Plasmodium falciparum, to assist in identifying the most appropriate strategies for control and to evaluate the impact of control interventions. This study aimed to determine the genetic diversity and the multiplicity of infection in Plasmodium falciparum isolates from Pointe-Noire, Republic of Congo.Molecular characterization of malaria parasites from different localities is important to improve understanding of acquisition of natural immunity to Plasmodium falciparum isolates were collected from 71 children with uncomplicated malaria; enrolled into the study for evaluating the therapeutic efficacy of artemether-lumefantrine combination. Both msp-1 and msp-2 genes were genotyped.msp-1 and 27 msp-2 different alleles were identified. For msp-1, RO33 family was poorly polymorphic. The K1 family has shown the trend of predominance (41%), followed by Mad20 (35%). Comparatively to msp-2, 49.6% and 48.8% fragments belonged to 3D7 and FC27 respectively. Taking together msp-1 and msp-2 genes, the overall multiplicity of infection has been increased to 2.64 and 86% harbored more than one parasite genotype. Parasite density was not influenced by age as well as the multiplicity of infection which was not influenced neither by age nor by parasite density.From 296 distinct fragments detected, 13 Plasmodium falciparum in isolates from patients with uncomplicated malaria in Pointe-Noire is high and consisted mainly of multiple clones. The overall multiplicity of infection has been largely increased when considering msp-1 and msp-2 genes together. With the changes in malaria epidemiology, the use of both msp-1 and msp-2 genes in the characterization of Plasmodium falciparum infection is recommended.Genetic diversity of  Plasmodium falciparum, remains a public health concern. Although massive interventions deployed in sub-Saharan Africa have reduced the global malaria morbidity and mortality at 212 million and 429,000 deaths respectively in 2015, some sub-Saharan Africa region had provided partial data regarding the impact of certain interventions [Plasmodium falciparum to chloroquine as well as the inefficacy of sulphadoxine-pyrimethamine and amodiaquine either singly or in combination for the treatment of uncomplicated malaria have been well documented [Plasmodium falciparum infection (MOI) in other localities, nine years after the implementation of artesunate-amodiaquine and artemether-lumefantrine in Republic of Congo.Malaria, a disease mainly caused by cumented -6. Thus,cumented . Studiescumented -10. Onlycumented . AlthougPlasmodium falciparum strain, the occurrence of variant forms of the parasite in different geographic areas and variation of MOI after recombination between genetically distinct gametocytes, constitute the major obstacles to the design of a malaria vaccine [Plasmodium falciparum and would assist in identifying the most appropriate strategies for control and also to evaluate the impact of control interventions [(msp-1) and merozoite surface protein-2 (msp-2) are asexual blood stage antigens that are considered prime candidates for the development of malaria vaccine, and also suitable markers used extensively to identify genetically distinct Plasmodium falciparum parasite sub-populations in many malaria endemic countries as well as to distinguish recrudescence to re-infection in anti-malarial drug trials and efficacy [msp-1 and msp-2 allele frequency and genetic diversity should be monitored regularly to ensure the reliability of the PCR (polymerase chain reaction)-adjusted treatment outcome [Plasmodium falciparum genetic diversity have been conducted in Brazzaville [msp-2 gene as marker [msp-1 diversity in Pointe-Noire. During the study aiming the efficacy assessment of artemether-lumefantrine combination in Pointe-Noire in 2015, we proposed to determine the genetic diversity of Plasmodium falciparum in the recruited population using both msp-1 and msp-2 markers.Genetic diversity of circulating  vaccine , 13. Conventions , 15. Theefficacy -18. It hzzaville . Howevers marker . ConversPlasmodium falciparum.The present study was conducted at the “Centre de Santé Intégré de Mbota” located in the eastern part of the city of Pointe-Noire, the economic capital of the Republic of Congo, separated by 510km from Brazzaville. Two rainy seasons are observed each year with the main one during the months of February to May, and a short one from October to December. Malaria endemicity is high with perennial transmission and peaks during the rainy season, which normally runs from October to May  and malaPlasmodium falciparum was collected from children under 13 years old enrolled into the study (study ACTRN12615001110572) for evaluating the therapeutic efficacy of artemether-lumefantrine combination from September to November 2015. Diagnostic of Plasmodium falciparum was confirmed by light microscopy on thick blood smears. Malaria parasites were quantified against 200 leucocytes. Parasite density was calculated for each patient assuming an average of 8,000 leucocytes per µL of blood using the proposed method of the WHO [Infected blood with  the WHO . Before Genomic DNA was extracted from samples collected on the Watman filter paper by QIAamp DNA mini Kit  according to the manufacturer’s instruction. Extracted DNA was stored at -20°C until use.Plasmodium falciparum was performed using the nested polymerase chain reactions (PCRs) technique. The merozoite surface protein-1 (msp-1) and merozoite surface protein-2 (msp-2) genes in their highly polymorphic loci, namely msp-1 block 2 and msp-2 central region were used as markers for this genotyping as described previously [msp-1, and FC27 and 3D7 allelic families for msp-2  value for each allelic family of msp-1 and msp-2 genes was calculated as described by Mohd Abd Razak et al. [msp-1 and msp-2 allele was considered as one parasite genotype. The multiplicity of infection was defined as the minimum number of Plasmodium falciparum genotypes per infected subject and estimated by dividing the number of amplified PCR fragments reflecting the parasite genotypes by the number of positive samples. Chi-square test was applied to compare proportions. Spearman’s rank correlation coefficients were calculated to assess association between multiplicity of infection (MOI) and geometric mean parasite density and age. Differences were considered significant if P values < 0.05.The frequency of k et al. . The detPlasmodium falciparum only were enrolled in the study, with 34 (47.9%) and 37 (52.1%) being males and females respectively. Twenty-six and 45 were children aged between 1 to 4 years old and 5 to 12 years old respectively. The geometric mean parasite density was 36,699.6 parasites/µl of blood with the range of 1,435-349,700 parasites/µl , Mad20 (35%) and R033 (24%) families respectively. Eight K1 type alleles , 4 Mad20  type alleles and only 1 RO33 type allele (150 pb) were identified, with no statistical significant predominance of any specific family  and 14 FC27 type alleles , with no statistical significant predominance of any specific family  and 61 belonged to FC27 (48.8%). Two genotypes (1.6%) could not be assigned to any specific family.All 71 samples were analyzed for polymorphisms on c family . The mspc family . Out of msp-1 and msp-2 genes separately, the MOI was 2.40 and 1.74 respectively, while 55 (77.46%) and 34 (47.88%) of isolates contained multiclonal infection at least with 2 clones respectively. Taking together msp-1 and msp-2 genes, the overall MOI was 2.64 and 61 (86%) isolates harbored more than one parasite genotype. No statistical significant difference was observed in the MOI between patients with less than 5 years old (2.44) and those with the age between 5 and 12 years old (2.40) with the p-value = 0.972. The combinations of msp-1 and msp-2 different parasite genotypes were not associated with the parasite density . Taking together, no statistical significant difference was observed in the parasite densities and the MOI according to the age (When considering  density , as well density . Moreove the age .Plasmodium falciparum genetic diversity using merozoite surface protein genes which are considered like robust and suitable markers [Plasmodium falciparum msp-1 and msp-2 allele frequency-genetic diversity [Plasmodium falciparum populations to employ both msp-1 and msp-2 gene markers, 9 years after the implementation of ACTs for the treatment of uncomplicated Plasmodium falciparum malaria in Pointe-Noire.The genetic diversity may be an important element for implementing malaria control strategies in the country, as elimination may influence genetic diversity . In the  markers , 25, 26  markers , 19, 27.iversity , despiteiversity , 28. To msp-1 and msp-2 genes showed a high genetic diversity in the Plasmodium falciparum population in the analyzed isolates. The degree of polymorphism found in the present study is also consistent with the previous findings in the same area using microsatellites markers [msp-2 gene has showed a high diversity as previously reported in Pointe-Noire [msp-1 gene was amplified in all isolates, genetic diversity still low compared to msp-2 gene as previously described in the country [msp-1, the presence of Mad20, K1 and R033 allelic families in the 71 isolates was noticed. RO33 family was poorly polymorphic with only one allele as reported in previous studies in Brazzaville and other countries such as Senegal, Iran, Nigeria and Brazil [msp-2, no predominance to any allelic family was found. Taking together msp-1 and msp-2, our findings are in contrast to those reported in previous studies in Brazzaville and other countries such as Gabon, Benin, Ghana, and Ethiopia where K1 for msp-1 and 3D7 for msp-2 were predominantly found in isolates from uncomplicated malaria [Allelic specific PCR typing of  markers . The mspte-Noire . Althoug country . With red Brazil , 30-32 i malaria , 33-36.Plasmodium falciparum infection in Pointe-Noire are needed to better characterize the allelic polymorphism of Plasmodium falciparum in this city. Understanding the diversity of Plasmodium falciparum has implications for vaccine since the major obstacles to the design of an efficient malaria vaccine is the large genetic diversity of vaccine targets allowing parasites with mutated genes to escape from the host’s immune response [Plasmodium falciparum for improving vaccine design.The trend observed in the prevalence of K1 and 3D7 families in the current study may be related to the limited sample size. Repeated studies including a large sample size and different sites including symptomatic and asymptomatic response . Thus, dmsp-1 and msp-2 genes of 2.40 and 1.74 respectively. The overall MOI value of 2.64 was found, when taking together msp-1 and msp-2 genes and this was higher than values obtained from Brazzaville, even before or after the introduction of ACTs [The presence of more than one parasite genotype was detected in 86% of isolates from Pointe-Noire, with the MOI values for  of ACTs , 19, 20. of ACTs , 33, 37. of ACTs , 39. Mul of ACTs , 13, 40. of ACTs , 41, 42, of ACTs , 39. ThePlasmodium falciparum in isolates from patients with uncomplicated malaria in Pointe-Noire, Republic of Congo is high and consisted mainly of multiple clones. By considering msp-1 and msp-2 genes together, the overall multiplicity of infection has been largely increased. Thus, with the changes in malaria epidemiology, this study supports the use of both msp-1 and msp-2 genes in the characterization of Plasmodium falciparum infection, when the MOI is considered like one of the major parameters to be evaluated for malaria control interventions.Genetic diversity of Malaria is still the leading cause of attendance in health facilities in many sub-Saharan countries;Plasmodium falciparum genetic diversity would assist in identifying the most appropriate strategies for control and also to evaluate the impact of control interventions as well as changes in malaria epidemiology;Plasmodium falciparum genetic diversity and the MOI using both msp-1 and msp-2 as molecular markers, particularly in Pointe-Noire, the Republic of Congo.Little data available regarding the Plamosdium falciparum is high and consisted mainly of multiple clones in Pointe-Noire;Genetic diversity of msp-1 and msp-2 are used together as molecular markers;Increase MOI when The epidemiological trend of malaria seems to be different between Pointe-Noire and Brazzaville in the Republic of Congo.The authors declare no competing interests.


