy, while in the Fulani, 14 children were having a malaria infection and 23 were healthy. Written informed consent was obtained from both the community and from the children's guardians before inclusion in this study. The study was approved by the institutional review boards of the University of Bamako Mali (N°08_64/FMPOS).Children between 2 and 10 years of age belonging to either the Fulani or the Dogon ethnic group was included in the study. In total, 40 children from the Dogon and 37 from the Fulani ethnicity were recruited. Healthy children and children diagnosed with asymptomatic malaria were included from both ethnic groups. A thick blood smear was made from each donor and the slides were stained in 3% Giemsa and examined for the presence of This study was conducted in a rural area of the Dogon valley located approximately 850 km from the capital of Bamako, in Mali. The malaria transmission in the area is meso-endemic with intense transmission during the rainy season that usually extends from June to October . Samples® Plasma Tube, 143 USP Units of Sodium Heparin freeze dried) and plasmas were obtained by centrifugation and were kept at -80°C and transported to Stockholm University and analysed for cytokine and antibody levels.Blood samples were collected from uninfected and infected children of both ethnicities. From each donor, 6 ml of venous blood were collected in heparin tubes . All samples were diluted 1:2 and tested in duplicates. The enzyme-substrate reaction was developed using p-nitrophenyl phosphatase  and the optical densities were measured at 405 nm in an ELISA plate reader . The concentrations were calculated from a standard curve obtained in a sandwich ELISA with eight dilutions of lyophilized native human IFN-α standard  and recombinant human IFN-γ standard .Levels of IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF, regulated on activation normal T cell expressed and secreted (RANTES), monokine-induced by IFN-γ (MIG), monocytes chemotactic protein (MCP)-1 and IFN-gamma-inducible protein (IP)-10 were measured in plasma using cytometric bead array (CBA)  according to the manufacturer's recommendation. Briefly, 24 μl of bead populations with discrete fluorescent intensities coated with cytokine capture antibodies, were added to 24 μl of plasma sample and the mixtures were incubated for 90 min in room temperature. At the same time, standards for each cytokine/chemokine (range 0-10.000 pg/ml) were also mixed with cytokine specific capture beads. After incubation, the samples and standards were washed to remove unbound material and 24 μl of phycoerythrin (PE)-conjugated anti-human inflammatory cytokine or chemokine antibodies were added and mixtures were incubated again for 90 min followed by washing an acquisition on a fluorescent activated cell sorter (FACS) Calibur (BD Biosciences). Samples and standards were analysed using FCAP Array software v1.0.1 . To determine levels of IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF samples were incubated with anti-cytokine capture beads for 90 min, washed and incubated with PE-detection reagent for another 90 min and after final washing acquisition was done on FACS. Calibration was performed on the flow cytometer before acquisition using BD FACSComp™ and BD CaliBRITE™. The lower detection limit for the various cytokines and chemokines was 3.6, 7.2, 2.5, 3.3, 3.7, 1.9, 1.0, 2.5, 2.7 and 2.8 pg/ml for IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70, RANTES, MIG, MCP-1 and IP-10 respectively.Plasmodium falciparum parasites of the strain F32 were held in continuous cultures as described elsewhere [lsewhere  and keptlsewhere . A contrLevels of malaria-specific antibodies were quantified using sandwich ELISAs as described by Bolad and colleagues  but withDifferences between the Dogon and Fulani were analysed for statistical significance using Kruskal-Wallis non-parametric test. If differences were seen, a Mann-Whitney U non-parametric test was performed, to distinguish intra- and inter ethnic differences between infected and non-infected Fulani and Dogon. Spearman rank correlation was employed when analyzing possible correlations. Multiple regression was employed to correct for age on antibody titres. Statistical significance was assumed when p < 0.05. The data were analysed using StatView software 5.0.1 and Stata 12.The results from study subject characteristics have been described elsewhere . There wBlood plasma samples from the Dogon and Fulani children were analysed for levels of IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF, IFN-α and IFN-γ Figure , IL-8 p, IL-12p7When comparing the levels in the infected children of the two groups, IFN-γ was the only factor out of all tested that showed a significant difference, where the levels were higher in infected Fulani as compared to infected Dogon (p = 0.04).P. falciparum infection, it was found that infected Dogon children had significantly higher levels of IL-6 (p = < 0.0001) and IL-12p70 (p = 0.0002) compared to the uninfected Dogon. Likewise, the only factor that showed significant difference between infected and uninfected Fulani was IL-8, which was lower in the infected Fulani (p = 0.048).When the levels were compared within the same ethnic group in relation to In most cases levels of IL-1β and TNF were under the detection limit for the kit and were therefore excluded from analysis.Blood plasma from the Dogon and Fulani children were analysed for levels of RANTES, MIG, MCP-1 and IP-10 Figure . When coWhen comparing the uninfected children from both groups, the uninfected Dogon children had higher levels of RANTES (p = 0.05) but lower levels of MCP-1 (p = 0.007) and IP-10 (p = 0.05) compared to uninfected Fulani. In contrast, when comparing infected Dogon to infected Fulani children, no such difference was seen for any of the factors tested.When comparing children from the same ethnic group, there were no differences for any of the factors tested between infected and uninfected Fulani children. In contrast, infected Dogon children had significantly lower plasma levels of RANTES (p = 0.003) and significantly higher plasma levels of both MIG (p = 0.006) and IP-10 (p = 0.001) as compared to uninfected Dogon. The infected Dogon also had increased levels of MCP-1 compared to uninfected Dogon, even though this did not reach statistical significance (p = 0.15).Levels of malaria-specific total IgG and IgM as well as the IgG subclasses; IgG1, IgG2, IgG3 and IgG4 antibodies were measured in blood plasma samples from Dogon and Fulani children Figure . When coWhen comparing the infected children of both ethnicities, the infected Fulani children had higher titres of malaria-specific IgG1 (p = 0.03) and IgG3 (p = 0.001) as compared to infected Dogon children Figure .When comparing infected and uninfected children within the ethnic groups, IgG3 was significantly higher in infected Dogon as compared to uninfected individuals (p = 0.034). The reverse pattern was seen in Fulani, in which uninfected Fulani had higher titres as compared to infected individuals (p = 0.045) Figure . No antiAvailable studies trying to resolve the reason for the Fulani's relative protection against malaria have so far focused on immunological responses mostly in adults and not so much is known about these responses in children. The previously established relative protection for Fulani seen in adults was in this study shown to be evident during early childhood in this ethnic group. Children from the Fulani ethnic group had higher titres of malaria-specific antibodies and pro-inflammatory cytokine levels as well as unchanged chemokine levels upon an infection, as compared to Dogon children, suggesting that these differences are