This study examines the relative importance of living in an urban versus rural setting and malaria in contributing to the public health problem of malarial anaemia (MA) and anaemia respectively in apparently healthy primary school children.A cross-sectional study was conducted among 727 school children aged between four and 15 years living in an urban (302) and rural (425) settings in the Mount Cameroon area. Blood sample collected from each child was used for the preparation of blood films for detection of malaria parasites and assessment of malaria parasite density as well as full blood count determination using an automated haematology analyzer. Based on haemoglobin (Hb) measurements, children with malaria parasitaemia were stratified into MA (Hb<11g/dL); mild MA (Hb of 8–10.9g/dL); moderate MA (Hb of 6.1–7.9g/dL) and severe MA (Hb≤6g/dL). Evaluation of potential determinants of MA and anaemia was performed by multinomial logistic-regression analysis and odds ratios used to evaluate risk factors.2 = 36.5, P <0.001; χ2 = 16.19, P <0.001 respectively) in children in the urban  than in the rural area . Majority of the MA cases were mild (88.9%), with moderate (5.6%) and severe MA (5.6%) occurring in the urban area only. The age group ≤6years was significantly (P <0.05) associated with both MA and anaemia. In addition, low parasite density was associated with MA while malaria parasite negative and microcytosis were associated with anaemia.Out of the 727 children examined, 72 (9.9%) had MA. The prevalence of MA and anaemia were significantly higher (χMalarial anaemia and anaemia display heterogeneity and complexity that differ with the type of settlement. The presence of severe MA and the contributions of the age group ≤6 years, low parasite density and microcytosis to the public health problem of MA and anaemia are noteworthy. Plasmodium falciparum causes the most severe anaemia, with a significant risk of death  x 100 where n0 = anaemic children without malaria, n1 = anaemic children with malaria, whereby n0 + n1 = n; m0 = non-anaemic children without malaria, and m1 = non-anaemic children with malaria, whereby m0 + m1 = m [Data was doubly entered into spread sheets using Microsoft Excel and validated using SPSS version 17. Analysis was done with the IBM statistical package for social sciences (SPSS) version 19 . Data was summarized into means and standard deviations (SD), and percentages were used in the evaluation of the descriptive statistics. Proportions were compared using the Chi-square test  in children who were malaria parasite positive  when compared with their negative counterparts .The AR of anaemia caused by malaria was 24.5%.Out of the 727 primary school children evaluated for malaria related anaemia in the urban and rural settings in the Mount Cameroon area, 443 were in the 7–10 years age group and majority  of them were of poor SES. The proportions of males  and females  in the study population were comparable as seen in Children in the urban set up had a significantly higher prevalence of malaria parasitaemia, AM, UM, anaemia, MA and microcytosis when compared with those in the rural area as shown in Parasitaemia of low density was significantly higher in children in the urban area  than the rural , while the prevalence of moderate and high parasite density was higher in children in the rural  than the urban area  as shown in 2 = 36.5, P <0.001; χ2 = 16.19, P <0.001 respectively) in children in the urban  than in the rural area . In the urban, but not in the rural area, the prevalence of MA decreased with improved SES . In children in the urban area, the AR of anaemia caused by malaria was 15.7% while in children in the rural area it was 16.1%.The prevalence values of MA and anaemia were significantly higher  and 0% while that of severe anaemia was 1.7% (CI = 0.7–3.8%) and 0.2% (CI = 0.04–1.3%) respectively. Although all the MA cases in the rural area were mild, the prevalence of mild to SMA did not differ significantly (P >0.05) between children in the urban and the rural areas. However, the prevalence of NSMA was significantly higher (P <0.01) in children in the urban than in the rural area .Malarial anaemic children in the rural area had significantly higher mean Hct (33.9%) and MCV (85.8 fl) than those in the urban areas (27.7% and 73.8 fl respectively). On the other hand children in the urban area had significantly higher MCHC (338 g/dL) than those in the rural area (302.7 g/dL) as shown in Children in the ≤6 years age group had the highest prevalence of malaria parasites  and GMPD  when compared with the other age groups. The differences were statistically significant  as seen in The prevalence of low, moderate and high parasitaemia in the population was 86.1% (CI = 81.2–89.9%), 9.8% (CI = 6.7–14.2%) and 4.1% (CI = 2.2–7.4%) respectively. The variations in prevalence of the different categories of parasite densities were comparable (P = 0.64) in the different age groups as shown in 2 = 12.5, P = 0.002) in children with high parasite density  had MA. Anaemia prevalence increased with the parasite density category. The prevalence of anaemia was significantly highest (χ density .Children with MA had a significant reduction (P <0.01) in mean Hct and RBC counts when compared with their NMA counterpart. On the other hand, the MCHC was significantly higher (P <0.001) in children with MA than those with NMA as shown in The different categories of age groups, sex, clinical status, parasite density, malaria parasite status, MCV, MCHC, SES and setting were entered into the stepwise multinomial logistic regression model. The age group ≤6years was significantly (P <0.05) associated with both MA and anaemia. In addition, parasitaemia density < 1,000 parasites/μL of blood (low) was associated with MA while malaria parasite negative and microcytosis were associated with anaemia .Data on malarial anaemia is lacking in most transmission settings even though anaemia has been reported  as a comUrbanization may alter the frequency and transmission dynamics of malaria. The higher prevalence of malaria in urban (60.6%) when compared with the rural (14.8%) area may be attributed to favourable climatic conditions such as high temperatures and rainfall which promote the rapid growth of the anopheline vectors. Malaria transmission in urban areas varies according to a location , climate, land use, human movement patterns, socioeconomic factors, local vector species, vector breeding sites, waste management, and local malaria intervention programs . The preObservations from the study revealed that children from the rural area had a significantly higher level of haemoglobin than their urban counterparts even though the difference was not statistically significant when only malarial anaemic children in the different settings were compared. The higher mean Hb concentration in children in the rural area coupled with a higher GMPD/μL of blood probably reflects the impact of lesser malaria transmission intensity when compared with the urban area. In areas of endemicity, the rate of exposure to infected mosquitoes has been correlated with the density but not the prevalence of parasitemia . In addiet al. [et al. [dMA, SMA, NSMA, moderate and severe anaemia were higher in children in the urban than rural area even though, the AR of anaemia caused by malaria were comparable in both areas. The presence of SMA in school children in the urban area with regular school attendance is a cause for concern as SMA increases the risk of death in children [The overall prevalence of anaemia (19.8%) was higher than that obtained by Kimbi et al.  in schoo [et al.  in Mozamchildren . Severe children ,33 and bchildren ,35. The children  or impaiet al. [et al. [The prevalence of MA decreased with improved socio-economic status in the urban area, but not in the rural highlighting the problem of unregulated rapid urban growth on the health of the population. Buea municipality has experienced a rapid growth in infrastructure, population and socio-economic changes within the last decade although the surrounding area from which the study population was drawn still remains largely rural. This has also been accompanied by the institution of a monthly clean-up campaign to improve on the environmental conditions. In line with Tatem et al.  and Kimb [et al. , the enhThe decreased prevalence of malaria parasite, GMPD/μL of blood, anaemia and MA with an increase in the age group is not unusual as age has been associated with a decrease in disease severity . The ageet al. [Parasite density may be used as a surrogate marker of transmission intensity in malaria endemic areas . The siget al. , the maiet al. . Also, tet al. . FurtherP. falciparum and anaemia has been highlighted by Biemba et al. [Although the prevalence of anaemia increased significantly with the parasite density category , the loga et al.  with sevet al. [Even though observation from the study suggests parasite density of the lowest category to be significantly associated with malarial anaemia, other causes of anaemia cannot be excluded. However, it is possible that the lower- parasitaemia density could have kept the children in a sub-optimal haematologic state that prevented them from attaining the more favourable Hb concentrations of parasite-free children. McElroy et al.  reportedP. falciparum. Thrombocytopenia and anaemia have been commonly observed in malaria infected individuals in other studies [The significant reduction in some red cell indices (RBC and Hct) and increase in MCHC observed in children with MA is not unexpected. Haematological abnormalities are considered a hallmark in infection with  studies ,42,43. Fet al. [et al. [Plasmodium falciparum and may reduce the risk of anaemia by other Plasmodium species, as well as other causes of anaemia. Also, they further stated other host polymorphisms that induce an increased erythrocyte count and microcytosis may confer a similar advantage.Findings from the study are consistent with previous reports that have associated microcytosis with lower haemoglobin levels , 45. Micet al.  who alsoet al. . However [et al.  assert tThe finding in the overall population that children who were malaria parasite negative were significantly associated with anaemia is unexpected and in contrasts with the main literature ,34,35. GMalarial anaemia and anaemia display heterogeneity and complexity that differ with the urban setting hence, its implication in control efforts. Malarial anaemia decreased with improved socioeconomic status in children living in urban areas accentuating the need for a regulated growth in urban areas. The presence of SMA and the contributions of age group ≤ 6 years, low parasite density and microcytosis to the public health problem of MA and anaemia in healthy children with regular school attendance are noteworthy. Nonetheless, further investigation on the contribution of microcytosis, iron deficiency and thalassaemia to these health problems are crucial.