Unfortunately after publication of the original article , it cameFigure 2 is incorrectly captioned as: “Kernel density estimates of PCR-positive malaria prevalence in the study sites”.Please be advised that the correct caption for Fig. 2 is “Fig. 2. Kernel density estimates of RDT-positive malaria prevalence in the study sites”.


Plasmodium falciparum and Intestinal Parasitic Infections (IPIs) - with the corresponding pathogenesis among children remain uncertain. This study aimed at determining the prevalence and the outcomes (including anaemia) of the respective infections and co-infections. Anaemia is a condition in which the number of red blood cells transporting oxygen to the various body parts is not sufficient to meet the needs of the body.The prevalence of This was a cross sectional study conducted among 476-refugee camp school children. Kato-Katz technique was used to screen stool samples for intestinal parasites. Microscopy was used for malaria testing while the portable Haemoglobin (Hb) calorimeter was used to measure haemoglobin concentration.Plasmodium falciparum was most prevalent of the single infections 262(55.04%) followed by Taenia spp. 14 (2.9%), Schistosoma mansoni 12(2.5%), Giardia lamblia 7 (2.9%), Trichuris trichiura 2(0.4%), Hookworm 2(0.4%) and Strongyloides stercoralis 1(0.2%). The odds of developing simple or uncomplicated malaria infection or anaemia was 14 times higher in individuals with dual co-infection with Plasmodium falciparum + Taenia sp. compared to single parasitic infection . Co-infection with Plasmodium falciparum + Taenia spp, was a strong predictor of Malaria and anaemia.The overall prevalence of the mixed infections was 63.03%. Plasmodium falciparum and Taenia spp. co-infections is a stronger predictor of malaria and anaemia. The prevalence of malaria and anaemia remains higher than the other regions in Uganda outside restricted settlements. The findings of this study underline the need for pragmatic intervention programmes to reduce burden of the co-infections in the study area and similar settlements.This study shows that The online version of this article (10.1186/s12879-019-3939-x) contains supplementary material, which is available to authorized users. Uganda currently hosts the highest number of refugees in the country’s history and also continues to receive simultaneous emergency influxes from South Sudan, Democratic Republic of Congo (DRC) and Burundi. The Kiryandongo refugee camp has been one of largest camps created to resettle the increasing number of Sudanese refugees in Uganda which has exceeded one million, and the daily arrival rate remains high , 2. MalaSeveral studies have had limited contribution of intestinal metazoan and protozoa co-infections with malaria in children . SimilarThe most common diseases in the Kiryandongo refugee camp have been previously reported and remain as malaria, upper respiratory tract infections and watery diarrhoea . No studThis study was carried out from October 3, 2017 to May 10, 2018, at Kiryandongo refugee settlement camp in Kiryandongo district, Mid-Western region of Uganda, as shown in Fig. The primary target population were divided into two groups aged < 5 and 5–14 years as per the local Uganda Ministry of health guidelines. The secondary study population were the care takers/guardians respectively residing within the refugee resettlement camp.The study included all children who provided both parental consent and assent in addition to providing blood and stool samples. Children below 3 years and above 14 years were excluded from the study.Plasmodium falciparum and intestinal parasitic co-infection of children. Eligible children aged 3–14 years were recruited and screened for malaria and intestinal parasitic infection. Parents/guardians were interviewed to provide socio-demographic information related to sanitary and malaria prevention practices.An analytical cross-sectional study design was used to investigate the relationship between 2 x P x (1-P)/d2. A minimum of 437 samples (n) was estimated as shown: n = Z2 P (1-P)/d2  by taking the proportion of metazoa and protozoa co-infection prevalence in the study area. However, since the prevalence of co-infection is unknown, was used 0.50 to have the largest sample size. The expected margin of error was taken to be 5% (d = 0.05). Using the formula, n = ZRandom sampling of children visiting the Panyadoli Health Centre III (HC III) based on the selection criteria was used to obtain blood film and stool sample by the law of voluntarism. Using roll numbers, random numbers were generated using Microsoft Excel 2013. This technique provided each participant with an equal chance of participating in the study. Along with sample collection, face-to-face interview of parents/guardians was conducted using a questionnaire constituting socio-demographic information. The data collection form has been included as Additional file A researcher developed structured questionnaire based on the objectives of the study, conceptual framework and guided by the literature review was used to collect data from the respondents. The data collection tool with the questionnaire has been included as Additional file The choice of diagnostic techniques was dependent on available equipment, reagents, experience of the technician and considerations of cost. The techniques used in this study for analysis of stool and blood samples included malaria microscopic examinations using Giemsa Staining technique, intestinal parasitic detection using Kato Katz and Calorimetric Hb estimation. Microscopy was used for detecting malaria because it is a gold standard putting in mind the limitation of rapid diagnostic kits (RDT) since it detects malaria antigen 4 weeks even after treatment and does not allow differentiation of malaria species.Completed questionnaires were serialized and coded. Data was entered and cross checked for errors using Microsoft Excel 2013 before running the analysis.Plasmodium falciparum infection, intestinal parasitic infection and co-infection. Chi-square test and Fishers Exact test was used to determine the association between the factors associated with Plasmodium falciparum and intestinal parasites. Statistical significance of the differences in the prevalence of single and co-infection with each factor was determined via Chi-square analysis and Fishers Exact test. Multivariate logistic regression analysis was used to predict the extent of the association with the different infection status as the dependent variable. The study was able to control for confounding factors during analysis by adjusting for age, sex and nutritional status, while the association between helminth and Plasmodium infections, and related outcomes was evaluated.The preliminary data was entered into Microsoft Excel 2013 and exported into STATA 14. Descriptive statistics was done to analyse the background characteristics of participants and the factors associated with Table Plasmodium falciparum accounted for the highest prevalence 262(55.04%).Nutritional status assessment using mid upper arm circumference (MUAC), showed no evidence of malnutrition of 100% of children who had median MAUC of 18.8 cm. Out of the 476 children examined, 300 (63.03%) had a mono-infection, 28(5.88%) were co-infected with malaria and IPIs whereas 148 (31.09%) were not, as shown in Table Plasmodium falciparum was (5–14 years) with the prevalence of 55.7%. The younger age group (< 5 years) had a prevalence of 52.7% and the difference was statistically significant (P = 0.000). A total of 256 (53.8%) males and 220 (46.2%) females were enrolled in the study. Overall, 72.27% (159/220) of the females were slightly more infected with all the intestinal helminths as compared to 55.1% (141/256) of the males but this and the difference was not statistically significant (P > 0.05).From table Plasmodium falciparum + Taenia spp. 5 (1.05%), Plasmodium falciparum + Giardia lamblia 2 (0.42%) and Plasmodium falciparum + Schistosoma mansoni 1(0.21%).As shown in table Plasmodium falciparum + Taenia spp. compared to single parasitic infection .The results above further indicate that the odds of developing simple or uncomplicated malaria infection was 14 times higher in individuals double infected with Plasmodium falciparum + Taenia spp.  showed a strong predictor of anaemia in the study population. No single infection was a strong predictor of anaemia.From table From table Plasmodium falciparum followed by Taenia spp. at 2.94%. However, studies conducted in North Western Uganda reported a lower prevalence of anaemia (34.4%) of children aged 1-14 years [More than half of the study population tested positive for one parasite (63.03%). The results of this study are similar to the previous report in Tach Gayint, Ethiopia  and in t14 years . This wa14 years ., 2011 among children living along shores of Lake Albert and in the Islands of Lake Victoria [Taenia spp. (2.9%). Therefore, the current finding suggests that the co-infection of Plasmodium falciparum and Taenia spp. is a stronger predictor of anaemia. These results differ from previous reports where the prevalence of the co-infection of 22% [The current finding of the higher prevalence of malaria and anaemia, is comparable to the previous report by Green et al.Victoria . It is nn of 22% . This isPlasmodium falciparum reported in this study is four times higher than previously reported Mid-Western region of Uganda [N = 4939) higher compared to our sample size.The high prevalence of f Uganda . These df Uganda . The difIn this study, the prevalence of intestinal parasitic infection (IPIs) was 7.9%, which is lower compared to the 34.7% reported in communities around Dschang, in the West Region of Cameroon , 36.5% iIt is important to note that low socioeconomic status, poor living conditions, insufficient knowledge and practice of correct sanitation, and limited access to safe water are known risk factors for IPIs, are also common in populations from these settlements. This argument is consistent with reports from Ethiopia  and Rwanda (54.5% in a remote area versus 20% in a peri urban area) , but conThe prevalence of intestinal protozoa was higher than that of intestinal metazoa (1.5 vs. 0.8%), which is in contrast with some studies but in agreement with others due to the