established already early in life.P. falciparum infection and the balance between pro- and anti-inflammatory cytokines may be important for the clinical outcome of malaria [P. falciparum infections, possibly due to higher baseline levels of these cytokines.Cytokines and chemokines are essential mediators during  malaria . Higher Plasmodium parasites compared to cells from Dogon [When comparing the infected children from both ethnic groups, IFN-γ was the only factor out of all tested that was significantly increased in infected Fulani compared to infected Dogon. This observation is in line with previous findings by McCall and colleagues who reported that mononuclear cells from Fulani are able to produce a 10-fold stronger IFN-γ response after stimulation with om Dogon . Availabom Dogon ,29. The When comparing the uninfected children from both ethnic groups, the levels of IL-8 were found to be higher in uninfected Fulani compared to uninfected Dogon. However, when comparing differences within the same ethnic group, the levels of IL-8 were significantly lower in infected Fulani compared to their uninfected peers and this difference was not evident between infected and uninfected Dogon. IL-8 is a chemoattractant cytokine known to be involved in recruiting neutrophils to inflammatory sites. Very little information concerning the role for IL-8 in malaria pathogenesis/protection exists. IL-8 has been shown to be elevated in a small group of severe malaria cases in adults . In addiP. falciparum-induced IL-6 production has previously been associated with increased incidence of clinical episodes [P. falciparum malaria [Further, the only cytokines that showed differences between infected and uninfected Dogon were IL-6 and IL-12p70. The results showed higher levels of IL-6 and IL-12p70 in infected Dogon compared to their uninfected counterparts, and these differences were not seen between infected and uninfected Fulani children. IL-6 is a major mediator of the acute phase response. Increased episodes  and othe malaria . The fac malaria ,35. The The role of chemokines and their distribution in plasma has not been investigated in the Fulani and Dogon ethnic groups. The results from this study show that all chemokines tested were unchanged upon an infection in Fulani children, while this was not the case in Dogon. Infected Dogon children undergoing an infection had lower levels of RANTES compared to uninfected Dogon. Low levels of RANTES have previously been reported to be associated with mortality in children with severe malaria , in partThe observed higher levels of MIG, MCP-1 and IP-10 in infected Dogon children compared to uninfected children. This marked up-regulation of chemokines was not evident in the infected Fulani children. Chemokines are involved in attracting different immune cell populations to the site of infections thereby aiding in the inflammatory response. Some of these factors have been implicated in severe malaria disease as well as in inflammatory diseases . High leTaken together it seems Fulani already have high baseline levels of these chemokines in circulation compared to Dogon since the levels do not change when they become infected, again suggesting that they are more prepared for combating infections. Since chemokines such as MIG, MCP-1 and IP-10 are involved in cell migration, the lower frequencies of these chemokines found in uninfected Dogon might imply that these cells are not able to migrate as well as cells from uninfected Fulani. Interestingly, Arama and colleagues  reportedIt has been known since the early 1960s that immunoglobulins (Ig) play an important role in malarial immunity . The resTo reassure that the malaria-specific antibodies are specific for the parasite, a control antigen of sonicated erythrocytes were included on all plates. There was no reactivity in any sera tested with sonicated erythrocytes confirming that the antibodies detected against the crude antigen are parasite specific.Growing evidence supports that not all immunoglobulin subclasses are associated with protection against malaria, but rather it is the cytophilic subclasses; i.e. IgG1 and IgG3, that are important. IgG1 and IgG3 antibodies have shown to be crucial in parasite clearance by binding to the surface of the infected erythrocyte or to mediate opsonization by cooperating with human monocytes . The obsP. falciparum malaria in individuals carrying a certain Fcγ receptor subtype [The result showed that the titres of malaria-specific IgG2 and IgG4 antibodies were relatively low in both Fulani and Dogon. These antibodies are thought to inhibit the protective effects mediated by the cytophilic antibodies (IgG1 and IgG3) and could thereby potentially inhibit their neutralizing effect on the parasite. However, the role of IgG2 antibody has shown somewhat conflicting results and recent studies have indicated that this antibody may have a protective role against  subtype . Further subtype . There a subtype .P. falciparum infection and that these differences are evident at an early age. The Fulani were shown to have higher levels of all tested inflammatory cytokines compared to the Dogon children, especially levels of IFN-γ, a cytokine that is crucial in determining the outcome of an infection. When children were further subdivided into infected and uninfected individuals, infected Dogon children had significantly lower levels of RANTES compared to uninfected Dogon, while significantly higher levels of MIG and IP-10 and also higher levels of MCP-1. Such patterns of differences in chemokine levels between infected and uninfected individuals were not evident in Fulani children whose levels remained unchanged upon an infection with P. falciparum. In addition, the results showed that the Fulani children had higher titres of malaria-specific antibodies compared to the Dogon children. This was true for levels of IgG1 and IgG3 antibodies, which have been shown previously to be involved in protection against malaria. The findings presented here suggests that there are marked differences in the cytokine and chemokine levels as well as in malaria-specific antibody titres between Fulani and Dogon that can be found at childhood age in these ethnic groups. Such difference might be important for the relative protection against malaria seen in the Fulani as compared to the Dogon. However, due to the small sample size in this study a prospective evaluation of a larger cohort of patients is required to confirm the results presented here. Further investigations are needed to elucidate the underlying mechanisms responsible for Fulani's relative resistance against malaria and hopefully, such insights may help in control strategies in malaria-endemic areas.Taken together, the results from this study demonstrate that there are marked differences between Fulani and Dogon immune responses to Plasmodium falciparum; RANTES: regulated on activation normal T cell expressed and secreted; TLR: toll like receptor; TNF: tumor necrosis factor.APC: antigen presenting cell; CBA: cytometric-bead array; DC: dendritic cell; FACS: fluorescent activated cell sorter; IFN: interferon; Ig: immunolglobulin; IL: interleukin; IP: IFN-gamma-inducible protein; MCP: monocyte chemotactic protein; MIG: monokine-induced by IFN-γ; PE: phycoeryhtrin; P. falciparum: The authors declare that they have no competing interests.PG, CA, VD, BT, AD, OD and MTB conceived, designed and coordinated the study. PG and CA participated in the sample collection and processing. SB and MTB designed and supervised the immunoassays. SB carried out the immunoassays. SB and JP performed statistical analysis. SB drafted the first version of the manuscript. All authors read and approved the final manuscript.