Plasmodium falciparum malaria. Insulin resistance and beta cell function were computed by homeostatic models assessment of insulin resistance (HOMAIR) and beta cell function (HOMAB) formulae, respectively. Body mass index (BMI) was computed. At baseline, diabetics had significantly (P < 0.05) higher levels of BMI, FBG, HbA1c, and HOMAIR but lower level of HOMAB than controls. Baseline insulin levels were comparable (P > 0.05) between the two study groups. During malaria, diabetics maintained significantly (P < 0.05) higher levels of BMI, FBG, and HbA1c but lower levels of insulin and HOMAB than controls. Malaria-induced HOMAIR levels were comparable (P > 0.05) between the two study groups but higher than baseline levels. Apart from BMI and HOMAB, mean levels of all the remaining parameters increased in malaria-infected controls. In malaria-infected diabetics, significant (P < 0.05) increase was only observed for insulin and HOMAIR but not the other measured parameters. Uncomplicated malaria increased insulin resistance in diabetics and controls independent of BMI. This finding may have implications for the evolution of T2DM in malaria-endemic regions.The study aimed at investigating the effects of adult uncomplicated malaria on insulin resistance. Fasting levels of blood glucose (FBG), glycosylated hemoglobin (HbA1c), and serum insulin were measured in 100 diabetics and 100 age-matched controls before and during  Malaria and type 2 diabetes mellitus (T2DM) continue to affect millions of people globally. Whereas T2DM can be considered a truly global phenomenon, malaria seems to be concentrated in relatively less developed economies of the globe. This implies that the economically less developed continents of the globe have to battle with the negative impact of malaria and T2DM separately or synergistically on the health of the populace.Malaria is an intricate disease viewed as the most detrimental and pervasive parasitic infection worldwide . It impiIronically, current report by the International Diabetes Federation (IDF) indicates that the incidence of diabetes is increasing globally . It is pInsulin resistance, which signifies reduced sensitivity of insulin-sensitive cells to insulin-mediated actions, has long been recognized as a key factor in the pathogenesis of T2DM . In a prApart from a study in Sudan that investigated insulin resistance in the context of childhood malaria , all othTo the best of our knowledge, no study has considered the effect of malaria on insulin resistance in adult diabetics and nondiabetic controls. In view of the predicted increased incidence of T2DM in sub-Saharan Africa, a continent that is still grappling with malaria-imposed health challenges, coupled with a recent finding that diabetics are more prone to getting malaria , we soug2 with Cape Coast as its capital [The study was carried out at the Diabetic Clinic of CCTH in the Cape Coast Metropolis. CCTH serves as the referral hospital for the various health facilities in the region. It has a well-structured and active Diabetic Clinic with patients from various parts of the region. The Central Region shares boundaries with Ashanti and Eastern regions to the north, Western region to the west, Greater Accra region to the east, and the Atlantic Ocean to the south. It covers an area of 9,826 km capital . The reg capital . Ecologi capital . The peo capital .The study was an outpatient-based comparative type that followed type 2 diabetics and nondiabetic controls over a two-year period for malaria infection. The inclusion criteria used in selecting study participants were being 40 years or older, nonalcoholic, nonsmoker, not suffering from any cardiovascular disease, hepatitis B, C, and HIV, not pregnant, not nursing a baby, with no evidence of hepatic dysfunction, with absence of severe arthritis, lupus, or inflammatory bowel disease, and not undergoing hormonal therapy or having any medical condition that can significantly influence the levels of any of the biomarkers measured.Diabetics were randomly selected from a database of registered diabetes patients attending the diabetic clinic at CCTH and invited to participate in the study. Controls from the general inhabitants of the Cape Coast metropolis were age-matched with the diabetic group. In all, 200 respondents consisting of 100 diabetics and 100 nondiabetic controls were enrolled for the study in 2011. Before and during malaria infection, 10 mL of venous blood sample was obtained after overnight fast. Blood sample was separated into plasma and serum for determination of various biochemical indices. Whereas plasma samples were analyzed the same day, serum samples were aliquoted appropriately and stored at −80°C till further analysis. Weight and height were measured for computation of body mass index (BMI) using standard protocol.Fasting blood glucose (plasma) and glycosylated hemoglobin were determined the same day by the BT3000 autoanalyzer with reagents from JAS diagnostics . Blood glucose determination followed the enzymatic protocol with that of glycosylated hemoglobin relying on the agglutination procedure.μL each of the various concentrations of insulin standards  was placed in appropriate wells. The same volume of sample was also placed in each well, followed by the addition of 100 μL of 1X enzyme conjugate prepared by diluting 500 μL of 20X enzyme conjugate concentrate with 9500 μL of assay diluent. After thorough mixing, the setup was then incubated for 1 hour. Using an automated microplate washer , the microplate wells were washed 6 times each with 300 μL of 1X wash buffer prepared by mixing 25 mL of 20X wash buffer concentrate with 475 mL of distilled water, after the incubation period. Exactly 100 μL of TMB substrate was then added to each well followed by incubation at room temperature for 15 minutes and addition of 50 μL stop solution. Absorbance was then read immediately at 450 nm using a Multiscan Microplate Reader . Appropriate standard curve was prepared from absorbance values of the insulin standards. The concentration of insulin in sample was subsequently determined from the standard curve.Serum insulin was measured by a commercially available quantitative enzyme-linked immunosorbent assay (ELISA) kit from Calbiotech  with reagents from the same manufacturer. The specific ELISA is the direct sandwich type. Briefly, samples, standards, and reagents were brought to room temperature. Exactly 25 μU) × fasting glucose (mmol/L)/22.5:HOMAIR and HOMAB were estimated from glucose and insulin levels by the formulae of Matthews et al.  as HOMAI falciparum malaria.Malaria was diagnosed by the CareStart Malaria HRP2Pf rapid diagnostic test kit manufactured by Access Bio . The CareStart Malaria HRP2Pf has been found to exhibit high sensitivity and specificity and correlates strongly with microscopy –16 in diDiabetics were already diagnosed type 2 diabetic patients receiving appropriate treatment at CCTH excluding exogenous insulin.t-test. Mean levels of parameters before and during malaria were compared across groups by one-way ANOVA with Tukey post hoc test. Pearson correlation test was used to assess linear relationship between any pair of measured parameters. A stepwise linear regression model was then applied to identify independent predictors of malaria-induced HOMAIR. A P value < 0.05 was considered statistically significant.Data obtained were analyzed by SPSS software version 17. Data are presented as mean ± standard deviation except the HOMAIR values that were log-transformed and presented as geometric mean ± standard deviation. Mean levels of measured parameters were compared between study groups and between genders with independent sample P > 0.05) between the defined study groups  mean level of all the measured parameters between the sexes in both study groups except FBG and BMI. FBG was higher (P = 0.023) in control males compared with their female counterpart with BMI being higher (P < 0.001) in diabetics females in comparison with their male counterpart (data not shown).Of the 200 respondents, 100 (70 diabetics and 30 controls) had malaria after a two-year follow-up. Age and baseline serum insulin level did not differ (y groups . HoweverP < 0.05) older and had higher levels of FBG, HbA1c, and HOMAIR but lower level of HOMAB than control males (P = 0.182) and higher (P < 0.001), respectively, in diabetic respondents (P > 0.05) show either intergroup or intragroup gender variation  higher mean levels of BMI, FBG, and HbA1c but lower levels of serum insulin and HOMAB than controls (P = 0.563) in their malaria-induced HOMAIR levels  across groups with and without malaria  in diabetic respondents during malaria, the mean levels of all the other measured parameters were comparable (P > 0.05) before and during malaria (P < 0.05) higher mean levels were observed for all the measured parameters except BMI and HOMAB  but strongly  with malaria-induced HOMAB and insulin, respectively, in the diabetic group. In a subsequent stepwise linear regression model, malaria-induced HOMAB and insulin were significant  independent predictors of HOMAIR, jointly explaining over 91% of the observed variation.Malaria-induced HOMAIR correlated positively and moderately , malaria-induced insulin , and baseline insulin level . Also, malaria-induced HOMAB correlated positively with baseline HOMAB  and malaria-induced insulin level . Above all, baseline HOMAB correlated positively with malaria-induced insulin level . As expected, a stepwise linear regression model revealed that malaria-induced HOMAB, malaria-induced insulin, and baseline insulin levels were independent  predictors of the observed HOMAIR level during malaria infection. The model explained over 99% of the observed variation in HOMAIR levels in controls who had malaria.Similar analyses in the control group showed positive correlation of malaria-induced HOMAIR level with malaria-induced HOMAB (P > 0.05) association was observed between BMI and any of the measured indices of insulin sensitivity or glycemia.Conspicuously, no