urbanization, the public investments in basic sanitation, and improvement of general living conditions, and the accessibility to health services , 22, 24.Taenia spp. were reported in studies conducted in Ghana [Taenia spp., which is similar to reports previously published across Africa demonstrating taeniasis prevalence ranging from 0 to 8.7%, although these studies do not use a standardized diagnostic protocol [Taenia spp. infection risk in humans.A higher prevalence of 13.15 and 9.6% in Ghana  and Nigein Ghana  respectiprotocol . This isprotocol . This suPlasmodium falciparum were significantly more likely to harbor Taenia spp. 5 (1.05%) and Schistosoma mansoni 1(0.21%) compared to individuals without malaria. The most prominent co-infection combinations were of Plasmodium falciparum + Taenia spp. 5 (1.05%), Plasmodium falciparum + Giardia lamblia 2 (0.42%) and Plasmodium falciparum + Schistosoma mansoni 1(0.21%). The factors that supported the co-infections in this study include humidity, surrounding water bodies of Lake Kyoga and Albert, bushes and poor sanitary disposal. The co-infection of Schistosoma mansoni and Plasmodium falciparum observed in this study has been previously observed [In this study, individuals infected with observed . The finobserved .Plasmodium falciparum + Taenia spp. compared with single parasitic infection. The co-infection of Plasmodium falciparum + Taenia spp. showed a stronger prediction of malaria and anaemia . The reason for this co-infection leading to malaria is due to the established immunological antagonism of Th1 and Th2 for Plasmodium falciparum and Taenia control as has been reported previously in a similar study with other helminthes. Plasmodium falciparum and Taenia infections are established risk factors of anaemia. Therefore, this co-infection is expected for the observed prevalence of anaemia. Helminthiasis has been shown to significantly contribute to the problem of anaemia in studies conducted in Rwanda, Kenya and Uganda [The current study shows that the odds of developing malaria and anaemia was 14 times higher in individuals co-infected with d Uganda –34.P > 0.05) possibly due to our sample size and the extremely low prevalence. This finding agrees with studies conducted in Uganda around the lake side and island communities of lake Victoria that reported declined prevalence of Schistosomiasis from 42.4 to 17.9% in 2005, while hookworm prevalence reduced from 50.9 to 10.7%, Ascariasis and Trichuriasis from 2.8 and 2.2% in 2003, to very undetectable levels in 2005 [In this study, we did not find statistical relationship between single helminthes infection and anaemia


Over the past 15 years, malaria mortality has reduced by approximately 50%. However, malaria still causes more than 400,000 deaths annually, most of which occur in African children under 5 years of age. Significant advances in understanding the pathogenesis of the disease provide a basis for future work to prevent severe malaria and its complications. Herein, we provide an overview of advances in our understanding of severe malaria in African children over the past 15 years, highlighting key complications and identifying priorities to further reduce malaria-associated mortality. Plasmodium falciparum accounts for the majority of malaria deaths, and is the predominant malaria species in Africa  [ica Fig.  1]. Sev. SevPlassitemia) . Furthersitemia) .Fig. 1SeP. falciparum erythrocyte membrane protein-1 (PfEMP1) is expressed on the pRBC membrane, where it binds to endothelial receptors . Recently, the discovery of endothelial protein C receptor (EPCR) as a novel and conserved host receptor for PfEMP1 binding transformed our understanding of SM pathogenesis  in vascular beds, leading to impaired tissue perfusion and lactic acidosis. Parasite ligand nd Smith ), providEndothelial activation is common in SM and is associated with upregulated cellular adhesion molecules on endothelium and their concomitant increase in plasma through ectodomain cleavage . The angSM is a multi-system disease driven by both host and parasite factors. CM is the deadliest form of SM. Children with CM often have malaria retinopathy, presenting with hemorrhages, retinal whitening, and vessel color changes, all of which can be visualized by trained observers using ophthalmoscopy. Interestingly, these changes mirror findings in the brain at autopsy . FurtherRespiratory distress is a common form of SM best characterized by deep acidotic breathing. Like other forms of SM, respiratory distress is multifactorial. Impaired tissue perfusion secondary to pRBC sequestration and reduced oxygen-carrying capacity in severe anemia contribute to acidosis . It is eFifteen years ago, renal failure was considered a rare complication in children with SM, yet it is now recognized as one of the strongest predictors of mortality in SM . The recSevere malarial anemia (SMA) is the most common form of SM. The etiology of SMA is complex, involving increased destruction and removal of infected and uninfected RBCs, and reduced RBC production due to bone marrow dyserythropoiesis (recently reviewed by White ). SMA caOne in four children prospectively enrolled in studies with CM develop neurocognitive impairments that persist at least 2 years following exposure . RetrospElevated cerebrospinal fluid (CSF) levels of TNF-α are associated with prolonged coma duration, neurologic deficits, and long-term cognitive deficits in children over 5 years . A stronFigure


Plasmodium falciparum malaria and treated with intravenous artesunate. The patient suffered from wide complex tachycardia after treatment with artesunate 170 mg (2.4 mg/kg) i.v. bolus, tachycardia was managed with amiodarone (150 mg i.v. for 10 minutes). Electrocardiographic abnormalities, including QT prolongation, are common in patients with sickle cell anemia. The mortality rate in sickle cell anemia patients due to cardiovascular and pulmonary complications remains high. The probability of precipitation of ventricular arrhythmias may increase in patients with sickle cell anemia, diagnosed with malaria and treated with artemisinin derivatives.Treatment of severe malaria with artemisinin derivatives in patients with comorbid conditions such as sickle cell anemia must be considered with precaution. We report here a case of possibly undocumented ventricular arrhythmia in a sickle cell anemia patient diagnosed with  Plasmodium species was P. falciparum (97%) followed by P. vivax (2%). There are members of four Anopheles species complexes in KSA including An. gambiae, An. fluviatilis, An. culicifacies, and An. subpictus complexes. The species of An. arabiensis from An. gambiae complex is the only member of the complex that extends outside of Africa into the southwestern and western areas of the Arabian Peninsula including in KSA and Yemen, where it is a major malaria vector. Other recorded vectors of malaria in KSA include An. fluviatilis, An. stephensi, and An. azaniae [Plasmodium falciparum malaria recommended by the WHO. In adults, parenteral artesunate remains the treatment of choice for severe P. falciparum malaria [Malaria remains one of the major public health issues in the modern world. Malaria is endemic mainly in the sub-Saharan Africa, Southeast Asia and Western Pacific regions. The WHO report shows that, in 2017, an estimated 219 million cases of malaria occurred worldwide, compared with 239 million cases in 2010 and 217 million cases in 2016. There were an estimated 435,000 malaria-related deaths in 2017 [P. falciparum malaria patient with sickle cell anemia, treated with intravenous artesunate. A written informed consent of the patient was obtained to publish his data.Chronic hemolytic anemia is the characteristic feature of sickle cell anemia. It is associated with high morbidity and mortality. Highest prevalence of sickle cell anemia in Saudi Arabia is in the Eastern Province, followed by the provinces of the southwestern region . Electro9/L ; hemoglobin 7.3 g/dL ; mean corpuscular volume 72 fL/red cell ; platelet count 62 × 103/μL ; reticulocyte 2.6% ; aspartate transaminase 89 U/L ; alanine transaminase 78 U/L ; total bilirubin 600 μmol/L ; direct bilirubin 200 μmol/L ; and sodium 149 mmol/L , whereas the following laboratory values were found to be in normal ranges: alkaline phosphatase 120 IU/L ; random blood glucose 7.2 mmol/L ; potassium 4.9 mmol/L ; magnesium 0.9 mmol/L ; calcium 2.3 mmol/L ; phosphate 1.4 mmol/L ; creatinine 78 mmol/L ; and urea 3.7 mmol/L .A 19-year-old male with sickle cell anemia, being treated with folic acid, presented to the emergency department of Samtah General Hospital, Jizan, Saudi Arabia, with fever, nausea, headache, and yellowish discoloration of sclera. Patient was living in Jizan, an endemic area of malaria, which raised suspicion of malaria infection. Patient looked unwell, conscious, and alert. His vital signs were normal except the temperature which was high (38.5 degree C). Chest examination was normal and the cardiovascular system showed normal first and second heart sounds with no added sound. Fundus examination was normal. Other systemic reviews were unremarkable. Patient was admitted in the medical ward, and baseline laboratory investigations revealed the following remarkable laboratory values: white blood cells 12 × 10P. falciparum (ring form) with parasitemia index 10%. His body weight was 74 kg and height 166 cm.ECG initially showed normal sinus rhythm, and chest X-ray was normal. The patient had no previous history of cardiac arrhythmias, and his baseline ECG was normal . PeripheP. falciparum malaria based on clinical presentation and laboratory investigations and started on intravenous artesunate 170 mg (2.4 mg/kg) bolus slowly over 2 minutes at 0 hour, 12 hours, and 24 hours, and every 24 hours thereafter for 5 days. The powder for injection was reconstituted with 5% sodium bicarbonate and diluted in an equal volume of normal saline. He was started on paracetamol 500 mg tablet orally as needed every 6 hours. Patient was clinically improving and temperature subsided. After 4 hours of the third dose of artesunate, the patient became sick, faint, and suddenly arrested. Resuscitation was done for 32 minutes, ECG was done and showed wide complex ventricular tachycardia, and QTc (Bazett) was 516 msec , the QTc