Cerebral malaria is a common cause of neurological sequelae and death in childhood. Information on persistent neurological sequelae post hospital discharge and their predisposing factors are scarce.This is a prospective study describing persisting neurological impairments post discharge among children treated for cerebral malaria. In addition the study was designed to investigate the frequency of persistent neurologic deficits and the risk factors for their persistence in these patients. The case records of 160 patients treated for CM at the Paediatrics Department of University College Hospital, Ibadan from January 2004 to November 2006 were reviewed to recruit cases. Recruited survivors were then followed up for information concerning the presence and persistence of neurological sequelae.A total of 160 children aged 9 months to 134 months were admitted and treated for CM during the study period. One hundred and thirty one (81.9%) survived while 29 (18.1%) died. The 131 survivors of cerebral malaria consisted of 64 boys and 67 girls. Neurological sequelae occurred in 13.7% of survivors of cerebral malaria at discharge and 4.6% at follow up. Six children with neurological deficits at discharge had persistence of deficits 6 months post-hospital discharge and one at 24 months. No associations were found between hypoglycemia, anemia, age, sex and multiplicity of convulsions, and persistence of neurologic sequelae. The persisting neurologic deficits among survivors at follow up were: memory impairment (1.5%), seizure disorders (0.8%), visual impairment (0.8%), speech impairment (0.8%), monoparesis (0.8%) and hyperactivity (0.8%) at follow up. The longest persisting sequelae lasted for at least 24 months.Neurologic deficits are not uncommon complications of CM. Neurologic sequelae may persist for as long as 24 months or more in survivors of childhood CM. There is no association between the risk factors for neurologic deficits and persistent neurologic sequelae. Plasmodium falciparum is the most common cause of malaria worldwide [orldwide . It is rorldwide –3. The sorldwide .Cerebral malaria is the most severe neurological presentation of acute falciparum malaria, the clinical hallmark of which is the presence of coma . It is aPlasmodium falciparum malaria significantly threatens child survival and long-term impairment of cognition often follows early insult and injury to growing brains, inadequate attention has been given to the impact of malaria on general cognitive and behavioural development of children after discharge from the hospital by previous reports [Despite the growing recognition that  reports , 11. Mos reports , 12–14.  reports , 9, 11. The present study is a cross sectional study. The subjects were children who were previously admitted and treated for cerebral malaria at least six months before the time of study, who were then followed up for presence or persistence of neurological sequelae. Ethical clearance was obtained from The Joint University of Ibadan and University College Hospital Ibadan Ethical Committee. Informed verbal consent was obtained from the parents or guardians of the children and explanation was made to the children as much as possible to ensure their optimal cooperation.All children with cerebral malaria admitted into Children Emergency ward between January 2004 and November 2006 were identified from admission records book and their case record files were retrieved from the Records Department of the University College Hospital. Baseline demographic and clinical data of patients at the time of diagnosis of cerebral malaria was extracted from the case notes using a structured case record form. The homes of affected patients were traced, using the contact addresses and mobile phone numbers in the case records. Enquiries were made to exclude other causes of brain injury between discharge and time of review. During the home visit, information was obtained from the caregivers. General clinical and detailed neurological examination  was carried out on the survivors of cerebral malaria and the information obtained was recorded in a structured case record form. Children with neurological deficits needing further evaluation were comprehensively reviewed at University College Hospital, Ibadan by specialists such as the ophthalmologist and physiotherapist in order to determine the exact deficit and its extent with the view of managing appropriately.Data was analyzed using SPSS 11.0 for Windows . The patients were grouped based on age and the presence or absence of neurological deficit. Data was presented as frequency tables and mean ± standard deviations (x ± SD). Selected clinical and laboratory factors such as duration of coma, nutritional status, multiple seizures, anaemia and hypoglycemia were compared in those with and without sequelae. Analysis for significance was by use of Chi square or Fishers exact test, where counts were less than 5. P values less than 0.05 was regarded as significant.Neurologic sequelae were defined as impairment of neurologic or cognitive function. Specifically they include: impairment or loss of function in musculoskeletal system, these includes paralysis or paresis manifesting as inability to walk, staggering, monoparesis, hemiparesis, quadriparesis; memory impairment , hyperactivity (excessive activity on the part of the child), seizure disorder , speech Impairment , hearing impairment , and visual impairment  .A total of 160 patients aged 9 months to 134 months were admitted with diagnosis of cerebral malaria during the study period; 131 (81.9%) survived while 29 (18.1%) died. Of the 131 survivors of cerebral malaria, 18 (13.7%) had neurological deficits at discharge. Four of the 18 survivors with neurological deficits at discharge were lost to follow up in the community while 6 (4.6%) survivors had persisting neurological sequelae for more than 6 months after discharge from the hospital.The 131 survivors were made up of 64 males and 67 females giving a male to female ratio (M:F) of 1:1.05. The age distribution of survivors shown in The survivors were followed up for a period that ranged from 6 months to 33 months with a median duration of 17.5 months from discharge to follow up. Eighteen (13.7 percent) of 131 survivors had neurological deficit(s) at discharge. Four of these were lost to follow up, giving an attrition rate of 22.2 percent among patients with neurologic deficit. Of the 131 survivors 6 had persisting neurological sequelae at 6 months, constituting 4.6 percent of all survivors and 33.3 percent of the 18 survivors with neurologic deficits at discharge. Thirty-eight neurologic deficits were identified among the 18 children at discharge. Of these thirty-eight deficits, 22 (57.9%) resolved completely while 9 (23.7%) persisted among 6 children. At follow-up, the most common persisting neurological deficit was memory impairment observed in 2 cases . All theThe 18 children with neurologic impairment at discharge had 38 neurologic deficits. The commonest neurological deficits at discharge were movement disorder and speech impairment in 9 children each, followed by visual impairment and memory impairment in 5 children each. Seizure disorder, hearing impairment, hemiparesis and quadriparesis occurred in one child each.No significant association was found between selected factors such as hypoglycaemia, severe anaemia, multiple seizures, under-nutrition, age, duration of coma among those with or without neurologic deficits at follow up. This result is shown in Neurological impairment following CM is not uncommon and a number of studies have documented these , 15, 16.The prevalence of persisting neurological deficits in survivors of cerebral malaria in this study was 4.6%. This is an improvement over 9.7% prevalence reported by Bondi in the same centre 30 years earlier but higher than 2.6% prevalence obtained by Meremikwu et al in Calabar, Nigeria , 15. TheThe persisting neurological deficits in this study were memory impairment, visual impairment, speech impairment, monoparesis, quadriparesis and hyperactivity. There was no persisting case of movement disorder which was the second commonest neurological deficit at discharge. Memory impairment was the most common persisting sequelae in this study. It usually becomes more obvious as survivors advance in age and face academic challenges. Our findings in the present study are similar to those of Cater et al at Kenya . Subtle The persisting neurological deficits found in this study, such as; ataxia, motor deficits, hearing impairment, visual impairment, speech difficulties, behavior difficulties, severe learning difficulties, seizure disorder, hemiplegia, spastic quadriplegia and cognitive impairment are very similar, with few exceptions, to those described in previous studies , 15, 16.The prevalence of seizure disorder in this study was 0.8%. This is higher than 0.2% reported by van Hensbroek et al in a study of 452 Gambian children who survived cerebral malaria . It is hA third of the survivors who had neurological deficit at discharge in this study continued to have deficit at follow-up. This is comparable to 34.4% reported by Brewster et al in The Gambia and 38% in Kenya by Carter et al but lower than 54.5% reported by Bondi in the same centre 30 years ago , 8, 9. TThere was no statistically significant association between selected factors such as hypoglycaemia, severe anaemia, multiple seizures, under-nutrition, age, duration of coma and the presence or absence of neurologic deficits at follow up. These are at variance to the commonly reported findings in other studies , 12, 17.It is concluded that, the present study shows that neurological sequelae is common among survivors of CM. A minority have neurological deficits persisting beyond 6 months with memory impairment being the most common. The most vulnerable age groups were the under-five.