significant (This study investigated malaria-induced insulin resistance in adult diabetics and controls in the Cape Coast metropolis of the Central region of Ghana. The comparable mean age of respondents between the two study groups in the current study appears contrary to a study in Kumasi that found higher age for diabetics than nondiabetic controls  though t1c than controls except that the observed mean levels for diabetic respondents were lower than those reported by other researchers in China [c is a marker for relative long-term blood glucose control, its appreciable increase due to Plasmodium falciparum malaria in the control group of the current study is an indication that the increased FBG was not transient. Interestingly, the comparable levels of FBG and HbA1c of diabetics before and during malaria could be due to treatment effect since most diabetics were on metformin, an antidiabetic drug with acknowledged suppression effect on hepatic gluconeogenesis [c. This view requires further studies to be proven in the light of a recent finding that associates HbA1c with intima-media thickness, a surrogate marker for CVD risk [c is associated with 1.67 risk of developing coronary heart disease in apparently healthy adults. Thus, with diabetics being already at risk for CVD, further investigations ought to be carried out to ascertain the clinical relevance of the slightly elevated FBG and HbA1c levels as a result of malaria found in the current study.As expected, diabetics in the current study recorded higher baseline mean levels of FBG and HbAin China , India [in China , Nigeriain China , and Uniin China . The ratin China , 21, 25 in China  and has ogenesis , 29. On CVD risk , and a lCVD risk  suggesti falciparum malaria. This observation, notably, in the nondiabetic group may have implications for the development of T2DM in malaria-endemic countries. With the possibility of multiple episodes of clinical malaria in the same individual, the corresponding insulin resistance per episode could predispose the affected people to the development of overt T2DM with time since insulin resistance precedes development of overt T2DM [Insulin resistance, defined as the reduced response of insulin sensitive cells to the action of insulin, has been amply demonstrated to be central to the development of T2DM  and CVD ert T2DM . This isInterestingly, the various measures of glycemia and insulin sensitivity did not associate with BMI, suggesting that general fat accumulation played no significant role in the observed insulin resistance in the current study.Based on the above we propose that malaria-induced insulin resistance may contribute to the predicted higher increased incidence of T2DM for malaria-endemic sub-Saharan African countries . Hence,  falciparum malaria caused insulin resistance in diabetic and nondiabetic semi-immune adults, independent of BMI. This finding has implications for the evolution of T2DM in malaria-endemic regions of the globe.Uncomplicated


Plasmodium falciparum may pose challenges in malaria treatment and prevention through chemotherapy and vaccination. We assessed Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) of P. falciparum infections and sort relationship of parasitaemia with P. falciparum msp2 genotypes as well as with the number of infecting clones. The study was carried out in Kibaha, Tanzania. Ninety-nine children under five years with uncomplicated malaria were recruited. Genetic diversity was analyzed by genotyping the msp2 gene using PCR-Restriction Fragment Length Polymorphism. Thirty-two different msp2 alleles were obtained. The msp2 3D7 allelic frequency was higher (48.1%) and more prevalent than FC27 (27.3%) (p < 0.05). Twenty-four percent of the infections were mixed alleles. The individuals with FC27 had high parasitemia compared to those with 3D7 alleles (p = 0.038). The mean MOI was low . The P. falciparum population among children at Kibaha is composed of distinct P. falciparum clones, and parasites having 3D7 are more frequent than those with FC27 alleles. Individuals with parasite having FC27 alleles have high parasite densities suggesting that parasites with FC27 alleles may associate with severity of disease in Kibaha. Low MOI at Kibaha suggests low malaria transmission rate.Genetic diversity of P. falciparum is a major characteristic and a factor by which the parasites survive the hosts' immune responses. It results from allelic polymorphism, recombination, chromosome rearrangements, and antigenic variation [Genetic diversity withinariation . Experimariation  establisariation –6. It is msp1, msp2, and glurp of P. falciparum can be used as genetic markers for genotyping of field parasite populations [ msp2 gene of P. falciparum is extremely polymorphic [ Plasmodium falciparum infections in endemic areas.The mechanisms controlling the genetic diversity within the parasite genome are many and complex. Studies have shown extensive polymorphism in some key parasite antigen during the parasites asexual blood stage (reviewed in ). The exulations . Studiesymorphic  and ther msp2 of alleles among symptomatic P. falciparum infections and assess the multiplicity of infection among children with uncomplicated malaria. The msp2 genotypes and multiplicity of infection were related to peripheral parasitaemia.In the present study, Polymerase Chain Reaction- (PCR-) Restriction Fragment Length Polymorphism (RFLP) was used to define the diversity and distribution of P. falciparum is the dominant malaria species. Malaria transmission in Kibaha peaks towards the end of the long and short rains.This study was carried out at Mlandizi, Kibaha, in the Coastal Region, Tanzania . MlandizPlasmodium falciparum isolates were collected during a monitoring programme of Sulphadoxine-Pyrimethamine (SP) to establish its efficacy using WHO 14-day protocol. One hundred and nineteen (119) children aged 6–59 months attending Mlandizi Health Centre were enrolled during the 2002 rainy season. Inclusion criteria were monoinfection with P. falciparum with at least 300 parasites/μL, no intake of antimalarials or sulphonamide-based drugs during the previous 4 weeks, absence of severe illness, presence of axillary temperature of  ≥37.5°C but <39.5°, no history of allergic reactions to the administered drug, and willingness to give consent to participate. Infected blood was taken from children presenting with nonsevere malaria at day 0, before drug administration. Upon presentation to day 0, thick and thin blood smears were collected by finger prick and stained with Giemsa for microscopic parasite identification and quantification. In the thick smear, parasites were counted against 200 white blood cell count, assuming this to be 8000/μL. Parasite density was recorded as the number of parasites per 200 white blood cells (WBC). Taking 8000 WBC as the conversion factor, densities were converted to the number of parasites/μL of blood. The parasites density was expressed as the number of parasites/μL. Assessment of the treatment response was based on the WHO 14-day in vivo protocol.Ethical approval was given by the National Institute for Medical Research, clearance ref. no. NIMR/HQ/R.8a/Vol.1X/107. Verbal informed consent was obtained from village leaders and written consent was obtained from all parents or guardians.2 was cut and placed in 1.5 mL capacity microfuge tubes containing 1 mL of PBS (pH 7.4) and 50 μL of 10% saponin and incubated at 4°C overnight. The tubes were then centrifuged to aspirate the reddish PBS/saponin. Thereafter, 1 mL of PBS was added to each tube inverted each time and then incubated at 4°C for two hours. The solution was then centrifuged for two minutes and again the aspiration of the liquid was done, drying the filter paper by removing the liquid. 100 μL of sterile water was added to each tube together with 50 μL of 20% chelex. Chelex was then transferred to the microfuge tube using a yellow tip, with its tapered end clipped off, inverting the chelex solution after every transfer. Then, the parasite DNA was extracted by incubating the tubes for 20 minutes on a 100°C heat block vigorously vortexing each sample every two minutes.At enrolment, finger prick blood was also spotted on 3 mm Whatman filter paper , dried, and kept separately to avoid contamination at room temperature. Parasite genomic DNA was extracted from 116 samples by chelex extraction as described elsewhere . Briefly P. falciparum in these samples, the polymorphic repetitive regions of the merozoite surface protein 2 (msp2) were amplified by a nested PCR assay [ P. falciparum and alleles determined by RFLP. The initial amplification using the outermost 5′ and 3′ primers, S2, and S3 , 1.5 mM MgCl2, 125 μM mixture of deoxynucleoside phosphates, 0.25 μM of primer pair (S2/S3), 0.02 U of Taq DNA polymerase, and 5 μL of genomic DNA. The PCR reaction involved 30 amplification cycles composed of initial denaturation at 94°C for 5 minutes, a denaturing step at 94°C for 1 minute, followed by annealing at 55°C for 2 minutes, and an extension step for 2 minutes at 72°C. In the second round amplification reaction, 2 μL of DNA amplicon from the primary PCR was used as a template in a 30 μL reaction volume containing same reaction mix as in the primary reaction, using nested conserved region 5′ and 3′ primers S1 and S4 ( msp2 gene. Same cycling parameters were used as in the primary PCR. The nested PCR products were then monitored on 2% agarose gel before restriction digestion. A Hinf I (New England Biolabs) restriction digest was performed to identify msp2 alleles in parasite isolates. Digestion was carried out overnight and digestion products were run on 10% PAA gel  using 1.5 mm spacers, stained with ethidium bromide, then observed under UV light, and photographed. A 1 Kb ladder was used as a DNA size was used as a marker on all gels. Hinf I restriction digestion of a nested PCR product of FC27-type alleles produced two conserved fragments of 115 and 137 base pairs (bp) and a repeat unit of 96 bp, which represent the 5′ and 3′ end