interval exceeded 0.5 seconds, but in both cases, an alternative explanation was plausible [Artemisia annua are also active against other diseases, such as, cancer, viral infections, schistosomiasis, and trypanosomiasis. Artesunate has been effectively and safely used as an add-on therapy to the guideline-based oncological therapy in patients with metastatic breast cancer [Artemisia annua demonstrated efficacy against non-small cell lung cancer cells [Malarial parasite is able to develop resistance against antimalarial drugs. Hence, WHO recommends the use of combinations of two drugs, having different mechanisms of action, as a strategy to overcome antimalarial resistance. Artemisinin is one of the vital components of antimalarial combination therapy, known as artemisinin-based combination (ACTs). It is now considered as first-line treatment for complicated and uncomplicated malaria in Saudi Arabia. Our case diagnosed as complicated i Arabia . It is ri Arabia . In a prlausible . In anotlausible . Alteratlausible . One replausible . Artesunnts n = 2, the QTclausible . It has lausible . There alausible  Moreovert cancer . In a reer cells . Thus, tDue to presence of other predisposing factors known to cause arrhythmias, it is presumed that artesunate induced the ventricular arrhythmia. Artesunate is a relatively new antimalarial drug from artemisinin derivatives. It is considered as first-line treatment in patients with malaria in many endemic areas. However, the possibility of cardiovascular effects of artesunate should always be kept in mind prior to its administration, especially in patients with predisposing factors for causing arrhythmias, such as sickle cell anemia and in malaria-endemic regions.


Malnutrition remains a serious health problem in Cambodia with over 10% of children less than five years of age suffering from acute malnutrition. In addition to the presence of nutritional edema, two indicators are recommended by the World Health Organization for the diagnosis of acute malnutrition: weight-for-height Z-scores  and mid-upper arm circumference . Earlier, we showed that WHZ and MUAC identified different subgroups of children with acute malnutrition. To explore factors associated with both indicators of acute malnutrition, we analyzed baseline data from a longitudinal study in three provinces in Cambodia: Phnom Penh , Kratie , and Ratanakiri . Data was available for 4381 children below 30 months of age. Malnutrition rates were higher in the two rural provinces than in the capital. Although both MUAC and WHZ showed gender bias, with MUAC identifying more girls, and WHZ identifying more boys with acute malnutrition, the gender effect was strongest for MUAC. The gender bias of MUAC diminished with older age, but remained significant up to 30 months of age. Only using both MUAC and WHZ as indicators resulted in gender neutral identification of acute malnutrition. WHZ alone always identified more children with acute malnutrition than MUAC alone. In Phnom Penh, MUAC alone identified only 11% with acute malnutrition in addition to WHZ. To conclude, both MUAC and WHZ showed gender bias in this cohort of Cambodian children. In Cambodia, implementation of a MUAC-only or a WHZ-only program for the identification of acute malnutrition would be unethical as it will lead to many children remaining undiagnosed. Acute malnutrition (AM), which includes both marasmus and kwashiorkor, is still a major health threat in children, associated with a remarkably higher risk for mortality and morbidity. Children who are wasted have an almost five times higher risk to die, a risk that increases to over 12 times when stunting is also present .In addition to the presence of nutritional edema, indicating kwashiorkor, the World Health Organization recommends using either weight-for-height Z scores (WHZ) and/or mid-upper arm circumference (MUAC) for diagnosing acute malnutrition in children above six months of age . WhereasHowever, as recently shown by us, MUAC and WHZ identified two different subgroups of Cambodian children suffering from malnutrition , with liBriend and colleagues have argued that genetic differences in fat distribution, for example, the different fat distribution in Asian populations, or differences in length or height could explain the differences between MUAC and WHZ to identify acute malnutrition . To idenThe MyHealth study is a longitudinal cohort study, conducted in three provinces in Cambodia: Phnom Penh , Kratie  and Ratanakiri . For the cohort study over 1000 children and 1000 women were recruited in each province.Lists of pregnant and lactating women, women in reproductive age (WRA) and children under three years were obtained from village health volunteers (VHSGs) and midwives in selected operational districts. In total 5419 households were recruited and teams visited each household to collect data on socio-economic data, dietary intake, water, sanitation and hygiene, and anthropometry of mother and child. In addition, in each province, pregnant women were recruited into the study also, but these are not considered in the present study. Eligible children were children aged between 0 and 30 months of age, and not suffering from a chronic health condition requiring medication . Data foChildren’s heights were measured to the nearest 1 mm. Height measurements (UNICEF measurement boards) were collected in triplicate from each child in the household under three years to ensure accuracy. Weight were measured to the nearest 0.1 kg . The nutritional status of children was defined length or height-for-age (L/HAZ), weight-for-height (WHZ), and weight-for-age (WAZ) z-scores, calculated according to the Child Growth Standard of the World Health Organization (WHO) using the WHO Anthro software. To ensure the accuracy of the data, extreme values were excluded from the analysis: WAZ < −6 or > 5; L/HAZ < −6 or >5; WHZ < −6 or > 5. Excluded values represented less than 5% of the total values. We used the following cut-offs to define wasting: weight for height < −2 Z-scores (according to the WHO growth charts) or MUAC < 12.5 cm, and to define severe acute malnutrition (SAM): weight for height < −3 Z-scores or MUAC < 11.5 cm. Stunting was defined as a height-for-age < −2 Z-scores.Ethical approval for the MyHealth study was obtained from the Cambodia National Ethical Committee for Health Research, National Institute of Public Health, Ministry of Health, Cambodia. Informed consent was obtained from all participants, with consent obtained from parents or guardians for participating children. All data collected is anonymized and stored on the Kobotoolbox server, with access to the data limited to the core team of researchers only.www.kobotoolbox.org). Anthropometrical indicators  were calculated according to the WHO growth standards [Data was collected on tablets, using the Koboltoolbox software , used as reference category. For the WHZ diagnosis, subjects were categorized in a similar way: children with a WHZ < −2 cm; children with a MUAC < 12.5 cm, but a WHZ ≥ −2; and non-wasted children (WHZ > −2 and MUAC > 12.5 cm). We then performed two multinomial logistic regression models using these categorical variables as a response, adjusting for all the selected covariates, and then plotted the average marginal effect of the gender variable on the indicator category for several values of age. To examine if there was any gender bias when one considers the two diagnoses together , we created a binary variable taking the value 0 if the child is not wasted and 1 if s/he is diagnosed by any of the two indicators. This variable was then used as a response variable in a logistic model including all the covariates, and the average marginal effect of the gender variable was estimated.We used Stata 13 for data management and Stata 13  and R software version 3.4.0 for analysis. The type I error risk was set to 0.05 and the complete cases analysis was used to deal with missing data for all the multivariate analyses.P < 0.05, chi-square). Overall prevalence of AM was lower in Ratanakiri (17.2%) than in Kratie .Data were available for 4381 children  when using both WHZ and MUAC as an indicator. The remaining 42 children with AM in Phnom Penh were below six months of age, and would not have been identified with MUAC, as MUAC is used only in children above six months of age. Hence, in Phnom Penh, only 1/3 of the malnourished children above six months of age was identified with MUAC alone. For Kratie and Ratanakiri, MUAC alone identified 146 and 136 children, respectively . In contrast, in children above six months of age, WHZ alone identified 121 (85.8%), 250 (81.4%), and 161 (73.5%) in Phnom Penh, Kratie and Ratanakiri, respectively.P = 0.021, P < 0.01), being stunted  and being male , being stunted  or living in a rural province (aRRR 2.3 and 2.0 for Kratie and Ratanakiri, respectively, The gender bias of MUAC and WHZ as indicator for acute malnutrition is reflected in the figures A–C belowTo explore the age relationship between acute malnutrition and the two indicators for acute malnutrition, we plotted the probability to be diagnosed with AM by the two indicators alone or combined. As can be seen in In urban Phnom Penh, WHZ alone always diagnosed more children with AM than MUAC alone, whereas in the two rural study sites, MUAC alone diagnosed most of the infants with acute malnutrition between 6 and 12 months of age, before rapidly becoming less important. In urban Phnom Penh and rural Ratanakiri, there was a steady increase in the prevalence of acute malnutrition between 6 and 30 months of age, which was almost exclusively driven by an increase in the number of children having a WHZ < −2. In contrast, in rural Kratie, the prevalence of AM picked around 18 months of age. For all sites, after 18 months of age, WHZ alone became by far the most important indicator to diagnose acute malnutrition with >50% of the cases being diagnosed through WHZ alone.To further explore the relationship between height and indicators of acute malnutrition, we made similar curves for HAZ scores .In general, there was a negative association between the probability of being diagnosed with AM and HAZ, that is, being taller reduces the likelihood for having AM. MUAC as indicator for AM tended to be more affected by length status than