Plasmodium vivax spreads out near demilitarized zone (DMZ). This study investigated the prevalence of P. vivax after the malaria transmission season in Gimpo-si where adjacent to DMZ of South Korea. An indirect fluorescent antibody test (IFAT) was performed to evaluate anti-malaria antibodies in blood samples.First reemerged malaria case was reported in 1993 after two decades absent in South Korea. Thereafter, P. vivax were detected using IFAT, and blood samples from antibody-positive cases were tested using a polymerase chain reaction (PCR) assay that detects malaria parasites.Microscopic examinations were performed to identify the presence of malaria parasites. Antibodies against P < 0.05). Sixteen of the IFAT positive samples  were positive for malaria DNA according to PCR. Blood samples with an antibody titer over 1:256 had high positivity rates in the PCR analysis (P < 0.05).A total of 5,797 blood samples were collected from residents in Gimpo-si. The positivity rate by IFAT was 2.16% (n = 125). Yangchon-myeon (3.28%) had the highest positivity rate of the seven administrative districts tested. Positivity rates increased with age (P. vivax in low endemic areas and could be used to detect asymptomatic patients. Finding asymptomatic patients is important in eliminating vivax malaria in South Korea.These results indicate that antibody titers obtained using IFAT may provide useful information about the prevalence of  Plasmodium vivax, a causative agent of relapsing benign tertian human malaria, is the second- most important human malaria and afflicts several hundred million people annually. This disease is a major public health problem, with associated socioeconomic ramifications, for many temperate and most tropical countries [ountries .et al reported two civilian patients infected with vivax malaria [For several centuries, vivax malaria has been prevalent throughout the Korean peninsula. The first scientific documentation of malaria was published in 1913. At that time, malaria occurred throughout the country without noticeable geographical distinctions . However malaria . Thereaf malaria . The norAnopheles sinensis, Anopheles kleini, and Anopheles pullus. Malaria transmission is expected to begin 3-4 weeks after the appearance of vector mosquitoes in mid-May. An. sinensis (63.3%) was the most abundant anopheline mosquito captured in malaria high-risk areas (northern Gyeonggi-do) near the DMZ in South Korea, followed by An. kleini (24.7%) and An. pullus (8.7%) [The rainy season usually begins in late June and extends through the end of July. In South Korea, the potential vectors of vivax malaria are s (8.7%) . The incGimpo-si is located in north-western South Korea and is surrounded by the Imjin River and the Han River, which are close to North Korea. The Korean National Institute of Health (KNIH) reported one case of malaria in 1995, one in 1996, 15 in 1997, and 65 in 1998. These cases led the malaria research team at the KNIH to investigate the prevalence of malaria in Gimpo-si using microscopy and indirect fluorescent antibody tests (IFATs). Seroimmunological diagnosis, in particular by IFAT, is an important tool for the detection of malaria, especially when microscopic evidence of the parasites is not available due to the several reasons -14. BlooThe study was conducted in Gimpo-si, Gyeonggi-do, South Korea, from late October to mid- December 1999. All participants were adult volunteers who were enrolled after the nature of the study was explained and verbal informed consent was obtained. Approximately 3 ml of blood was collected from each individual. Thin and thick blood smears were prepared for microscopic examination (magnification 7 × 100). The blood samples were transferred to the KNIH, Korea Centers for Disease Control and Prevention (KCDC), where the sera and blood were separated and stored at -20°C for future antibody and polymerase chain reaction (PCR) analyses. The study protocol was reviewed and approved by the Human Ethics Committee of the KNIH.P. vivax [P. vivax indigenous patients. After removing the plasma, the cells were suspended in phosphate-buffered saline  and centrifuged for 5 min at 2,500 rpm. The supernatant was discarded, and the cells were resuspended in fresh PBS. The wash step was repeated three more times. Finally, an appropriate amount of PBS was added to maintain the parasitemia at no less than 1%. Cells were added to each well of Teflon coated slides. After being dried at room temperature for 12 hrs, the slides were stored at -70°C. To determine the antibody titers against P. vivax for each patient, the antigen slides were fixed in pre-cooled acetone (-20 °C) for 10 min and washed with PBS; then 20 μl of 1:32 to 1:8,192 (vol/vol) was diluted sera was added to each well. Positive and negative controls were spotted onto each slide and incubated in a humidified chamber for 30 min at 37°C. The reactions were stopped by washing the reacted sera with PBS. The slides were immersed in PBS for 6 min and then dried at room temperature. Diluted FITC-conjugated anti-human IgG  was added to each well, incubated, and washed using the same method described above. Several drops of buffered glycerol were added to the samples, and the slides were covered with coverslips. The slides were examined under a 40× objective of a fluorescence microscope.To test for antibodies against malaria, an IFAT was performed with whole blood antigen against P. vivax -17. BrieGenomic DNA was extracted from the blood samples using a QIAamp Blood Kit (Qiagen). PCR was performed with AccuPower PCR Premix (Bioneer), 50 ng of purified genomic DNA, and 40 pmol each of each forward  and reverse  primer to amplify interspecies conserved block 10 (ICB10). The final volume was adjusted to 20 μl with distilled water. The cycling conditions were as follows: (1) denaturation at 94°C for 5 min, (2) 40 cycles of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C, and (3) incubation at 72°C for 5 min -21. PCR The annual parasite index (API) was calculated as the incidence of malaria per 1,000 residents.Relationships between antibody titer and PCR-positivity rate, positive rates of IFAT and age groups, and dates of blood collection and PCR-positive cases were analysed by correlation. Analysis of the annual parasite index (API) was carried out using Kruskal-Wallis test. Relationship between APIs and IFAT positivity rates of villages were analysed by Two-way ANOVA. Data analyses were performed using GraphPad .The location of the study area is shown on the accompanying map. Four villages, Gimpo 1-dong, Gimpo 2-dong, Sau-dong, and Pungmu-dong, are adjacent to each other and are collectively considered as Gimpo-dongs, resulting in seven sites located on the map .The criteria for a positive IFAT result were established in a previous experiment. Briefly, when 86 individuals and 58 vivax malaria patients were tested, normal individuals had a serum dilution under 1:16 (range from 0 to 1:16), and the 58 vivax malaria patients had a serum dilution over 1:256 (range from 1:256 to 1:4096). Therefore, a positive antibody response was defined against vivax malaria as a serum dilution above (≥ )1:32 . A totalP = 0.03), furthermore similar result was observed in API analysis (P = 0.003) , followed by 70-79 years . The lower positivity groups were 10-19 , 20-29 , 30-39 , and 90-99 years . These results indicate that the positivity rate of IFAT increased with age groups (P < 0.05). It showed a significant reduction of PCR-positive cases according to the dates of blood collection (P = 0.0013). Four PCR-positive cases were detected at first date of blood collection , 5 cases at second date , and no case at the end of blood collection . PCR-positive cases were detected until mid-November. After that time, no additional positive cases were found. Eleven PCR-positive cases (68.75%) were collected in October, and 5 cases (31.25%) were collected in November  antibody-positive cases were positive by PCR. Eight PCR-positive cases had an antibody titer of 1:32, four had a titer of 1:64, one had a titer of 1:256, one had a titer of 1:512, one had a titer of 1;1024, and one had a titer of 1:2048 Figure . The PCRP < 0.0001). APIs were increased by age groups. Age group 0-9 showed API as 0.06 ± 0.04, age group 10-19 as 0.41 ± 0.31, age group 20-29 as 0.57 ± 0.37, age group 30-39 as 0.81 ± 0.67, age group 40-49 as 0.96 ± 0.74, age group 50-59 as 0.95 ± 0.61, age group 60-69 as 0.84 ± 0.46, age group 70-79 as 0.99 ± 0.83 .Annual parasitic index (API) of Gimpo-si showed 0.54 ± 0.29 during ten years (from 1997 to 2006) after reemerging vivax malaria in Gimpo-si. Among them, Wolgot-myeon (1.61 ± 0.93) showed highest API, Haseong-myeon (1.42 ± 0.65) ranked second. These areas are closed to DMZ. Yangchon-myeon (0.62 ± 0.36) ranked third. Gochon-myeon (0.19 ± 0.16) and Gimpo-dongs (0.16 ± 0.07) have a lower APIs than other areas. These areas located far from DMZ among seven study areas  were shown to be infected with the parasite by nested PCR. They had no clinical signs or symptoms associated with P. vivax although one of the soldiers showed symptoms after a few weeks. These