of amplification product, respectively. Members of the 3D7-type alleles produced two conserved restriction fragments of 70 and 108 bp in length [After incubation, tubes were centrifuged for 2 minutes and then 10 minutes at 14,000 rotations per minute to remove chelex resin, leaving the parasite DNA in the supernatant. The parasite DNA was then stored in microfuge tubes at −20°C, for PCR amplification. To genotype different forms ofCR assay  using ol, and S3  was carr1 and S4  to ampli msp2 alleles. To analyze differences in the frequency of occurrence of 3D7 and FC27 alleles in sample isolates, Z score statistical test was used. Two-way ANOVA was used to compare mean parasite densities between children with FC27, 3D7, and mixed allele using Prism 5 (Graph Pad software). Multiplicity of infection was calculated as the highest number of alleles of each P. falciparum isolate.Shannon winner index was used to calculate diversity index of P. falciparum msp2 genotypes 99 parasite isolate (blood samples) were analysed. Polymerase chain reaction analysis showed that (82) 81% samples were positive for P. falciparum. All 82 positive samples were subjected to RFLP analysis to define msp2 genotypes/multiple infections in parasite isolates. The Hinf I enzyme resulted in genotype-specific pattern of bands that allow discriminating a multitude of different msp2 alleles. Individual alleles were named according to the size of a further large Hinf I fragment, which varies between alleles.To assess msp2 alleles were grouped according to similarities in their PCR-RFLP patterns. The frequency of occurrence of the two msp2 genotypes is shown in  msp2 genotypes in different age groups showed that the 3D7 allelic types were the most prevalent in each age group  with high parasite densities in children carrying P. falciparum parasites belonging to the FC27 alleles (H′). The diversity of FC27 allele was higher (H′ = 1.15) than that of 3D7 (H′ = 1.08) but the difference was not statistically significant .There were significant differences between mean parasite densities between individuals with FC27, 3D7, and mixed genotypes  of infected blood samples contained single infection, 47% (n = 36) double infections, and 3% (n = 2) triple concurrently infecting genotypes. The mean number of concurrent infections  was 1.4 (95% CI 1.2–1.5) .The multiplicity of infection represents the actual number of coinfecting genotypes circulating in an individual. It was calculated as the highest number of alleles of each1.2–1.5) . The hig P. falciparum infections. We found 51 different msp2 alleles of P. falciparum-msp2 gene in blood samples collected from children under five years with clinical malaria. In this study, P. falciparum harboring the 3D7 parasites were more frequent than the FC27 parasites in blood samples collected from children under five years with uncomplicated malaria in Kibaha. Our findings are in agreement with other studies carried out in children with uncomplicated malaria in various malaria endemic areas [The analysis of malaria-infected blood revealed by PCR-RFLP in our study has shown considerable heterogeneity amongic areas , 15, sugp = 0.038). Association of FC27 parasites with high parasite density may suggest lack of antigen-specific antibodies amongst children due to lack of exposure and parasite immunosuppressive effects [Although the FC27 parasites were less frequent in children under five years in Kibaha, children having these parasites genotypes had higher mean parasite density  compared to other holoendemic areas including some parts of Tanzania. Felger et al. 1999)  reported99  reporThere was a weak-negative association between MOI and parasite density but the relationship was not significant see . The wea P. falciparum isolates from children under five years at Kibaha is extensively illustrated by greater polymorphism that exists in the parasite msp2 gene. In addition, we found that the 3D7 genotypes are more frequent than the FC27 genotypes in children under five at Kibaha. However, the FC27 genotypes are associated with higher parasite densities in children of 1 ≤ 4 years in Kibaha. Our data are important in understanding the molecular epidemiology of P. falciparum malaria in Kibaha, indirectly demonstrating the immune status of children with uncomplicated malaria and transmission intensity of malaria, which is spatially different in endemic areas.We have demonstrated that the genetic diversity of


For this reason it is necessary to find other indicators that can be measured in these patients.Nutritional screening is a fundamental aspect of the initial evaluation of the hospitalised patient. Body Mass Index (BMI) in association with other parameters is a good marker of malnutrition (<18.5 kg/m2.1) Analyse the relationship between BMI and Mid-Upper Arm Circumference (MUAC); 2) establish a cut-off point of MUAC equivalent to BMI <18.5 kg/mThe anthropometric data of patients hospitalised over the period 2004–2013 were retrospectively revised. The following variables were collected: weight, height, BMI, MUAC, sex and age.2; MUAC: 26.95±4.50 cm; age: 56.24±16.77. MUAC correlates suitably to BMI by means of the following equation (simple linear regression): BMI = − 0.042 + 0.873 x MUAC (cm) (R2 = 0.609), with a Pearson r value of 0.78 (p<0.001). The area under the curve of MUAC for the diagnosis of malnutrition was 0.92 . The MUAC value ≤22.5 cm presented a sensitivity of 67.7%, specificity of 94.5%, and a correct classification of 90%. No significant statistical differences were found in the cut-off point of MUAC for the diagnosis of malnutrition based on sex (p = 0.115) and age (p = 0.694).1373 patients were evaluated, who presented a mean weight of: 65.04±15.51 kg; height: 1.66±0.09 m; BMI: 23.48±5.03 kg/m2, independent of the age or sex of the patient, although there are other alternatives. MUAC constitutes a useful tool as a marker of malnutrition, fundamentally in patients for whom weight and height cannot be determined.1) MUAC correlates positively and significantly with BMI. 2) MUAC ≤ 22.5 cm correlates properly with a BMI of <18.5 kg/m Hospital Malnutrition (HM) is highly prevalent. It is estimated that around 30 million people in Europe present HM, and furthermore, it is associated with elevated costs of some €170 billion annually , in whic2, and is a good marker of malnutrition, present in the majority of existing nutritional screens. However, it must be taken into account that many inpatients cannot be weighed and measured. Consequently, there is a need for markers easier to measure to detect malnutrition in habitual clinical practice. Currently, a BMI of 18.5 kg/m2 is considered a marker of malnutrition when associated with other risk factors, although, per se, this is not of much predictive value as it does not exclude patients with constitutional underweight [Body Mass Index (BMI) corresponds to weight in kg/height in merweight ,8.Mid-Upper Arm Circumference (MUAC) is a simple measurement which has been used for many years in nutritional evaluation, being an indicator of protein and energy reserves of the individual. Diverse studies have employed MUAC as a nutritional parameter in different population groups  –16.In every day hospital practice, it is often impossible to weigh or measure the patient, thus frustrating BMI calculation. In this case, estimated weight and height can be obtained through indirect measures that have been demonstrated to correlate with direct measures. In this sense, the height could be measured in an indirect way through the length of the arm and the height of the knee, whereas the estimated weight is more difficult to calculate, because this requires several anthropometric values, and larger deviations from real weight can occur. These indirect measures undeniably lead to an inherent margin of error in the process of estimating height or weight, being more relevant in the case of height as the latter is squared in the BMI equation. For this reason, another anthropometric measurement that relates to BMI would have to be used. MUAC could be one, especially as several studies have examined this association ,10,17.2, one of the major problems observed was that a large part of the patients could not be weighed or measured. For instance, certain pathologies or conditions impeded correct height measurement , while others hampered determination of body weight (inability to stand) or might even have affected real weight . Therefore, BMI was calculated by means of methods which diminish the reliability of the test .During the design on the part of our team of a hospital nutritional screen termed CIPA –19 that 2.Through this study, MUAC will be studied to determine whether it can be a useful parameter to categorise the nutritional state of the patient for whom BMI cannot be measured. To this end a relationship between MUAC and BMI in inpatients has been observed, and concretely, an adequate cut-off point for MUAC that correlates to a BMI of <18.5 kg/mA retrospective study was carried out of 1,373 patients assessed by the Clinical and Dietetic Nutrition Section of Hospital Universitario Nuestra Señora de Candelaria (HUNSC) in the period comprising 2004 to 2013.The ethics committee of HUNSC gave it's approval for the carrying out of this study on the date 27 March 2014. Given that this was a retrospective study of patients screened by the clinical and dietetic nutrition section, it was not possible to obtain informed consent due to the peculiarities of the study. The patient’s information was anonymized and de-identified prior to analysis.All inpatients were included whatever their basic pathology, and who had received a nutritional assessment by Nutrition Department staff with anthropometric data . All patients with oedema in the extremities or ascites, absence of any limb, as well as patients with data missed in the anthropometric assessment were excluded. The following data were collected: age, sex, weight, height, BMI and MUAC.2.To obtain MUAC a flexible, inelastic measuring tape model