WHZ. However, only in Ratanakiri was WHZ as an indicator for AM not related to the HAZ score at all.The present study confirms the earlier study from Cambodia that MUAC and WHZ identify different sub-sets of children  and it hThe present study has some important practical implications. It has been argued that children with a low MUAC are at an especially higher risk of death , and thaAnother important policy implication of the current study is that unfortunately, programs using WHZ alone, are not suitable either. In Ratanakiri, MUAC alone identified 58 additional cases of AM, or 24.9% of the total. Hence, using a WHZ-alone program would have left almost 25% of the children with wasting unidentified. Earlier, we proposed the use of different cut-offs for MUAC for screening for AM, and for initiating treatment for AM [In our current study, almost 18% of the children was identified as being wasted, with around 3% of SAM. In Cambodia, and in most other countries, no program exists for the treatment of MAM, and only cases of SAM are being treated. Indeed, it has been proven difficult to reach most children with SAM, with <10% of the estimated 60,000 SAM cases receiving treatment in 2014. However, an integrated approach, trying to improve the nutritional status of all children with AM, with standard treatment for children with SAM, and supplementary foods for children with MAM, might increase awareness in the Cambodian population for this serious health problem.In the present study, and as reported earlier as well, a low MUAC was also associated with younger age and being stunted ,14. It iA weakness of the current study is that only cross-sectional data was available, with no long-term follow-up on subsequent risk for death or morbidity. As noted above, MUAC has been preferred above WHZ as an indicator for malnutrition, as MUAC was associated with a higher risk for death than a low WHZ . HoweverTo conclude, the present study showed large within country differences in a relative small country as Cambodia, in the ability of MUAC and WHZ to identify children with acute malnutrition. Both indicators showed gender bias, but MUAC did to a far larger extent than WHZ. Overall, WHZ identified more children with acute malnutrition than MUAC. Linear growth status affected MUAC more than WHZ but, even in stunted children, WHZ identified more children overall with acute malnutrition than MUAC. To ensure that no child with acute malnutrition is left without proper treatment and follow-up, both MUAC and WHZ should be used as indicators for nutritional status in children < 5 years of age.


Cases on concurrent infection of dengue and malaria are uncommon in Pakistan. Dengue and malaria are the two very common, mosquito-borne infections, which may have significant morbidity and mortality if not managed properly. Concurrent infections of dengue and malaria are rare due to the different habitats of its vectors and activities of different bearer mosquitoes. The first case was reported in 2005. Since then, several co-existing infections have been reported consisting of the dengue virus (DENV) and the malaria protozoans. Symptoms of each infection may be disguised by a coincident second infection, resulting in delayed treatment and severe complications. We report a case of concurrent malaria and dengue viral hemorrhagic fever in a 19-year-old male. Several tropical vector-borne infections continue to cause a rising incidence of morbidity and mortality in resource-restricted nations. Two of those infections are: a parasitic disease malaria and a viral disease dengue. Malaria is caused by a female mosquito, Anopheles; while dengue is caused by Aedes aegypti mosquito. In the past, the presence of a co-infection in an individual is sporadically reported , 2. BothA 19-year-old male, a resident of Mirpur with no known co-morbidities, presented to the emergency department (ED) of Dr. Ruth KM Pfau, Civil Hospital Karachi (CHK) in September 2018 with a history of fever, dizziness, generalized weakness and bleeding of gums since the past three days. According to the patient, he suddenly developed a continuous fever of 102°F, which was sporadic in nature and associated with chills and rigors. The fever temporarily alleviated with intake of antipyretics. He also experienced occasional bleeding from gums and dizziness with generalized weakness, for which he sought symptomatic treatment from a local health care facility, but the symptoms worsened. Therefore, he was then referred to CHK. No history of bleeding from any other site, nor hematemesis or black tarry stools was present. The patient revealed a decreased appetite and past addiction to tobacco.On examination (O/E), the patient was of average height and built, comfortably lying on the bed, and well oriented to time, place and person. Initial vitals included blood pressure (BP) 120/70 mmHg, a regular pulse of 90 beats/min and a respiratory rate of 20 breaths/min. The patient was anemic and dehydrated. He had a soft, non-tender, non-distended abdomen without hepatosplenomegaly, and bowel sounds were audible with a rate of 3-4/min. All other systems were unremarkable.Blood investigations revealed a hemoglobin (Hb) of 4.5 gm/dl, mean corpuscular volume (MCV) of 108.5 fl, mean corpuscular hemoglobin concentration (MCHC) of 35.4 gm/dl, total leukocyte count (TLC) of 2.7 x 109 L, hematocrit (HCT) of 12.7%, platelet count (PLT) of 12 x 109/L. The clotting profile showed an international normalized ratio (INR) of 1.11, while prothrombin time (PT) and activated partial thromboplastin time (aPTT) were 11.1 and 21.7 seconds, respectively. The various lab investigations conducted, including those for hepatitis B surface antigen and hepatitis C antibody, both came out normal, as did his X-ray chest, renal and liver function tests, spot urine examination and ultrasound of the abdomen. The need for conducting specific investigations like hepatitis and chest X-ray were to exclude any other likely cause of the fever and presence of an infection. The electrolytes were within the normal range as well. Upon serology testing, dengue antigen came out to be reactive while that of dengue virus-specific antibodies, immunoglobulin M (IgM) and immunoglobulin G (IgG), were not reactive. Malarial parasite (MP) and MP immunochromatographic test (ICT) came out to be positive for Plasmodium (P) vivax with 7% reticulocytes (RET). Therefore, a diagnosis of concurrent malaria and dengue infection was established.The patient was treated with an oral combination of artemether and lumefantrine 80/480 twice daily for three days, along with oral acetaminophen two tablets if needed. Additionally, he was intravenously (IV) given tranexamic acid 5 mg when necessary and 1000 ml sodium chloride at the rate of 80 ml/hour. Two units of packed cells and six units of platelets were transfused alongside. Malaria and dengue were treated as separate entities with the above-mentioned treatment.Dengue and malaria are the most prevalent arthropod-borne diseases with an estimated global incidence of 390 million and 214 million cases a year, respectively [Although usually their co-infection is masked as a mono infection; however, in 2005, Charrel et al. were successful in publishing the first dengue and malarial coherent infection . Once thA recent study showed that, in the last 15 years, the most number of malaria and dengue co-infection cases were reported from Pakistan. This is typical because the exponential rise in the cases is due to the rapidly changing environment, leading to an increased transmission period for both dengue and malaria. The most common species present in cases of malaria co-infections are P. falciparum and P. vivax, whereas, for dengue co-infections, dengue serotype 4 is the most common horrifying serotype .A case series study conducted in Jinnah Postgraduate Medical Centre, Karachi from September 2007 to January 2008 showed that the diagnosis of dual infections could be made on the basis of history and clinical examination with the help of hematological results. This study included a total of 114 patients, out of which, 23.21% had dual dengue and malaria co-infection (dengue IgM and MP positive), whereas 30.35% patients were negative for both the markers, but were strongly suspected for dengue hemorrhagic fever (DHF) and malaria on the basis of hematology and clinical results.Therefore, it is recommended that all patients suspected for dual infections should be treated collaterally for dengue and malaria in the endemic areas . AccordiA cross-sectional study conducted in Brazil showed the prevalence of malaria and dengue co-infection in a large sample taken. Out of the 1578 patients admitted to the hospital in a span of three years, 176 patients and 584 patients had P. vivax malaria and dengue fever mono-infection, respectively; while 2.8% (n = 44) patients were co-infected with both malaria and dengue. This study also explained the basis of diagnosing dengue virus, concluding the importance of diagnosing such infections precisely. Firstly, the IgM antibodies were detected; secondly, non-structural (NS1) protein was detected using Platelia Dengue NS1 antigen kit and finally, reverse transcriptase-polymerase chain reaction was used to identify the viral serotype .General practitioners (GPs) in Pakistan lack the necessary knowledge regarding prevalent concurrent infectious diseases and their treatments, and misdiagnose the cases as separate malaria or dengue attack. The severity of co-infections is much more than single infections as the patients have a higher risk of being anemic and thrombocytopenic . TherefoThe need for reporting this case is to highlight the presence of co-existing infections of malaria and dengue affecting the health of many people in endemic areas of Pakistan. Lack of knowledge regarding specific investigations and management may lead to catastrophic consequences. Thus, modern monitoring vector dynamics will be useful to determine the presence of such vectors and to formulate strategies with a huge emphasis on inhibition of vectors. This may lead to complete eradication in the future and consequently, reduce the transmission of infections. Prompt actions should be taken for educating the concerned masses about how to manage and treat these co-infections efficiently.