results indicate asymptomactic cases are present in the Korean population. Out of 1,713 soldiers who served in high-risk areas during P. vivax transmission season, 15% had antibodies for Pv200, merozoite surface protein-1 (MSP-1). Eleven of the 40 soldiers who later developed symptomatic malaria had elevated levels of anti-Pv200 antibody up to three months prior to the onset of symptoms. This result indicates that the P. vivax blood stage antigen may have increased during the early erythrocytic stage of infection, which indicates that the antibody test could be used in seroepidemiologic studies as an efficient tool for monitoring the prevalence of malaria in large cohorts. Considering the challenges in detecting malaria due to its long incubation period and the sometimes mild symptomatology of Korean P. vivax, anti-Pv200 ELISA and nested PCR could be valuable tools in a programme to systematically eradicate malaria from Korea [Previously, it was observed that 16.67% (4/24) of seropositive cases exhibited malaria symptoms in the following year (authors' unpublished data). This observation suggests that some seropositive cases may be asymptomatic. Those seropositive residents that did not exhibit malaria symptoms have a history of exposure to malaria parasites. It is not clear how many seropositive cases are asymptomatic. During the winter season, malaria parasites can survive mainly but not all in the liver as hypnozoites in human. Parasites cannot survive for long periods of time in human blood due to the host immune system; therefore, they reside in the liver, which allows the parasites to evade the immune system. Park om Korea .P < 0.05). PCR-positive cases were correlated with the pattern of the incidence of malaria. Patient cases were reported from March to November, with a peak in August [P < 0.05). Additionally, the positivity results for IFAT were similar to the API for 1999 (Table P < 0.05). In addition, the APIs from 1997 to 2006 supported the observation that the positivity rate for IFAT increased with age . During malaria transmission season, adults of this age often work from early in the morning to late at night in the fields without protection from mosquitoes. Children in the under-9 age group showed a high positivity rate (2.34%). The children are most likely to be exposed to malaria vectors with their parents during evening activities. However, the 10 to 39 age group showed a relatively low positivity rate; students and business workers normally belong to this age group. Therefore, this age group would be exposed less than other age groups to malaria vectors due to their lifestyles . 5,797 blood samples were collected from residents of Gimpo-si in 1999. 125 seropositive cases were detected, 12.80% (16 cases) of which were also positive by PCR. These results indicate that the antibody level titers obtained by IFAT may increase the likelihood of identifying asymptomatic patients. This technique will be helpful in the early eradication of malaria in Korea. Additional studies should be examine the relationship between antibody titer and PCR results, specifically whether high antibody titers obtained by IFAT increase the chances of finding PCR positive cases, as shown in Figure n August ,24, becay rate 2.4%. The cP. vivax malaria infections in epidemic areas and could be useful in diagnosing asymptomatic patients. It is important to find asymptomatic patients to eliminate vivax malaria, and the addition of a serological method to normal surveillance methods can increase the accuracy of the interpretation about the prevalence of malaria.The main finding from this study was that antibody detection using IFAT might provide information about the prevalence of malaria in certain areas. Thus, this information might help us to understand the prevalence of malaria in the past and in the future situations. Additionally, IFAT might provide useful information about The authors declare that they have no competing interests.HWL, HHK, and WJL conceived and designed the study and participated in the research. HWL wrote the manuscript. SMH, MYP, NRK, SHC, TSI, JYK, HK, and YS collected the blood samples in the field. MYP, HK, YS, HHK, and TSI performed the IFAT and PCR assays. HWL read all IFAT slides. JS, HWL, JKL, and WJL helped to design the study. All authors have read and approved the final manuscript.


Plasmodium, the apicomplexan parasite responsible for this disease, challenges efforts to develop new strategies to control the disease. Proteolysis is a fundamental process in the metabolism of malaria parasites, but roles for proteases in generating vasoactive peptides have not previously been explored.The malaria burden remains a major public health concern, especially in sub-Saharan Africa. The complex biology of Plasmodium parasites  internalize and process plasma kininogen, thereby releasing vasoactive kinins  that may mediate haemodynamic alterations during acute malaria. In addition, it was demonstrated that the P. falciparum cysteine proteases falcipain-2 and falcipain-3 generated kinins after incubation with human kininogen, suggesting that these enzymes have an important role in this process. The biologic activity of peptides released by Plasmodium parasites was observed by measuring ileum contraction and activation of kinin receptors (B1 and B2) in HUVEC cells; the peptides elicited an increase in intracellular calcium, measured by Fluo-3 AM fluorescence. This effect was suppressed by the specific receptor antagonists Des-Arg9[Leu8]-BK and HOE-140.In the present work, it was demonstrated by mass spectrometry analysis that In previously undescribed means of modulating host physiology, it was demonstrated that malaria parasites can generate active kinins by proteolysis of plasma kininogen. Plasmodium species and appear to be involved in several important aspects of parasite biology -BK (4 μM) and HOE-140 (10 μM). The antagonists were incubated for 10 min before the addition of: 20 μg kininogen (HK), 10 µL of culture supernatant after culturing 104P. falciparum for 30 minutes without HK (Pf - HK); 10 μL of culture supernatant after culturing 104P. falciparum for 30 minutes with HK (1.9 μg/μL) (Pf + HK); or 5 μM THG. Fluorescence data were normalized as F/Fmax, where Fmax represents maximal intracellular fluorescence obtained with Ca2+ released from ER with addition of THG (Ca2+ATPase inhibitor).Human umbilical vein endothelial cells were cultured in RPMI 1640 (GIBCO) containing 10% heat-inactivated fetal bovine serum (FBS), 24 mM sodium bicarbonate, 40 mg/mL gentamicin, 10 mM HEPES, pH 7.4 at 37°C in a 5% COPlasmodium falciparum and Plasmodium chabaudi isolated trophozoites were incubated with a synthetic fluorogenic kininogen fragment, which contains the bradykinin sequence , according to their calculated molecular mass  were measured. When HK alone or d Figure . When th9[Leu8]-BK and the B2 receptor antagonist HOE-140 before stimulus and then measured intracellular fluorescence . This processing is inhibited by the cysteine protease inhibitor E-64 and reproduced in vitro by isolated falcipain-2 and falcipain-3, suggesting roles for these proteases in kininogen processing. Kininogen peptides were biologically active, as demonstrated by their ability to induce ileum contraction and activate endothelial cells via calcium signaling, both principally through B2 receptor activation.The results reported here show that The processing of plasma macromolecules by the malaria parasites has already been reported -35. Howein vitro suggests a second physiologic function for these proteases in addition to the hydrolysis of haemoglobin [Plamodium falciparum aminopeptidase PfAPP was shown to localize to the food vacuole and cytosol of the parasite [The processing of a plasma protein by falcipains moglobin . Interesparasite , suggestparasite . The mas2+ release in endothelial cells and this process was also blocked by HOE-140. Thus, proteolysis of kininogen by malaria parasites generates biologically active peptides. This effect may explain clinical features of malaria, which is commonly accompanied by alterations in vascular haemodynamics.It was of interest to determine if peptides generated by parasites and isolated falcipains possess biological activity and exert a specific physiologic effect. Alternatively, they might be generated simply for acquisition of nutrients, as appears to be the case for haemoglobin. Indeed, generated peptides promoted guinea pig ileum contraction, which was abolished in the presence of the B2 receptor antagonist HOE-140. Further, the peptides stimulated CaAnother potential role for BK and B2 receptor stimulation is attenuation of host systemic damage during infection by anti-apoptotic or other protective effects. Similar protective effects were observed in cardiac, renal and liver damage models . For insPlasmodium infection. This process appears to depend on endothelial cell activation, which may be promoted by proinflammatory agents such as cytokines (e.g. Tumor Necrosis Factor), chemokines and infected erythrocytes [In the late 60s, some studies suggested that kinins could be involved in the pathological manifestations of malaria -29. Howehrocytes ,35,38. Throcytes . Nitric hrocytes  and may Plasmodium is shown in Figure Taken together, the results reported point to a potential role for the hydrolysis of HK by malaria parasites in the pathophysiology of malaria. The hydrolysis of plasma proteins suggests an intricate relationship between host and pathogen, and may represent a key adaptation by malaria parasites to facilitate host infection. A schematic model of endothelial cell activation by kinin peptides generated by Piero Bagnaresi, Nilana MT de Barros, contributed equally to this workThe authors declare that they have no competing interest.PB carried out the confocal microscopy experiments, participated on the design of the study and drafted the manuscript. NMTB carried out the ileum contractions assays, participated on the design of the study and drafted the manuscript. DMA carried out the spectrometry analysis. PMSM participated in spectrometry assays and microscopy. RPG, JBP, MAJ, LJ and PJR participated in the design of the study. AKC and MLG conceived and coordinated the study, and helped to draft the manuscript. All authors read and approved the final manuscript.