Seca 201 was used, and the measure was taken in the patient's non-dominant arm, just at the mid-point between the acromion and the olecranon, in sitting or standing posture. The value obtained was expressed in centimetres. MUAC was taken twice and the averages were recorded. Weight and height were obtained with a model Seca 220 scale, placing the patient with their back to the instrument, measuring the height and weight. BMI was calculated by analysis software using the formula: weight (kg)/ height (m)t-test is used. An analysis of covariance (ANCOVA) was carried out with the aim of evaluating the effect of sex and the presence of malnutrition based on BMI on the averages values of MUAC. For the continuous variable MUAC a Receiver Operating Characteristic (ROC) curve was plotted with the aim of obtaining a global measure of the accuracy of the test for the combination of all the possible cut-off points. The area under the curve (AUC) was calculated along with its confidence interval (CI) to 95%. Based on the coordinates of the curve, a cut-off point is selected for the variable MUAC, and the sensitivity, specificity, likelihood ratio for positive and negative test, as well as the percentage of correct classification were carried out. A logistic regression model is adjusted with the aim of evaluating, using the interaction term whether the selected cut-off point presented a different effect from the classification of malnutrition based on sex and age. Null hypothesis was rejected by a type I error minor than 0.05. The statistical analysis was carried out using the STATA statistics package, version 12.0.The qualitative variables are presented with their frequency distributions. The quantitative variables are summarised by mean and standard deviation (SD). The linear relationship between MUAC and BMI was studied via the calculation of the Pearson linear correlation coefficient and the estimation of the simple linear regression coefficients of the equation. The determination coefficient (R2) is calculated as a goodness-of-fit index. For the comparisons of the quantitative variables between two independent groups the Student's parametric 2.The parameters of 1,373 patients (43.3% women) were collected, whose initial data can be found listed by subgroup in 2), being statistically greater in women (20.2%) than in men (14.3%) (p = 0.024)  greater than women (p<0.001). As for BMI, the distribution is different between men and women  .MUAC shows a significant relationship with BMI by means of the equation BMI = −0.042 + 0.873 x MUAC (cm) (R2 = 0.609), with a Pearson correlation coefficient of 0.78 (95% CI: 0.76–0.80), while that in men was of 0.83 (95% CI: 0.80–0.85) and in women 0.74 (95% CI: 0.70–0.77), as set out in The mean MUAC of the group of patients with malnutrition (21.4 (3.1)) was statistically inferior (p<0.001) to the group of patients without malnutrition (28.1 (3.9)). When stratified by sex, malnourished women presented in absolute terms a mean MUAC of 7.04 cm less , while in men the difference was 6.05 . Based on the ANCOVA results no statistically significant interaction was observed (p = 0.064) between sex and the presence of malnutrition in relation to MUAC.The AUC of MUAC for the detection of malnutrition in the whole sample was 0.92 . The results in women and men were as follows: AUC: 0.94  and AUC: 0.91 , respectively . Based oAccording to the results of the logistic regression models, the selected cut-off point for MUAC did not show statistically significant differences in relation to sex (p = 0.115) or age (p = 0.694).2).The results of the present study suggest that MUAC serves as a predictor of BMI. Likewise, the selected cut-off point of ≤22.5 cm presents a high percentage of correct classifications   advised 2, and thus the patient is found to be in a state of risk of malnutrition. This study also deals with weight loss over time and values of MUAC. In this respect they estimated that if MUAC changed by at least 10%, weight and BMI would probably also change by 10% or more.On the other hand, Malnutrition Universal Screening Tool (MUST)  suggestsIn turn, the Mini Nutritional Assessment (MNA) screening, designated particularly for a population over 65 years of age, also comprises MUAC data, including values of 22–21 cm as parameters to detect malnutrition .Sultana T et al.  recently2, the authors concluding that a MUAC <25 cm is identified with said BMI in males, and <24 cm in females. It should be taken into account, however, the differences that exist between the study population of this work and that of ours, not only for the interracial variability that could justify the differences encountered, but also for the greater chronic profile of our patients, for instance greater average age, which is almost double. This is probably a reflection of the important socioeconomic differences between the two countries, for which reason our work may be more representative of general hospitals in first-world countries. For this reason, people over 60 years of age were not excluded from our work  since l2.A retrospective study, also with patients suffering from acute pathology, identified a good correlation between BMI and MUAC, and even the patients with a MUAC <25 cm presented a greater length of stay during hospitalisation and a higher incidence of mortality. In thisThe sample of patients in our work was not only large but also very heterogeneous, scattered between the medical as well as surgical departments of a general hospital, thus reflecting the characteristics of usual clinical practice. The study was performed in Western Europe, understanding that the results would not coincide with the majority of studies carried out in developing countries due to interracial differences in body composition.We identified that there is a good correlation between MUAC and BMI, just like that occurring in the work of Sultana et al , as much2, the relationship of the results to patient age was studied, finding that a value ≤22.5 cm is applicable to any inpatient.To facilitate the evaluation of the results obtained by measuring MUAC, we sought to analyse the results in a grouped sample of men and women. Thus, it was found that the differences in MUAC between malnourished and non-malnourished patients were somewhat greater in women than in men, but without reaching statistical significance, being able to employ said common value to both sexes in the analysis of this measure. Furthermore, to be able to generalise the threshold of MUAC that represents a BMI of <18.5 kg/mThrough this analysis, and observing that the possible cut-off points of each sex are very close to one another, the conclusion was reached that it is impractical to use a different value for men and women. It gains nothing in terms of classification capacity, and moreover, the intention in our case is to introduce the measure into a hospital nutritional screen where a high number of patients will be subjected to it. For this reason the greatest simplicity possible must be sought with optimum results. Understanding that the classification of BMI is the same for women as for men, it is reasoned that there is no clinical justification for it being different for MUAC, based on the observations in this sample.Our team specifically chose the cut-off point of MUAC ≤22.5 cm based on the results found in It is possible that to use MUAC in a different setting, other authors choose a higher cut-off point which gives greater sensitivity, or to choose various cut-off points according to sex or age, but we think that for its inclusion in a nutrition screening tool that facilitates to the maximum possible degree the functions of healthcare personnel, a single common cut-off point must be used. Moreover, based on the reduced muscle mass, it may be more convenient to choose a lower cut-off point in an elderly population. However, our analyses did not reveal significant differences for subjects aged over 65 using the selected cut-off point. With the value we have chosen of ≤22.5 cm included in the CIPA screen, we have been able to to predict a worse clinical prognosis in those patients that test positive, increasing mortality more than four-fold and hospital stay by a week .Our work reveals various limitations which should be explained. Firstly, it deals with a retrospective study with the attendant limitations that involves, albeit with a significant number of patients. As we have commented on in the discussion, different cut-off points can be obtained according to sex and age, but the objective of this study was to find a single common cut-off point for a nutritional screen.We believe, in any case, that the data obtained in our study are significant, since it is the first to be undertaken with a sample of inpatients from a general hospital in the first world. Furthermore, in Spain as well as in Europe there is a body of work in combating malnutrition related to illness in which many inpatient nutritional screens are been designed and applied, and so we believe that the results obtained in our article contribute somewhat more objectivity to the anthropometric data included in those studies.2  that provide different sensitivities, specificities, and correct classification, so that, depending on use and context, can serve in one or another value. We believe that for the introduction of this variable into a hospital nutritional screen, it would be ideal for said cut-off point to be common to both sexes and independent of patient age, to facilitate the task of those performing the nutritional screen, a key aspect of its implementation in a general hospital setting.In conclusion, this work authenticates MUAC as a parameter that correlates very well with BMI and which can be used as a complement to it, or as a substitute for the same in situations in which the patient cannot be weighed or measured. Furthermore, different cut-off points for MUAC are presented which correlate with BMI <18.5 kg/m


Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds.Species of avian malaria parasites  methods.One individual of Eurasian siskin All exposed birds developed malaria infection, survived the peak of parasitaemia, but suddenly died between 30 and 38 days post exposure when parasitaemia markedly decreased. Numerous phanerozoites were visible in histological sections of all organs and were particularly easily visualized after ISH processing. Blockage of brain capillaries with phanerozoites may have led to cerebral ischaemia, causing cerebral paralysis and is most likely the main reason of sudden death of all infected individuals. Inflammatory response was not visible around the brain, heart and muscle phanerozoites, and it was mild in parenchymal organs. The endothelial damage likely causes dysfunction and failure of parenchymal organs.Plasmodium homocircumflexum caused death of experimental passerine birds due to marked damage of organs by phanerozoites. Patterns of phanerozoites development and pathology were similar in all exposed birds. Mortality was reported when parasitaemia decreased or even turned into chronic stage, indicating that the light parasitaemia is not always indication of improved health during avian malaria. Application of traditional histological and ISH methods in parallel simplifies investigation of exoerythrocytic development and is recommended in avian malaria research. Plasmodium  are widespread on all continents, except Antarctica . Blood was taken for microscopic examination and PCR-based testing during the course of the experiment see Fig. . ApproxiBrain, heart, kidney, liver, lungs, spleen, and a piece of the pectoral muscle of the experimental birds were dissected after the birds’ death. The samples were fixed in 10 % neutral formalin and embedded in paraffin blocks. Histological sections of 4 µm were prepared, stained with haematoxylin-eosin (H&E) [An Olympus BX51 light microscope equipped with Olympus DP12 digital camera and imaging software Olympus DP-SOFT was used to examine slides and prepare illustrations. To trace the parasitaemia, each blood slide was examined for 15–20 min at medium magnification (×400), and then at least 100 fields were studied at high magnification (×1000). Intensity of parasitaemia was estimated as a percentage by actual counting of the number of parasites per 1000 erythrocytes or per 10,000 erythrocytes if infections were light [b (cyt b) gene of Plasmodium, Haemoproteus and Leucocytozoon species. The reaction mix for the first PCR consisted of 12.5 µl of Dreamtaq Master Mix , 8.5 µl of nuclease-free water, 1 µl of each primer and 2 µl of template DNA. The thermal conditions for the first PCR were according to Helgren et al. [b gene [Plasmodium sample, which was positive by microscopic examination of blood films) were used to determine possible false amplifications. No case of false amplification was found.Total DNA was extracted from blood samples using the standard ammonium-acetate protocol  with a mn et al. . For the [b gene . The reab gene fragments were sequenced from the 5′ end using the HAEMF primer [b lineages of detected DNA sequences.The cyt F primer . Dye terF primer . The ‘BaPlasmodium spp. [Turdus merula free of avian malaria parasites as well as application of an irrelevant oligonucleotide probe (designed for Leishmania spp.) on the experimental samples [see Chromogenic in situ hybridization ISH was carried out according to Dinhopl et al. . In brieium spp. . The digium spp. . Tissuesples see  were useExperimental procedures of this study were approved by the International Research Co-operation Agreement between the Biological Station Rybachy of the Zoological Institute of the Russian Academy of Sciences and Institute of Ecology of Nature Research Centre (25-05-2010). All efforts were made to minimize handling time and potential suffering of animals. None of the experimental birds suffered apparent injury during experiments.All control birds survived and remained non-infected during this study. The exposed Eurasian siskin, common crossbill and common starling got infected, and PCR-based testing confirmed presence of the parasite lineage pCOLL4 in these birds.According to microscopic examination of blood films, the prepatent period was 6, 6 and 8 dpe in Eurasian siskin, common crossbill and common starling, respectively. Parasitaemia remained during entire observation time Fig. . The higAll exposed birds survived the acute parasitaemia stage, but suddenly died when parasitaemia decreased and was 15.5, 0.2, 0.1 % in the Eurasian siskin, common crossbill and common starling, respectively; these birds died 30, 38 and 36 dpe, respectively  magnification Figs. a, d it wPlasmodium homocircumflexum (pCOLL4) caused lethal malaria in three species of experimental passerine birds; (2) mortality occurs suddenly during decreased or even chronic stage of parasitaemia 4–6 weeks after exposure, most likely due to damage caused by phanerozoites; and (3) dynamics of parasitaemia were different, but patterns of phanerozoites development and tissue pathology were similar in all exposed birds. It is also important to note and worth discussion that ISH is an effective method in determining tissue stages of avian malaria parasites, but traditional histology remains essential for determining the structure of exoerythrocytic meronts.The key results of this study are that: (1) Plasmodium homocircumflexum has been reported in several species of birds. The lineage pCOLL4 of this parasite along with the synonymous lineages pU12 (GenBank nr. DQ241519) and BOBO20085 (GenBank nr. KC867664) were detected in the red-backed shrike, domestic canary, collared flycatcher Ficedula albicollis, red-rumped warbling finch Poospiza lateralis, lark-like bushrunner Coryphistera alaudina, curve-billed reedhaunter Limnornis curvirostris, chopi blackbird Gnorimopsar chopi, chalk-browed mockingbird Mimus saturninus, diademed tanager Stephanophorus diadematus, bobolinks Dolichonyx oryzivorus, Eurasian siskin, common crossbill and common starling [Plasmodium relictum and the lineage pGRW2 of Plasmodium ashfordi [starling  along wiashfordi , 28–30. P. homocircumflexum (pCOLL4) has not been reported in Europe, where this infection has been observed only in adult far-distance migrants after their arrival from African wintering grounds [Culex pipiens (forms pipiens and molestus) and Aedes vexans [P. homocircumflexum remain unknown. However, it is also difficult to rule out that transmission might occur at some sites in Europe, but infected birds are dying, as was the case during the experiments. Additional experimental studies combined with post-mortem examination of naturally infected dead birds collected in wildlife are needed for the better understanding of epidemiology of this malaria infection.Transmission of  grounds , 26. It s vexans . VectorsP. homocircumflexum phanerozoites were similar in all exposed bird species (this study) and in domestic canaries [Plasmodium gallinaceum, Plasmodium cathemerium, Plasmodium durae, Plasmodium lophurae, Plasmodium matutinum, Plasmodium octamerium, and some other species [Patterns of development of canaries , indicatcanaries , 7. Beca species .Inflammatory changes in the brain cannot be expected in avian malaria, and this is supported by many reports , 4, 14. Plasmodium falciparum, but the underlying functional pathology in humans and birds is different [P. falciparum malaria, severe pathology is caused by adherence of infected erythrocytes to the endothelial cells of microvascular blood vessels in the brain, leading to blockage of the circulation and resulting in ischaemic brain changes.Brain lesions leading to an ischaemic brain changes are among the most severe conditions caused by the human malaria parasite ifferent , 31, 32.Plasmodium infections have been reported both due to high parasitaemia and damage caused by phanerozoites [Plasmodium parasite lineages are responsible for mortality in common European birds [Mortality of birds due to rozoites , 34. It rozoites , 35, 36.an birds ; howeverPlasmodium spp. is crucial for estimating of the true impact of these parasites on wild bird populations.Better understanding of pathology caused by phanerozoites and other exoerythrocytic meronts of Plasmodium pinotti: only roundish merozoites (micromerozoites) develop in the majority of phanerozoites, but phanerozoites containing elongate merozoites (macromerozoites) were also described [P. pinotti and other avian malaria parasites, but this finding likely is important epidemiologically and might be related to peculiarities of persistence of malarial infections in avian hosts [The number of “positive spots” after ISH treating is very high in some organs Fig. d–f. Thatescribed , 37. Thean hosts . IdeallyPlasmodium homocircumflexum (pCOLL4) caused lethal malaria in at least four species of experimentally infected passerine birds due to marked damage of organs by phanerozoites. It is likely that this infection is markedly virulent in non-adapted wild birds and it worth more attention in bird conservation projects. The patterns of parasitaemia development were different, but patterns of phanerozoites development were similar in all tested exposed birds. Mainly, sudden mortality occurred during decreased or even light chronic parasitaemia stages in all exposed birds, indicating that the chronic parasitaemia is not necessarily an indication of improved health during avian malaria. In other words, solely testing of blood samples is an insufficient method to understand avian malaria virulence, which might be underestimated in many Plasmodium spp. due to lack of information about their exoerythrocytic development. During investigation of exoerythrocytic merogony of haemosporidians, application of an ISH method for detection of tissue meronts in bird organs, and then processing the ISH positive organs using traditional histological methods is recommended. Application of both these tools in parallel would speed up the search for tissue meronts and also provide information about morphological characters of the parasites and their host cells.