Plasmodium falciparum is considered an important target for disrupting malaria transmission. Indications are that various demographic groups, such as children and pregnant women may differ in risk of harbouring gametocytes, which may be crucial for targeted control. In this study, the relationship between the prevalence and multiplicity of P. falciparum, asexual parasite infections and gametocytaemia was assessed in three different demographic groups in an area of southern Ghana with low malaria endemicity. Levels of antibody responses to Pfs230 were also assessed as a proxy for the presence of gametocytes.The gametocyte stage of Pfs25-real time PCR. Multiclonal P. falciparum infections were estimated by msp2 genotyping and an indirect ELISA was used to measure plasma IgG antibodies to Pfs230 antigen.The study involved multiple cross-sectional sampling of children , male and non-pregnant female adults  and pregnant women  from Asutsuare in the Shai Osudoku District of Greater Accra Region in Ghana. Asexual parasitaemia was detected by microscopy and PCR, and gametocytaemia was assessed by P. falciparum having both Pfmsp2 3D7 and FC27 parasite types  compared with those infected with only 3D7 or FC27 parasite types. In agreement with the parasite prevalence data, anti-Pfs230 antibody levels were lower in gametocyte positive adults  compared to children.Overall, children and pregnant women had higher prevalence of submicroscopic gametocytes  compared to adults (17.4%). Multiplicity of infection observed amongst children (3.1) and pregnant women (3.9) were found to be significantly higher (P = 0.006) compared with adults (2.7). Risk of gametocyte carriage was higher in individuals infected with P. falciparum submicroscopic gametocyte reservoirs and represent important focus groups for control interventions. The number of clones increased in individuals carrying gametocytes compared to those who did not carry gametocytes. The higher anti-gametocyte antibody levels in children suggests recent exposure and may be a marker of gametocyte carriage.These findings suggest that children and pregnant women are particularly important as The online version of this article (10.1186/s12936-018-2479-y) contains supplementary material, which is available to authorized users. Recent reports on malaria have indicated a 41% decline in incidence rate in sub-Saharan Africa, and a 37% decline globally between 2000 and 2015 [Plasmodium falciparum gametocytes develop through five stages (I–V) with different morphological features compared to the asexual parasites. The early stage gametocytes (I–IV) usually sequester in the bone marrow, spleen and other organs to avoid immune clearance [learance –7. The mP. falciparum gametocytes may persist at submicroscopic levels even after treatment with artemisinin-based combination therapy (ACT) and may be effectively transmitted [With the exception of primaquine, most of the currently available anti-malarial drugs target the disease-causing asexual stage parasites and have very limited effect on the liver stage parasites and gametocytes. Studies have shown that nsmitted , 9. EffeChildren in malaria endemic areas have been shown to have generally higher parasite densities compared to adults –12, and Malaria infection during pregnancy has been demonstrated to result in asexual parasite sequestration in the placenta as an immune evasion strategy , 16. ParP. falciparum parasites. The presence of multiple asexual parasite clones may enhance the chance of some clones to evade the immune response, persist in the host and promote gametocyte development [P. falciparum sexual stage antigens have been identified and specific antibodies to these have been detected in host plasma [Pfs230, expressed on matured gametocytes, is one of the leading transmission reducing vaccine candidate antigens and natural anti-Pfs230 antibody responses have been demonstrated [Plasmodium infected individuals.Another important determinant of gametocyte carriage is infection with multiple clones of elopment , 18. Degelopment , 19. Sucelopment , 21. A nt plasma –22. Pfs2nstrated , 21. ThePfs230 antibodies as a marker of previous exposure to gametocytes in the three study groups. Since these factors most likely influence gametocyte carriage, which impacts on transmission, it is important to know which demographic group(s) serves as infectious reservoirs for malaria transmission in this low transmission area.This study used sensitive molecular tools to investigate the association between asexual parasites and gametocyte prevalence and multiclonal infections among children, adults and pregnant women living in a low malaria endemic area. The study further investigated the levels of anti-The study was conducted in Asutsuare in the Shai Osudoku District of the Greater Accra Region of Ghana. The study area and its surrounding villages including Mafi Korpe, Avakpo, and Volivo, described in details elsewhere (2), are about 120 km north-east of the national capital, Accra , May 2014  and September 2014  in which finger prick blood (300 µl) for detection of asymptomatic parasitaemia was taken during follow-up visits. Clinical malaria was defined as microscopy slide positive for any parasitaemia plus fever (axillary temperature > 37.5 °C), together with at least one other symptom such as malaise, vomiting or diarrhoea. Spotted filter papers were stored at − 20 °C in zip lock bags with silica gel desiccant for molecular analysis. For gametocyte detection, 50 μl whole blood samples were preserved in 250 μl RNAlater® solution  and stored at − 20 °C until use. Weekly active surveillance was conducted by home visit, with a standardized questionnaire to capture clinical information for all participants. Participants who became ill during follow-up periods were referred to the community health centre for treatment per the Ghana Health Service guidelines. In between visits, participants were advised to go on their own to the health centre when they felt ill.At enrolment, 5 ml of EDTA anti-coagulated venous blood was collected from each participant at baseline (November 2013). A small aliquot (50 µl) was used to prepare thick and thin blood films for microscopy. Plasma was separated from the rest of the whole blood by centrifugation at 450×Blood haemoglobin levels were measured using the Hemocue-Hb 201 . The ABO blood type was determined using a commercial anti-sera blood grouping kit . All participants were screened for their sickle cell status using the sodium metabisulphite method.Thick and thin blood smears were prepared and stained with 10% Giemsa for detection of both asexual stage and sexual stage parasites by microscopy under oil immersion (100× magnification). Parasite densities were assessed by counting 200 leucocytes, and converted to number of parasites per μl of blood by assuming a standard leucocyte count of 8000/μl. A slide was considered negative if no parasites were seen after examination of at least 200 fields.Plasmodium species were determined by nested polymerase chain reaction (PCR) methods as previously described [Plasmodium genus were detected using the outer genus-specific primers (rPLU 5 & 6) targeting sequences of their small subunit ribosomal (ssrRNA) genes. The initial outer reaction contained 4 mM of MgCl2, 200 µM DNTPs, 0.0625 μM of each primer and one unit of Taq DNA polymerase . The PCR cycling conditions for the primary reaction were an initial denaturation at 94 °C for 15 min, denaturation for 1 min at 94 °C and annealing at 58 °C for 2 min . Extension was at 72 °C for 2 min, a final annealing at 58 °C for 2 min and final extension at 72 °C for 10 min. The inner PCR reaction was used for detection of all Plasmodium species as described in [DNA was extracted from blood samples blotted on filter paper by the Chelex method as previously described . Plasmodescribed , with mo® plus mini kit  according to the manufacturer’s protocol for selected RNA-preserved samples that were asexual parasite positive by microscopy and/or PCR. Gametocyte detection was done by Pfs25 real time quantitative PCR [Pfs25 mRNA is transcribed only by female gametocytes, gametocyte sex ratios are female biased and the marker has therefore been used as a sensitive marker for circulating matured gametocytes [Pfs25 transcripts was performed with the primer sequences pfs25_fw 5′-GAA ATC CCG TTT CAT ACG CTT G-3′, pfs25_rev 5′-AGT TTT AAC AGG ATT GCT TGT ATC TAA.