Malaria is a major cause of pediatric morbidity and mortality. No clinical features clearly differentiate malaria from other causes of pediatric febrile illness. Lack of laboratory equipment and expertise makes malaria diagnostics a challenge, leading to overdiagnosis and overtreatment of malaria. Molecular methods (PCR) are emerging but have so far not proven practical for routine clinical use. Emerging antimalarial and antibiotic resistance calls for precise diagnostics and treatment of febrile illness, and has led WHO to recommend laboratory confirmation of malaria in children before starting treatment.Children admitted with fever were recruited consecutively among admissions at the general pediatric wards at Muhimbili National Hospital (MNH) in Dar es Salaam Tanzania from January-June 2009. Clinical, demographic and laboratory features were registered. Microscopy of thick blood smears was done as part of the routine at MNH, and thin blood smears were stained and examined later. Retrospectively, genus-specific PCR of Plasmodium mitochondrial DNA was performed on DNA extracted from whole blood for all patients and species-specific PCR was done on samples positive by genus-specific PCR. Univariate and multivariate statistical analysis was performed using IBM SPSS Statistics version 19 .P. falciparum. PCR results confirmed positive routine microscopy in only 52,5% and research microscopy in 100%. Almost every fifth febrile child (55/304) had positive PCR but negative research microscopy. High parasitemia on routine microscopy was associated with positive research microscopy and positive PCR. The true prevalence of malaria in the population remains unknown as none of the diagnostic methods can be interpreted as a true gold standard. Palmar pallor, low hemoglobin and low platelet count were significantly associated with both positive PCR and positive research microscopy (p<0.001). In hospital, 65.1% received antimalarial treatment. Clinically determined severity of palmar pallor was clearly associated with hemoglobin level.The study included 304 children. Within four weeks before admission 62.6% had received antimalarials. Forty children had positive routine thick blood smears upon admission and twenty had positive research thin blood smears upon retrospective examination. Twenty-five percent had positive PCR, all positive for The study identified discrepancies between routine malaria microscopy, research malaria microscopy and PCR. Almost half of routine microscopy positive cases were negative on PCR, indicating prevalent overdiagnosis of malaria. PCR was positive for many research microscopy negative cases. This may in part be due to prevalent treatment with antimalarials before admission. Palmar pallor and low hemoglobin levels were predictors for malaria in this study. The current routine diagnostic method for malaria appears to lead to overdiagnosis of malaria and, consequently, overuse of antimalarials. Conversely, children with false positive malaria diagnosis may die because they do not receive treatment for the true cause of their illness. Malaria is still a prominent health issue in Tanzania, and the uncertainty of both clinical and routine laboratory tests may lead to misuse of antimalarials and antibiotics and consequently contribute to emerging drug resistance. Diagnostic algorithms employing new methods such as rapid diagnostic tests (RDTs) may contribute to improving malaria diagnosis and treatment.


Malaria contributes to high childhood morbidity and mortality in Nigeria. To determine its endemicity in a rural farming community in the south-south of Nigeria, the following malariometric indices, namely, malaria parasitaemia, spleen rates, and anaemia were evaluated in children aged 2–10 years. This was a descriptive cross-sectional survey among school-age children residing in a rubber plantation settlement. The children were selected from six primary schools using a multistaged stratified cluster sampling technique. They were all examined for pallor, enlarged spleen, or liver among other clinical parameters and had blood films for malaria parasites. Of the 461 children recruited, 329 (71.4%) had malaria parasites. The prevalence of malaria parasitaemia was slightly higher in the under fives than that of those ≥5 years, 76.2% and 70.3%, respectively. Splenic enlargement was present in 133 children (28.9%). The overall prevalence of anaemia was 35.7%. Anaemia was more common in the under-fives (48.8%) than in those ≥5 years (32.8%). The odds of anaemia in the under fives were significantly higher than the odds of those ≥5 years (OR = 1.95 [1.19–3.18]). Malaria is highly endemic in this farming community and calls for intensification of control interventions in the area with special attention to school-age children. Malaria remains a leading cause of illness and death in sub-Saharan Africa with the greatest risk seen in children under the age of five, pregnant women, and people living with HIV/AIDS , 2. AbouAn understanding of the malaria burden in a given setting is important for health planning, policy development, and control interventions. This study aimed at determining the malaria burden at Ikot-Omin, a rural rubber plantation settlement, using malaria parasitaemia, spleen rate and anaemia as malariometric indices.Anopheles gambiae and Anopheles funestus. Parasite resistance to chloroquine and sulfadoxine/pyrimethamine was over 80% ) (A positive film was reported in 329 (71.4%). All had 9–3.18]) .t test = 0.02; P value = 0.98) as shown in There was no significant difference in the mean haematocrit value between the parasitaemic and aparasitaemic children . The results of a more recent study, National Malaria Indicator Survey (MIS), have shown an improvement in this situation with a national average prevalence of malaria parasitaemia in preschool children of 42% [The high prevalence of malaria and n of 42% . The res The overall spleen rate in this study is 28.9%. The under fives had a marginally higher spleen rate (31%) compared with those of 5−10 years of age (28.4%). These findings buttress the fact that under fives have a more pronounced immunological response (including splenic enlargement) to malaria than the older children . The splP. falciparum infection than others with severe and potentially fatal complications ensuing [Anaemia occurred in 35.7% of the study population with the under fives having a higher prevalence (48.8%) compared with children of five years and above (32.8%). The under fives were about twice more likely to be anaemic compared with those of five years and above. These observations agree with the fact that this age group is more vulnerable to  ensuing . The pre ensuing . The dif ensuing . Studies ensuing , 23. Die ensuing , 23. This study shows a marked disparity in the malariometric indices assessed in the children. The highest being the prevalence of malaria parasitaemia (71.4%), followed by anaemia (35%), and then spleen rate (28.9%). The prevalence of malaria parasitaemia may have been influenced by antimalarial drug use in the community, anaemia by helminthic infestation and dietary intake and spleen rate by the extent and duration of exposure to the parasite , 25. MalThere was no statistically significant difference in the mean haematocrit value between parasitaemic and aparasitaemic children in this study, though some studies have reported such a difference between them , 23. TheThis study shows that malaria remains a major health problem among school-age children residing in agricultural settlements in the south-south of Nigeria. It is therefore necessary to intensify malaria control efforts with special attention given to school-age children residing in at-risk environments. Further studies on aetiology of anaemia in children using a uniform methodology are necessary for the development of effective preventive and therapeutic interventions for anaemia in school-age children residing in such environments.