P. falciparum infection was 7% in 2003, close to the pre-elimination threshold of 5%. The aim of this work was to determine the prevalence of P. falciparum infection in the same community a decade later. A cohort of 370 individuals aged from 3 to 85 years living in Dienga was investigated for P. falciparum infection; during six passages (P) in 15-month period. Demographic data were collected, along with behaviors and attitudes towards malaria. Plasmodium infection was diagnosed by microscopy (ME), followed by PCR to detect submicroscopic infection. The prevalence of P. falciparum infection in P1, P2, P3, P4, P5 and P6 was respectively 43.5% ; 40.9% , 52.7% ; 34.1% , 57.7% ; and 46.2%  with an overall average of 45.9% . P4 and P5 prevalences were statically different throughout the six passages. Microscopic prevalence was significantly higher than that observed ten years ago . Asymptomatic infections were the most frequent (96%). Gametocytes were detected in levels ranging from 5.9% to 13.9%. Insecticide-treated nets, indoor residual insecticides, and self-medication were used by respectively 33.2% (95%CI [29.0–37.4]), 17.7% (95%CI [15.5–19.9]) and 12.1% (95%CI [10.6–13.6]) of the study population. A near-threefold increase in P. falciparum infection has been observed in a rural area of southeastern Gabon during a 10-year period. Most infections were asymptomatic, but these subjects likely represent a parasite reservoir. These findings call for urgent reinforcement of preventive measures.Control strategies implemented a decade ago led to a marked reduction in the prevalence of malaria in many countries. In Dienga, southeastern Gabon, the prevalence of microscopic  Plasmodium falciparum and P. vivax are responsible for most morbidity and P. falciparum for most deaths, mainly among children under the age of 5 years living in sub-Saharan Africa.In 2013 there were 135–287 million new cases of malaria and 473 000–789 000 deaths , [7–10 y], [11–14 y], [15–18 y] and [19 y≥]. There were slightly more males than females in July 2013 (53.7%) and June 2014 (54.5%)  were included in this longitudinal study from April 2013 to June 2014. The prevalence of  (54.5%) .P. falciparum infection was 161/370 (43.5%) in April 2013 (P1), comprising 93 ME + (25.1%) and 68 (18.4%) PCR+ ME—participants. The corresponding data were 109/267 (40.9%), 72 ME + (27%) and 37 PCR+ (13.9%) in June 2013 (P2); 115/218 (52.7%), 57 (26.1%) ME + and PCR+ (26.6%) in July 2013; 87/255 (37.7%), 36 ME + (14.1%) and 51 PCR+ (20%) in October 2013; 134/232 (57.7%), 59 ME + (25.4%) and PCR+ (32.3%) in March 2014 (P5); and 67/145 (46.6%), 31 ME + (21.4%) and PCR+ (24.8%) in June 2014 (P6). The microscopic prevalence was significantly higher during the first three passages  than in P4 (P = 0.007). There was also a significant difference between the prevalence of PCR positivity between June 2013 (P2) and July 2013 and March 2014 (p<0.005). No difference in the overall prevalence of falciparum infection (ME + plus SMI) was observed across the six passages (p = 0.13) (The frequency of  = 0.13) .falciparum infection did not differ across the age groups or between genders (p>0.05). Nevertheless, a significant difference was observed between July 2013 (P3) and March 2014 (P5) among children aged 3–6 years (p<0.05); between these same children and also adults [≥19 years] in October 2013 (P4) (p<0.05), or the group of over five years older children [7–10 years] also in 2013 (P4) and [15–18 years] in June 2013 (P2) (p = 0.04)  .P. falciparum infection was significantly higher in individuals less than 18 years old than in older individuals   and whom of 15–18 years than in adults of 19 years old and more (p<0.05) . The PCRp<0.001) .The prevalence of both ME+ and PCR+ was slightly but not significantly higher in males than in females .Although peripheral blood parasites were almost all trophozoites (50 to 150 000 parasite per μl), several gametocytes were identified in 35 ME + individuals (9 symptomatic and 26 asymptomatic) during all the periods except P6, with 11 (11.8%) in P1; 5 (6.9%) in P2; 8 (14.0%) in P3; 5 (13.9%) in P4; and 3 (5.9%) in P5). Gametocyte density ranged from 50 to 700 per μl.P. falciparum was detected in 23 of these 30 febrile subjects . Eight of the 17 ME + patients had at least 5000 parasites per μl  of individuals, with no significant difference between clinical groups (infected and uninfected), genders or age groups  and IRS by 18.26% (95%CI [13.8–23.8]). No significant difference was observed in the use of ITNs between e groups .P. falciparum infection, respective odds ratios were 0.60 (0.34–1.05), 1.38 (0.69–2.75) and 1.57 (0.68–3.59). When we analyzed according to age, protection against P. falciparum infection was associated with the use of ITNs: adults tended to be more protected than younger individuals, with an odds ratio of 2.43 (1.36–4.34)  .Plasmodium-positive slides was reported to have fallen considerably in Libreville, the capital of Gabon [The decline in the prevalence of malaria observed in many countries, including Gabon, has been attributed to artemisinin-based combination therapy (ACT), monitoring of pregnancies, and distribution of insecticide-treated nets. For example, in 2008 the proportion of of Gabon , as wellof Gabon ,28. HoweP. falciparum infection. The study was designed to determine the true prevalence of P. falciparum infection during six passages (P) involving 370 individuals. The overall P. falciparum carriage rate (ME + and PCR+) ranged from 57.7% in P5 to 34.1% in P4, with an average of 45.9% (23.2% ME + and 22.7% PCR+). This study provides one of the most accurate field estimates of P. falciparum prevalence across all age groups in Gabon. Indeed, previous studies focused only on children [falciparum infection among asymptomatic individuals was found in Dienga, confirming the results of previous studies conducted in the same location [P. falciparum infection in Dienga (23.2%) was very similar to that found in 2011 among febrile children in Libreville (24.1%), an urban area of northwest Gabon [P. falciparum carriers have a five-fold increase in the risk of developing malaria [This longitudinal study revisited the situation in Dienga 10 years later, focusing on the hidden reservoir of children ,21,29 orchildren ,31. A hilocation , as welllocation , and Kenlocation . The pre malaria , such caP. falciparum across our six survey periods. In 2001, Elissa et al. reported that malaria transmission was perennial in Dienga, with a major peak in December to March and a minor peak in July to August; malaria transmission was almost undetectable during the rest of the year, owing to the disappearance of the main vector, Anopheles gambiae sensu lato, in the dry season [A. gambiae s.l. during the 10-year period in question. Entomological investigations will be necessary to confirm this hypothesis, as well as the possible introduction of other vectors.No significant difference was observed in the overall prevalence of y season ,36. It iP. falciparum infection was similar in children  and adolescents (15–18 years) but higher than in adults  (p<0.05). This increase could be due to several factors. First, in 1994 CIRMF established a fully equipped field base with three permanent workers (one nurse and two technicians) ensuring full-time cohort monitoring. In addition, the base was supplied monthly with food and drugs and was visited by a CIRMF physician. All febrile patients in Dienga were examined, and those with malaria were treated free of charge. The antimalarials used at the time were mainly chloroquine and sulfadoxine pyrimethamine (SP). In 2006, CIRMF research activities in Dienga were reduced, and in 2008 they were shifted to other topics. Antimalarial drugs were no longer distributed to the population and patient management was provided only by the local health center in Dienga, that had limited resources. Following the change in the national antimalarial policy, with a switch to ACT, in 2005 the Gabonese Malaria National Control Program (MNCP) launched a nationwide campaign of ITNs distribution, intermittent preventive treatment (IPT), and educational messages. These interventions have been evaluated in many parts of the country [In February 2003, a prospective cohort of 319 subjects 13–70 years old was followed for malaria and hepatitis virus co-infection during a one-year period in Dienga. The overall prevalence of malaria parasitemia was 20.8% (7.2% ME + and 13.6% PCR+) . This mi country ,46,47 bu country .The study found that ITNs and IRS were used by respectively 33.47% and 18.26% of respondents, and self-medication by 12.22%. In 2003, the rate of self-treatment was 11.3% in Dienga .P. falciparum malaria in Dienga  was observed in March 2014 (The comparison in the use of ITNs between youngers (3–15 years old) and individuals of 16 years old and more has shown a significant difference (p = 0.003) , the adun Dienga . Howeverrch 2014 . ITNs coThese results show that the MNCP campaign has not yielded the hoped-for results. The increase in malaria prevalence among febrile children observed in several pediatric units has been attributed to the decline in the preventive activities of the MNCP . This maIt would have been interesting to perform, as planned, passages every two months for a full year. Unfortunately, because of logistic constraints, it was not possible to achieve this goal. Consequently, the six passages were carried out irregularly during a 15-month period.P. falciparum infection in Dienga, southeastern Gabon, over a recent 10-year period. Although a large proportion of infected subjects were asymptomatic, the risk of older children and adolescents of developing falciparum infection becomes higher. These results reveal an urgent need for reinforcing intervention strategies.In conclusion, the study showed a near-threefold increase in the prevalence of S1 Annexe(DOC)Click here for additional data file.S1 Questionnaire(DOC)Click here for additional data file.