-3′ and Pfs25_probe HEX-TGT AAG AAT GTA ACT TGT GGT AAC GGT-BHQ1. The RT-PCR reaction mixture contained an enzyme mixed reagent, a master mix solution, Pfs25-specific forward and reverse primers at concentrations 800 nM, Pfs25_probe at 200 nM, and 2 µl of RNA template. The RNA template for the reaction included 3D7 gametocyte-positive standard RNA generated from culture with concentrations starting from 10,000 to 0.005 gametocytes/μl and a parasite-negative sample, as well as a no template control. The reaction plate was loaded into an ABI 7300 real-time PCR instrument . The cut-off threshold (CT) for positivity was set to ≤ 37.0 CT value by using a purified gametocyte standard from a 3D7 parasite culture.RNA extraction was done using RNeasytive PCR . Even thetocytes , 28. BriP. falciparum positive samples were selected for the detection of multiple parasite clones by nested PCR using msp2 family-specific primers [P. falciparum laboratory strains were used as positive controls and a negative control consisting of only working reagents was used. The amplified products were run on a 2% ethidium bromide-stained agarose gel and visualized using an ultraviolet transilluminator. Multiplicity of infection was calculated by dividing the number of msp2 bands obtained from the gel by the total number of positive samples run. Samples with single bands were considered as monoclonal infections while samples with multiple bands were termed multiclonal infections.The  primers . BrieflyPfs230D1M (GenBankTM accession number XP_001349600.1) was characterized from Pfs230 domain 1 (Pfs230D1H) and non-structured domain 1–2 (Pfs230D1-2). The recombinant protein was refolded by removing the His6 fusion tag and expressed in Pichia pastoris using good manufacturing practice conditions and purified by RP-HPLC as previously described [Recombinant escribed .Pfs230-specific antibody levels were measured by indirect ELISA. Briefly, 96-well microtitre  plates were coated with 1 µg/ml of recombinant Pfs230D1M in carbonate buffer (pH = 9.2) and incubated overnight at 4 °C. Plates were washed 4 times with washing buffer (PBS with 0.05% Tween-20) and blocked in 3% skimmed milk powder in PBS, 0.05% Tween-20 for 1 h. Plasma samples were diluted 1:300 in dilution buffer (1% non-fat dry milk in PBS with 0.5% Tween 20) and incubated for 2 h at room temperature. A positive control of pooled hyper immune plasma was titrated as a standard and plasma from malaria-naïve US volunteers as negative controls were included on each plate as internal controls. Bound antibodies were detected with 1:3000 diluted peroxidase conjugated goat anti-human IgG (H+L)  in dilution buffer and incubated for 1 h at room temperature. After washing 4 times, plates were developed with tetramethylbenzidine substrate  for 15 min in the dark and the reaction stopped with 0.2 N H2SO4. Absorbance was read at 450 nM on an ELISA plate reader .msp2-3D7 and msp2-FC27 clones was assessed using quasi-Poisson regression. For the study period, samples that were P. falciparum positive at least one-time point throughout the entire study period were included in data analysis. The ELISA OD values were transformed into arbitrary antibody units (AU) using the Auditable Data Analysis and Management System for ELISA . Pairwise comparison amongst median antibody levels across groups and within groups at various time points was done using Bonferroni comparison test following Kruskal–Wallis analysis. The mixture model [Study data were entered into a Microsoft Access database and exported into Microsoft Excel for statistical analysis. The difference in proportions of covariates including age, sickle cell, bed net use, haemoglobin levels and blood group among the groups were compared using Pearson’s Chi squared test and ANOVA. The association between asexual parasitaemia and/or gametocytes and multiple infections were tested independently using multiple logistic-regression analyses. Gametocyte prevalence in relation to the proportion of re model , 31 was A total of 463 samples from the three study groups were analysed at baseline, consisting of 184 children, 154 non-pregnant adults and 125 pregnant women ; Plasmodium malariae and Plasmodium ovale species were less than 5% and were detected either as single infections or co-infections with P. falciparum. The prevalence of asexual P. falciparum parasitaemia among the adults at baseline (November 2013) was generally lower (6.4%) compared to that in children (8.0%) and pregnant women (16.4%), . Based on the RT-PCR data, children had high median gametocyte densities compared to pregnant women and adults in the study population  by 5/38 by PP. falciparum positive samples (87.1%) had multiple alleles (≥ 2 PCR bands) and few infections were single alleles (12.9%).During the entire study period, asexual parasite-positive samples detected by PCR were used for the detection of multiplicity of infection (MOI). Most of the From the study, overall MOI amongst children and pregnant women were found to be higher compared with adults  individuals separately. Age was significantly associated with increased anti-Pfs230 antibody levels in both children  and the older individuals .Antibody levels for each group varied at different time points, however that of adults remained relatively stable at the different follow-up periods except for follow-up 3 in September 2014 . However, the seroprevalence of antibodies were relatively lower in children (3%) and in pregnant women (6.6%)  Fig. a. DuringPfs230 was not significant. When Pfs230 antibody responses were compared between gametocyte negative and positive individuals, the levels were significantly different in the adults  and pregnant women  but not in the children , it malaria , 33.The current study investigated the association of asexual parasite density and multiple parasite clone infections with gametocyte carriage in three different demographic groups in a low malaria transmission area. Using PCR, a relatively high prevalence of asymptomatic infections and gametocytaemia were detected among children and pregnant women compared to adults. The immune systems of children and pregnant women have been shown to be less robust compared to that of non-pregnant adults , 34 and The overall MOI observed in this study was high (3.1) for such low transmission area. This finding was strange and unexplainable, since studies have shown higher MOI in high transmission areas due to more parasite diversity and associated with transmission intensity, compared to low transmission areas with limited parasite diversity , 39. ThiThere was a high proportion of multiclonal infections among the children and pregnant women groups in this study compared to adults. Pairwise comparison revealed that pregnant women had higher mean number of clones than children and adults. This is an indication that they carry a greater diversity of parasites, possibly reflecting infection with both placenta-specific and non-specific parasites, especially in primigravids –43. InteP. falciparum parasite variants may influence gametocyte carriage differently, as was observed in this study where individuals carrying both 3D7 and FC27 allelic types of parasite had a higher risk of harbouring gametocytes compared to single 3D7 or FC27 allelic types. This findings may suggest the possibility of limited immune responses against these parasite variants due to antigen-specific immunosuppression or immune tolerance, hence their ability to persist for longer periods and commit to gametocyte formation [The 3D7 allelic types predominated in all the three study groups as compared to the FC27 allelic types, indicating its dominance in this geographical area. A number of studies conducted in Ghana and Tanzania have identified the 3D7 allelic parasite type to be dominant among the parasite populations , 45, 46.ormation . Howeverormation . This unPfs230 antigen among different study groups and found the adult study group with the highest anti-Pfs230 seroprevalence as compared to children and pregnant women at baseline. Difference between adults and children may probably reflect repeated exposure in adults, as has been observed in other studies [Pfs230 seroprevalence observed in the adults group between February and May could be due to reduced levels of circulating submicroscopic gametocytes over the period. Interestingly, antibody responses to Pfs230 were significantly lower in adults and pregnant women who had gametocytes compared to children, meaning the responses in children might have been boosted by recent exposure to Pfs230 antigens since children were found to harbour more gametocytes. For the adults, the lower gametocyte densities may indicate some form of anti-gametocyte immunity as proposed by other studies [Pfs230 levels among the groups, suggesting the length of gametocyte exposure among the groups. This age-dependent association of sexual stage antibody levels have been observed in some studies [This study also investigated the seroprevalence of antibody responses to  studies , 49, 50. studies , 51, 52. studies , 53. For studies , 54. How studies . In this studies , 53, 56,Pfs230 in children was associated with gametocyte carriage, an indication of it being a marker for current gametocyte exposure.Multiplicity of infection was higher in gametocyte carriers than non-gametocyte carriers, suggesting an association between simultaneous presence of multiple parasite clones and gametocyte carriage. This needs to be further evaluated in other transmission areas since it may impact on transmission. Antibody responses to Additional file 1. Asexual parasite prevalence data among the groups at various time points by Microscopy and PCR. Baseline, November 2013; F1-3 are follow-ups 1-3  respectively.