The diagnosis of malaria can be difficult in non-endemic areas, such as the United States, and delays in diagnosis and errors in treatment occur too often.A nationwide survey of laboratories in the United States and its nine dependent territories was conducted in 2010 to determine factors that may contribute to shortcomings in the diagnosis of malaria. This survey explored the availability of malaria diagnostic tests, techniques used, and reporting practices.The survey was completed by 201 participants. Ninety percent reported that their laboratories had at least one type of malaria diagnostic test available on-site. Nearly all of the respondents' laboratories performed thick and thin smears on-site; approximately 50% had access to molecular testing; and only 17% had access to rapid diagnostic tests on-site. Seventy-three percent reported fewer than five confirmed cases of malaria in their laboratory during the 12-month period preceding the survey. Twenty-eight percent stated that results of species identification took more than 24 hours to report. Only five of 149 respondents that performed testing 24 hours a day, 7 days a week complied with all of the Clinical and Laboratory Standards Institute (CLSI) guidelines for analysis and reporting of results.Although malaria diagnostic testing services were available to a majority of U.S. laboratories surveyed, very few were in complete compliance with all of the CLSI guidelines for analysis and reporting of results, and most respondents reported very few cases of malaria annually. Laboratories' difficulty in adhering to the rigorous CLSI guidelines and their personnel's lack of practice and proficiency may account for delays and errors in diagnosis. It is recommended that laboratories that infrequently process samples for malaria seek opportunities for practice and proficiency training annually and take advantage of available resources to assist in species identification. Malaria continues to be endemic in more than 100 countries worldwide, where it remains a leading cause of morbidity and mortality . An averIn a case series of imported malaria in the late 1990s in Canada, up to 92% of patients who presented to physicians without expertise in tropical medicine experienced delays in diagnosis due to physician failure to consider malaria as a diagnosis on initial presentation, laboratory error in recognition and species identification, or administration of incorrect therapy to treat the parasite(s) identified . A decliThe Clinical and Laboratory Standards Institute's (CLSI) recommendations for laboratories performing malaria diagnostic testing include :1. Make diagnostic testing available 24 hours a day, seven days a week.2. Prepare at least three thick and thin smears.Plasmodium vivax and Plasmodium ovale and can play a crucial role in identification of these species. Although parasites are visible by Wright's and Wright-Giemsa stains, these stains do not allow visualization of stippling.3. Use Giemsa stain for definitive diagnosis. It allows proper visualization of stippling (Schűffner's dots) in 4. Examine at least 300 fields using the 100× oil immersion objective.5. Report microscopy results immediately to the requesting physician or ward.6. Examine at least 10 fields on the thin film to determine percent parasitaemia; many more fields should be examined for patients from the U.S. who typically have lower parasitaemia (CDC recommends counting parasitized red blood cells (RBCs) among 500-2,000 RBCs on the thin smear ).A preliminary report of positive or negative should be available within four hours; percent parasitaemia should be reported within six hours and species identification within 24 hours.The focus of this survey was to identify laboratory practices that could contribute to delayed or incorrect diagnoses. The goal was to determine the proportion of laboratories in the United States that perform diagnostic malaria testing using procedures that might contribute to diagnostic delays.A convenience sample of laboratory-related personnel was obtained through a nationwide survey of U.S. laboratories to determine the practices for malaria diagnostic testing, including availability of diagnostic tests, time required for reporting, and test methodologies used . This study was submitted to the Institutional Review Board of Emory University and deemed exempt from review. Informed consent was implied by response.A total of 278 participants initiated the survey and 201 (72.3%) completed the survey. Respondents were from laboratories located in 46 of the 59 (78%) states and territories, including 30 respondents in California, 22 in New York, 14 in Texas, and 12 in Ohio  reported that their laboratory provided diagnostic testing either in-house or via on-call personnel, 24 hours a day, seven days a week. This number included 96% of respondents from university hospital laboratories, 83% from commercial referral laboratories, 82% from community hospitals, and 100% from other laboratories. The remaining 15% worked in laboratories with personnel who could perform diagnostic testing only during the 8- to 12-hour work day.Sixty-nine respondents 32%) noted that their laboratory received <10 specimens for malaria diagnostic testing within the 12 months preceding the survey. Twenty-seven (13%) received a large volume, with >100 specimens sent for analysis; however, most of these specimens were negative. Only 10 (~5%) had >15 confirmed cases, while 158 (73%) respondents noted that their laboratory had five or fewer confirmed cases of malaria during the 12 months preceding the survey. The type of diagnostic testing available on-site is shown in Figure 2% noted Of the 202 respondents in laboratories where light microscopy was performed on-site, nearly half (47%) used Giemsa stain in at least one step of the analysis of thick and thin smears; several others used more than one of three staining techniques: Giemsa, Wright-Giemsa, and Wright stain. Two respondents reported using acridine orange stain; in one laboratory, this was the only stain used, and in the other, positive staining with acridine orange was followed by Wright-Giemsa staining.Eighty-three respondents (46%) reported that their laboratory reviewed ≥three slides from a single patient's sample before a specimen was considered negative; this was true for 44% of university and 53% of community hospital respondents. A minority, 16 (~9%), reported that their laboratory reviewed only one slide to determine a negative test. The majority of respondents, 112 (63%), reported that their laboratory reviewed ≥300 high power fields (HPF) prior to determining that a slide was negative. This was the case in 75% of respondents from commercial referral laboratories, 68% from university hospitals, and 67% from other laboratories; 12 (~7%) reviewed <100 HPF to come to this conclusion.The majority of respondents, 91 (58%), reported that 500-2,000 RBCs were counted to determine percent parasitaemia. This included 67% of respondents from public health laboratories, 60% from university hospital laboratories, and 71% from other laboratories. Fourteen percent of respondents reported their laboratories counting >2,000 cells, while 14% reported counting <500 cells.The majority of respondents, 115 (64%), noted that a preliminary result was reported within 4 hours of receiving the specimen. This was the case for 74% of respondents from university hospitals, 71% from public health laboratories, and 62% from community hospitals. Approximately seven in 10 respondents (126) reported that at least two personnel  reviewed a slide prior to reporting a negative result; the remaining respondents reported that only a single technologist reviewed the slide. In contrast, most respondents, 127 (71%), reported that positive results were confirmed by a pathologist or laboratory director/supervisor. A little over half of the respondents, 93 (52%), noted that their laboratory reported percent parasitaemia within six hours. A majority of respondents from university (59%) and public health (71%) facilities stated that percent