Haemophilus influenzae type b and pneumococcal disease are contributing to improved child survival in Africa. However, increased parasitological testing for malaria is associated with increased use of antbiotics to which resistance is already high.The replacement of “presumptive treatment for malaria” by “test before treat” strategies for the management of febrile illness is raising awareness of the importance of knowing more about the causes of illness in children who are suspected to have malaria but return a negative parasitological test. The most common cause of non-malarial febrile illness (NMFI) in African children is respiratory tract inflection. Whilst the bacterial causes of NMFI are well known, the increasing use of sensitive techniques such as polymerase chain reaction (PCR) tests is revealing large numbers of viruses that are potential respiratory pathogens. However, many of these organisms are commonly present in the respiratory tract of healthy children so causality and risk factors for pneumonia remain poorly understood. Inflection with a combination of viral and bacterial pathogens is increasingly recognised as important in the pathogenesis of pneumonia. Similarly, blood stream inflections with organisms typically grown by aerobic culture are well known but a growing number of organisms that can be identified only by PCR, viral culture, or serology are now recognised to be common pathogens in African children. The high mortality of hospitalised children on the first or second day of admission suggests that, unless results are rapidly available, diagnostic tests to identify specific causes of illness will still be of limited use in guiding the potentially life saving decisions relating to initial treatment of children admitted to district hospitals in Africa with severe febrile illness and a negative test for malaria. Malaria control and the introduction of vaccines against  Interest in non-malarial febrile illness (NMFI) has increased following more widespread use of parasitological testing for malaria and, in particular, since 2010 when the World Health Organization (WHO) recommendation for antimalarial treatment for any unexplained fever was replaced by the recommendation that, wherever possible, antimalarial treatment should be restricted to children with a positive parasitological test . Over thPlasmodium parasitaemia may occur in the neonatal period but is usually asymptomatic due to the presence of maternal antibody and rarely meets our definition of NMFI [The definition of NMFI is pragmatic but it remains a useful term in a period of transition from presumptive to more targeted treatment of febrile illness in African children. The term describes the situation of a patient with suspected malaria and a negative parasitological test. Like many causes of fever, malaria is an illness with clinical features that often overlap with other common causes of fever . The mos of NMFI . Thus neThis review describes the main causes of NMFI in African children, with an emphasis on acute lower respiratory tract infections. Systematic testing of children for human immunodeficiency virus (HIV) is still not commonly practiced in Africa so there are few studies comparing causes of fever in children with or without HIV. The prevalence of HIV in hospitalised children varies between 1% and 17% in areas highly endemic for malaria but is considerably less in children presenting to outpatient clinics ,14. In tPlasmodium parasitaemia may be present as an incidental finding in a child with fever due to another cause. Determination of parasite density, which influences the attributable fraction of fever due to malaria, can be of help: The presence of parasites at high density make malaria more likely to be the cause of a child’s illness than a low density infection, but this relationship is influenced by the level of malaria endemicity [P. falciparum infections due to either parasite sequestration or very low density parasitaemia, although true “slide-negative malaria” is probably rare. RDTs for malaria are not affected by parasite sequestration since they measure water-soluble parasite antigens but, in areas of high transmission, devices that detect histidine-rich protein-2 (HRP-2) may lack specificity due to the persistence of HRP-2 in blood for up to 6 weeks following infection [Salmonella (NTS) and other Gram-negative infections [A definitive diagnosis of malaria is often difficult due to a number of reasons , the besdemicity ,17. In anfection ,19. Finanfection ,20. Whilfections ,22.Pneumonia is the most common alternative diagnosis to malaria in a febrile child in Africa ,9. The gThe study of pneumonia suffers from, among other things, difficulties in obtaining a culture specimen from the site of infection and variation in case definitions , the forThe selection of case definition depends on available diagnostic resources and priority for either high specificity  or high sensitivity (needed for treatment of sick children) Table . In mostA number of studies, summarised by Rudan et al , have deEstimating incidence and causes of pneumonia in Africa is difficult due to the problems already mentioned, compounded by the few high quality laboratories in Africa and the changing epidemiology created by vaccines and availability of polymerase chain reaction (PCR) methodologies. For more than a decade, the Child Health Epidemiology Reference Group (CHERG)—a group of independent technical experts on global child morbidity and mortality estimates co-sponsored by WHO and UNICEF—has grappled with these problems. The 2013 CHERG estimates are based on a model using estimates of prevalence of known risk factors, incidence data from control arms of pneumonia clinical trials , and resTable Streptococcus pneumoniae or Haemophilus infuenzae type b (Hib) is changing as vaccines against these infections are now being introduced across the continent. Currently, all African countries have introduced Hib conjugate vaccine in infants and all but 2 sub-Saharan African countries have introduced or are in the process of introducing infant vaccination using a 10- or 13-valent pneumococcal conjugate vaccine (PCV-10 or PCV-13) [The incidence of pneumonia due to  PCV-13) .The high efficacy of these vaccines in African children has been established in clinical trials ,36,44. TS. pneumoniae [The impact of PCV-10 is still being assessed and no large studies have so far been published. In a district hospital on the Kenyan coast, paediatric admissions for invasive pneumococcal disease (IPD) from the nearby demographic surveillance system have been continuously monitored since 2003. There was a 50% reduction in admissions with IPD between 2003 and 2007 that may have been due to the reduction in malaria incidence that occurred between these years ,47. PCV-eumoniae .Staphylococcus aureus and Gram-negative infections are important, albeit in the minority, causes of bacterial pneumonia. Staphylococcal pneumonia is often very severe and may affect very young children. Nosocomial transmission of methicillin-resistant S. aureus is a particular problem in intensive care units and has been described in Ghana, although its routine detection in Africa is almost certainly hampered by lack of laboratory capacity [S. aureus carrying the Panton-Valentine leukocidin genes cause a particularly severe form of pneumonia; while most reports of this organism are from developed countries, there is at least one case report of a fatal infection in a traveller returning from Senegal [capacity . Strains Senegal .Gram-negative infections, and in particular NTS, are associated with malaria (as described below) and many of these qualify for the WHO definition of severe pneumonia. While this definition lacks specificity, there is clear evidence that these infections are responsible for true pneumonia defined by isolation of the organism from consolidated lung .The study of viral pneumonia has been greatly enhanced by the use of PCR. A large case control study of paediatric hospital admissions for severe or very severe pneumonia (by WHO criteria) in Kenya in 2007 identified a number of viruses from a nasal wash specimen . ConsistA number of case-control studies have documented that both viral and bacterial respiratory pathogens are frequently isolated from the upper respiratory tract of healthy children and this underlines the importance of including well-matched controls in any study of the causes of respiratory illness in children. RSV and influenza virus are most consistently found to be significantly associated with respiratory illness compared to controls, although the odds of association may not be strong enough to be diagnostically useful –54. WithThere is evidence from various sources of the association between viral and bacterial pneumonia. There are anecdotal reports of lobar pneumonia in association with the 1918 influenza pandemic and the H1N1 epidemic . In a raSalmonella enterica (of which 58.4% were due to NTS) and 1,031 (18.3%) were due to S. pneumoniae. The commonly reported organisms isolated from febrile children in Africa include S. pneumoniae (18.3%), NTS (17%), S. aureus (9.5%), and Escherichia coli (7.3%).A study to identify bacterial causes of illness in sub-Saharan Africa identified 22 published studies since 1980 of children admitted to hospital with a blood culture result . OverallThe available evidence suggests that bacteraemia is uncommon in children with non-severe illness since the prevalence of a positive aerobic blood culture is approximately 1% compared to an incidence of up to 15% among children needing hospital admission ,13,20,59P. falciparum infection as a risk factor for blood stream infection with NTS and other Gram-negative infections. This association was first investigated in The Gambia where it was observed that children with malaria whose fever did not settle on quinine treatment often grew NTS from their blood culture and these infections were limited to the malaria transmission season while carriage of NTS by domestic animals was perennial [Plasmodium parasites, suggesting that vulnerability to these infections may persist for several weeks after an episode of malaria [S. typhi, the latter appearing to infect older, non-HIV-infected children [As alluded to above, a number of studies have described erennial . Studies malaria ,20. The  malaria ,61. Immu malaria . A recenchildren ,64.In the context of this review, “atypical infection” is defined as infections not commonly isolated from aerobic blood culture. Knowledge of these infections in African children is limited, although increasing with the use of molecular diagnostics over the last decade. To investigate the frequency with which reports of these infections have been made, we used methods broadly the same as used by Reddy et al  and searThe currently increasing attention to diseases that until now have been relatively neglected is encouraging, especially since a number of studies have shown that among hospitalised children with fever a negative malaria slide is generally associated with higher mortality. However, the recent attention to NMFI might lead some to think that a new epidemic has been discovered while in truth this is an “epidemic of awareness” of infections that have been present but neglected for a long time. In reality, the decline in malaria incidence has been associated with a reduction in invasive bacterial disease, particularly NTS infection. Falling incidence of malaria is by no means universal ,67. In mThere are a number of important reasons to know the local diagnostic probabilities of common causes of fever, the main one being the need for a guide to pragmatic treatment. However, there are very few effective interventions available for the treatment of sick children in African hospitals; the key treatment decision based on the likely pathogen is still limited to the use of an antibiotic and, if so, which one. More specific diagnostic information is usually limited to the results of a blood culture .Biomarkers to discriminate between blood stream bacterial compared to viral infection are still of limited clinical use. The reasons are that current tests that are available and affordable to most African hospitals lack sufficient sensitivity and specificity for routine use . In non-There are additional public health reasons for a much more comprehensive knowledge of what infections are prevalent and where. These include the need to guide the deployment of vaccines or other preventive strategies. In addition, sentinel sites can identify emerging infections and, in particular, epidemic or pandemic threats to sub-Saharan Africa. This is increasingly important with growing global travel both into and out of the African continent.Child mortality is declining in Africa due to a number of reasons but the decline in malaria transmission intensity and the introduction of vaccines against Hib and pneumococcal disease are important contributors. There is an additional benefit to malaria control in that up to half of all blood stream bacterial infections in African children are caused by NTS and these infections become rare in children at low levels of malaria transmission, although may still be seen in HIV-infected adults ,63. ThisThese trends are very positive but one worrying effect of improvement in the diagnosis of malaria has been an increase in the prescription of antibiotics, which is likely to increase the already high levels of resistance to affordable antibiotics in Africa . There i