In Nigeria, recent reports suggest that dengue viruses could be a major cause of acute fevers. We sought to make a cross-sectional estimate of the prevalence of current and previous dengue infections in patients presenting with fever to healthcare centres in Cross River State Nigeria.Onsite Duo dengue Ag-IgG/IgM lateral flow immunoassay cassettes was done. Samples which tested positive were further confirmed using the RecombiLISA dengue IgM and IgG enzyme linked immunosorbent assay kits and classified into primary and secondary dengue infection. Malaria testing was carried out using microscopy. Between 4 January 2017 and 24 August 2017 a total of 420 participants were sampled across 11 health centres. The mean age was 34 (range = 1–99), 63% were female, 49% reported sleeping under a treated mosquito net in the past week and 44% reported taking an antimalarial prior to seeking care. The mean number of days fever was present prior to seeking care was 8, and many of the participants presented with symptoms indicative of respiratory or urinary tract infections. Testing indicated that 6%  had either a primary or secondary dengue infection with or without co-existing malaria, while 4%  had either a primary or secondary dengue infection without co-existing malaria. 52%  had a malaria infection with or without any dengue infection, and 50%  had a malaria infection without any dengue infection.This cross-sectional health facility survey recruited persons with temperature ≥38°C. Dengue virus immunoglobulin M (IgM)/immunoglobulin G (IgG) antibody testing using Our study confirms the presence of dengue at not insignificant levels in patients attending health centres with fever in this south eastern province of Nigeria. These data highlight the danger of the common presumption in this setting that fever is due to malaria. Surveillance for dengue is vital in this setting. Flaviviridae family [Aedes aegypti and Ae. albopictus) are known to be well established [Dengue is the most important arboviral infection of humans caused by four dengue virus serotypes, namely dengue virus 1,2,3, and 4 (DENV 1–4), which belong to the e family ,6. Howeve family ,8. In Nie family , althougablished –13. HoweIt is clearly important that the burden of dengue be accurately defined, ideally across relevant spatial scales for public health planning. This is particularly important given that arboviral surveillance programmes are not yet well established in Nigeria. We therefore sought to make a cross-sectional estimate of the prevalence of current and previous DENV infections in patients presenting with fever to health-care centres across Cross River State, Nigeria, and to understand the risk factors associated with DENV infection.2. The vectors of dengue–Aedes aegypti and Aedes albopictus–have been identified in parts of Cross River from a previous entomological survey [In Cross River State there are 576 primary health-care centres (PHCs) and 157 secondary health-care centres, distributed across eighteen local government areas (LGAs), covering a population of approximately 3,866,300 . These PThis cross-sectional facility survey was conducted between January and August 2017 in health facilities (mostly PHCs) across Cross River State. It was not possible to obtain a probability sample because of the need to recruit patients presenting at facilities with fever. We therefore used random sampling methods to first select a probability sample of health facilities, but then had to take a non-probability sample of patients from those health centres. Specifically, in the first stage of sampling 10 LGAs were selected with probability proportional to the number of health centres across the three districts. In the second stage of sampling 1 health centre per LGA was randomly selected. Field teams then visited the health centre, initially for 2–3 days, and consecutively recruited patients into the study with the aim of reaching the sample size per facility in all facilities. However, in a number of facilities recruitment was much slower than anticipated. Therefore, in those facilities where recruitment was faster more patients were recruited than the necessary number per facility to reach the overall sample size, and in facilities where recruitment was very low another health centre from the same LGA was randomly selected and patients sampled there as well.Based on our resources we estimated that we could recruit a total of 396 participants , which we calculated would allow us to estimate the prevalence of DENV infections with a precision  of at most ± 7.8%. This made the conservative assumption that we would be estimating a prevalence of 50%, and that there would be a design effect of 2.5, which was a conservative inflation of the design effect found in the Nigeria 2015 Malaria Indicator Survey for the estimated prevalence of malaria in children (aged 6–59 months) based on rapid diagnosis testing .The study population was consenting febrile patients presenting to sampled health centres who met the following eligibility criteria: age ≥1 year and fever (axillary temperature of ≥ 37.5°C) for< 10 days.Following strict aseptic technique and using sterile needles and 10 ml syringes, 6ml of blood was withdrawn by venepuncture. Five mls of blood was delivered into a plain serum tube while 1 ml was put in an EDTA bottle, both labelled with patient hospital and study numbers. Thick blood smears were made on two grease-free slides and stained with 10% Giemsa for 10 minutes. The films were viewed using a compound light transmission microscope at a magnification of X1000. In this study, positive slides were determined by the presence of merozoites or ring forms of the malaria parasites. No schizonts or gametocytes were seen in this study. Two hundred fields were viewed before a film was declared negative.Onsite Duo Dengue Ag-IgG/IgM lateral flow immunoassay cassettes , according to the manufacturer’s instructions. Samples which tested positive were further confirmed using the RecombiLISA Dengue IgM and IgG 96-well enzyme linked immunosorbent assay (ELISA) kits , according to the manufacturer’s instructions. Detection of IgM alone or an IgM:IgG optical density (OD) ratio ≥1.2 was designated as primary dengue, while the detection of IgG alone or an IgM:IgG OD ratio <1.2 was designated as secondary dengue [Blood samples were transported in a cold chain to the University of Calabar Teaching Hospital (UCTH) for serum separation and storage. Serum was obtained from the plain bottle sample by centrifugation at 5000 rpm for 10 minutes, and was used for the rapid detection of DENV immunoglobulin M (IgM)/immunoglobulin G (IgG) antibody using y dengue –19.Data was collected from eligible and consenting participants or their caregivers using a proforma that captured the individual-level variables of interest including age, sex, occupation and use of mosquito preventive measures , home roof type, household location , household water storage and travel history. The information obtained was then entered into a data management system, including restrictions over the type and range of values allowed to reduce data entry errors. Quality control checks were performed on 10% of entered records to reduce the occurrence of entry errors. Also, 5% of the laboratory tests were repeated by an independent pathologist to corroborate the laboratory results obtained.The primary outcome was the prevalence of a primary or secondary dengue infection among participants with or without malaria. The secondary outcomes were the prevalence of1) primary or secondary dengue without malaria, 2) primary dengue with malaria, 3) primary dengue without malaria, 4) secondary dengue with malaria, 5) secondary dengue without malaria, 6) malaria with/without primary/secondary dengue, 7) malaria with primary/secondary dengue, and malaria without primary/secondary dengue.survey package [survey package svyciprop function’s logit method, which “fits a logistic regression model and computes a Wald-type interval on the log-odds scale, which is then transformed to the probability scale”. Multivariate logistic regression was also used to evaluate the direction, magnitude and statistical significance of associations between a range of risk/protective factors and dengue (primary or secondary) positivity , via adjusted odds ratios and their 95% confidence intervals, with two-sided hypothesis testing conducted at the 5% level. All independent variables were chosen before constructing the model based on evidence from the literature. Observations with missing outcome and/or covariate data were omitted from the model. Given the small size of the dataset this analysis should be treated as strictly exploratory.Data were analysed using R’s  package –22 commaEthical clearance was duly obtained from the Health Research and Ethics Committee of the UCTH with number UCTH/HREC/33/324. Written informed consent was obtained from either the participants or their caregivers (in the case of minors) prior to the recruitment and sample collection. Participation was voluntary and the cost of the tests were not be borne by the participants.A total of 11 health centres were sampled, with an additional health centre selected in one LGA due to a very low recruitment rate. Dates of patient recruitment varied across health centres. Across all health centres the earliest date of patient recruitment was 4 Jan 2017 and the latest was 24 Aug 2017. Across all health centres the range in the number of days between first and final patient recruitment dates was 0–232 (median = 97).There was also a large amount of variation in the number of patients recruited per facility, with a median sample size of 34 and a range of 2-96.There were a total of 420 participants with a mean age of 35 and 63% being female.Twenty-s6%  of participants had antibody test results consistent with either a primary (first infection) or secondary dengue infection with or without malaria, and 4%  were positive for primary/secondary dengue alone . Fifty percent  of participants who had a positive malaria test were negative for both primary and secondary dengue, and 52%  of the participants were positive for both malaria and primary/secondary dengue .The logistic regression model showed that age, gender, educational level, place of residence, presence of waste around house and the use of any household malaria protection were not clearly associated with having either primary and/or secondary dengue . HousehoWe found that 6% of patients tested positive for primary or secondary DENV infections either with or without a malaria co-infection. In these undifferentiated fever cases 50% tested positive for malaria but not dengue, while just 2% had co-existing primary dengue and malaria infections .Symptoms indicative of urinary tract and respiratory infections were commonplace among this cohort and may account for some proportion of the fevers experienced. Dengue seroprevalence from a survey done in Maiduguri  NorthernNigeria is a West African country in which dengue is reported to be endemic . HoweverDengue IgM levels begin to rise by the third day of a primary infection and peak at 2 weeks after the onset of fever and may remain detectable for up to 6 months or longer following disease resolution. IgG is detectable at the end of the first week of illness and can persist for life. ELISA tests can assay for IgM and IgG levels and the IgM: IgG ratio is useful in distinguishing primary from secondary dengue virus infections. An IgM: IgG ratio of ≥ 1.2 is indicative of primary dengue infection while an IgM: IgG ratios of < 1.2 is indicative of secondary dengue infection .The diagnosis of dengue is further complicated by malaria co-infection as demonstrated in our study. A recent survey done in the inland western Nigerian city of Ibadan put the number of persons with active dengue infection among confirmed malaria cases at 10%. Among this cohort of malaria cases, all of them were found to be positive for dengue IgG antibodies suggestive of a past dengue infection and consistent with endemicity of dengue virus in this area . All theAedes-borne diseases such as dengue, Zika, yellow fever and chikungunya [The use of mosquito nets has been recognised to be the most effective strategy for malaria control , but thekungunya . Intereskungunya . Therefokungunya . Up to 7kungunya . This iskungunya ,33. HoweRapid population growth, unplanned urbanization, increased international travel, agricultural development, possible global climate changes are some of the factors that have been put forward to explain the extensive transmission of dengue in these areas . Other eClearly there is need it for algorithmic guidelines, including universal malaria and selective dengue testing, for the diagnosis and management of patients with fever presenting to primary and secondary care in Nigeria. This pragmatic framework for triage and testing of undifferentiated cases of fever in resource limited settings is critical in determining the clinical outcomes of patients with dengue. These guidelines will need to reflect the fact that about half of these undifferentiated cases are neither due to malaria nor dengue, but may rather be related to respiratory or urinary infections. Such guidelines will need to include methods of identifying dengue related complications to promote optimum management and referral of relevant cases.Our study confirms the presence of DENV infections in patients attending PHCs in this south eastern province of Nigeria. Surveillance for dengue is vital in this setting for identifying outbreaks and initiating an early response.This study has several limitations. First, our survey did not use a probability sampling method, given that although we took a probability sample of health facilities we had to take a non-probability sample of patients from within those facilities. This therefore prevented the calculation of weights and limits the robustness and generalisability of the estimates, given that inferential statistics formally assume the data come from a random probability sample. Second, this is a facility-based survey, and so we cannot generalize our results to the community or wider population outside of those attending health facilities for fever, which would require a population survey. Third, dengue shows seasonal trends, and the data were collected over a large proportion of the year and importantly with uneven effort across that time period. Hence, the overall percent prevalence estimates do not represent the prevalence from any single part of the year, nor do they represent an evenly sampled average across the whole year. The results should therefore be treated cautiously as likely indicative of the broad level of dengue infections in patients attending PHCs with fever. Fourth, we were unable to definitively determine the proportion of patients with other febrile conditions.S1 Table(DOC)Click here for additional data file.S2 Table(SAV)Click here for additional data file.