parasitaemia was reported within this time period.A species-specific diagnosis was possible in-house for 144 (89%) respondents. For 133 (70%) respondents, a species-specific diagnosis was made within 24 hours; half of these were made within 6 hours after receipt of specimen. Species identification was available within 6 hours according to 67% of respondents from public health laboratories, 40% from university hospitals, 29% from community hospitals, and 57% from other laboratories. Twenty-eight percent reported that a species diagnosis took >24 hours.In 102 (63%) respondents' laboratories, species identification was performed in-house by either a microbiology director/supervisor or a pathologist. Approximately 40% noted that species identification was performed in-house and subsequently confirmed at an outside laboratory, such as the state or local health department or CDC. Eleven percent reported that species identification was done only at an outside laboratory.Of the 149 respondents who reported that their laboratory provided diagnostic testing either in-house or via on-call personnel, 24 hours a day, seven days a week, using the strict definition of immediate preliminary reporting within four hours, percent parasitaemia within six hours and species identification within 24 hours only five were in complete compliance with all six CLSI guidelines  respondents reported that their laboratory provided only send-out diagnostic testing for malaria. Seventeen (60%) were affiliated with community hospitals, five (18%) with public health laboratories, and two each with commercial referral and other laboratories. Of the 27 respondents whose laboratories offered only send-out testing for malaria who completed the survey, 85% reported receiving <10 specimens for malaria diagnostic testing within the 12 months preceding the survey. Only one respondent stated his laboratory received >100 specimens. Twenty-five (93%) reported ≤five confirmed cases of malaria; half of these respondents had seen no positive cases of malaria in their laboratory in the past year.The diagnostic tests available to the laboratories where only off-site testing is performed are shown in Figure It is undeniable that there are significant challenges in diagnosing malaria, especially in areas where it is not endemic. Laboratory challenges in the diagnosis of malaria include patients' low parasite density, altered parasite morphology due to the patients' use of chemoprophylaxis, empiric therapy, mishandling of specimens, and inexperience in identifying malaria parasites due to the small number of cases seen . The comet al analysing the proficiency of U.S. laboratories to identify malaria parasites reported a failure rate of 11.2% for identification of Plasmodium falciparum [P. falciparum, improvement is needed.A recent retrospective review by Edson lciparum . Althouglciparum ,14. HoweIn this study, the majority of laboratories surveyed reported using both thick and thin smears; very few respondents reported the use of thin smears alone, an inferior technique. It is important that both thick and thin smears be evaluated in the examination of blood specimens for malaria. Thick smears are useful for detecting parasites, while thin smears are useful for species identification and quantification of parasites.A few respondents reported that diagnostic testing was available only during the eight- to twelve-hour work day with no access to on-call personnel during the off-hours. Malaria is a potentially life threatening disease: therefore, it is recommended that all blood films be read immediately. CLSI recommends that 'any laboratory providing the expertise to identify malarial parasites should do so on a 24-hour basis, seven days a week' . TherefoP. falciparum is present) but none are able to provide quantification of parasitaemia. There are many brands and types of RDTs worldwide, but only one is FDA-approved for diagnostic use in the U.S.: BinaxNOW Malaria® . This test targets both the histidine-rich protein II (HRPII) antigen specific to P. falciparum and a malarial aldolase common to all human malaria species; it is therefore able to provide a diagnosis of malaria as well as determine specifically if P. falciparum is present[Very few respondents reported availability of serologic testing in their laboratories; several other respondents reported its availability as a send-out test. Serologic testing is used only for retrospective diagnosis of past malaria infection  and is ns present. HoweverThe detection of parasites in non-immune individuals who may be symptomatic at very low parasite densities is an additional barrier; unless enough fields are counted, these specimens may be mistakenly read as negative. In such cases it is imperative that repeat blood smears be performed. Standard guidelines recommend that a total of three specimens be obtained before declaring a patient does not have malaria. It is important to note that ensuring that an adequate number of specimens are sent is the responsibility of the physician; however, the laboratory may assist the physician with a reminder that three specimens must be sent to guarantee an accurate result.Any laboratory with the capacity to perform a complete blood count with manual differential has the tools necessary to make a diagnosis of malaria and to report the percent parasitaemia , it is impossible to calculate the true response rate, and our results may not be reflective of practices at all laboratories across the U.S. As with any survey, there is the potential for respondent bias. Although the responses received were from multiple different types of laboratories in a wide geographic distribution, it is possible that laboratories with more sophisticated testing for malaria or those who receive more samples for malaria may have been more likely to respond. Therefore, this data may represent the best case scenario rather than what occurs at the majority of laboratories.The names of laboratories and affiliated institutions were not obtained; this was intentionally done in order to maintain the confidentiality of the respondents. However, this poses a limitation, as more than one member of a laboratory may have submitted answers to the survey. It is for this reason that the results are reported based on the number of respondents rather than the number of laboratories.The results of this survey attest to the availability of services for malaria diagnostic testing in U.S. laboratories, showing that the delays and accuracy of diagnosis may be due to lack of practice and proficiency as well as the difficulty in complying with the rigorous guidelines set by the CLSI. Even in laboratories with large volumes of samples processed for malaria parasite detection, each individual staff member likely sees very few cases per year; consequently, it is imperative that all laboratories, especially those that infrequently process samples for malaria seek opportunities for practice and proficiency training annually (Additional file CDC: Centers for Disease Control and Prevention; CLSI: Clinical and Laboratory Standards Institute; DPDx: Division of Parasitic Disease Diagnosis; ELISA: Enzyme-linked immunosorbent assay; FDA: Food and Drug Administration; HPF: high power fields; HRP II: histidine-rich protein-2; IFA- indirect fluorescent antibody; PCR: polymerase chain reaction; RBCs: red blood cells; RDT: rapid diagnostic test.The authors declare that they have no competing interests.FA produced and disseminated the surveys, collected and analyzed the data, and drafted the manuscript. PA participated in the design of the survey and participated in drafting the manuscript. JG conceived of the study, participated in the design of the survey, and participated in drafting the manuscript. All authors read and approved the final manuscript.Malaria Diagnostic Survey Questionnaire.Click here for fileMicroscopic Procedures for Diagnosing Malaria.Click here for fileSelect Opportunities for Practice and Additional Training for Laboratory Diagnosis of Blood and Tissue Parasites.Click here for file