This study aims to evaluate the CD4+/CD8+ T cells counts were enumerated by automated hematology analyzer and flow cytometry, respectively.This case-control study involved blood samples collected from 165 children (between 5 and 12 years). This comprised 45 children with mild malaria, 40 each with moderate, severe malaria and apparently healthy (control). Serum cytokines, ferritin, malonaldehyde (MDA), ascorbate, α-tocopherol levels were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). Leucocytes differentials and CD4Plasmodium falciparum. The male to female ratio of children with mild malaria was 1.5:1 (mean ± SD age of 8.5 ± 1.9 years). However, other groups had 1:1 male to female ratio and mean ages of 9.2 ± 2.3, 9.8 ± 2.2, 8.5 ± 1.5 for children with moderate, severe malaria and control, respectively. There was a positive but not significant association of neutrophils and monocytes with the 3 grades of malaria parasitemia (p>0.05). There was a negative and significant correlation between severe malaria and lymphocyte count . However, there was positive and significant correlation between eosinophil with moderate  and severe malaria . There was a significant decline in serum ascorbate with increased malaria density (p<0.0001). However, there was no difference in the serum α-tocopherol concentration within the 4 groups of children (p = 0.182). Serum ferritin and MDA significantly elevated with an increase in malaria density (p<0.0001). There was a significant decline in CD4+ T and CD8+ T cells counts with an increase in malaria densities (p<0.0001). Serum IL-10 and IL-6 significantly elevated with increased malaria density (p<0.0001).All malarial children had only + and CD8+ T cell counts, upregulation of IL-6, and high serum levels of oxidative stress biomarkers.Based on these findings, severe malaria was significantly associated with declined CD4 Plasmodium falciparum  accounted for most morbidities and consequently, highest mortality rates in Africa and other high-risk regions of the world [falciparum requires quick recognition, proper treatment and effective patient management [The World Health Organization (WHO) affirms that malaria constitutes a global health threat with 228 million cases and 405,000 deaths in 2018 . It was he world . Therefonagement .Malarial infection can potentially lead to death if not treated promptly due to its progression into severe malaria anemia (SMA) . In SMA,Cellular and antibody-mediated immunity, innate, and adaptive cells play a crucial role in the immune responses to the stages of the malaria parasite life cycle and the development of immunopathogenesis of malaria ,10. ImmuP. falciparum interferes with maturation and proliferation of antigen-presenting cells (APCs) in addition to the impairment of dendritic cell activity, thereby resulting in the proliferation of “tolerant” T-cell phenotype [In malarial infection, innate and acquired immune responses are stage-specific and complex; the preerythrocytic (skin and liver) responses against sporozoites is distinct, while the erythrocytic responses against the merozoites are clearly different ,10. ThroOxidative stress in malaria can be attributed to two main mechanisms, viz; increased number of parasites in the erythrocyte as a result of high metabolic rate, and the host’s potential immune response to the infection . There i2O2) through the activity of SOD and catalase, capable of reacting with several molecules to form free radicals, there is a need to remove these radicals using substances with anti-oxidant properties such as the ascorbic acid. In inflammation, leukocytes, alpha-1-acid glycoproteins, IL-6, and Vitamin C have similar concentrations [While some studies suggested that oxidative stress during malaria plays a protective role, others opposed that it plays a deleterious role by worsening the pathological outcome of the infection . In malatrations . Vitamintrations . Therefotrations . Aside f+ T cell counts and induction of anemia through CD8+ T cell-dependent plasmodial clearance and red cells removal in the Spleen [In children, previous studies have reported varying findings on the roles of pro- and anti-inflammatory cytokines in the pathogenesis of malaria and disease outcomes –17. Howee Spleen ,17. Henc+ and CD8+ T cells, leucocytes subpopulations, IL-6, IL-10 and biomarkers of oxidative stress among children infected with varying grades of malaria attending the University of Abuja Teaching Hospital and National Hospital, Abuja, Nigeria.Given that a very larger proportion of the Nigerian population are at high risk of contracting malaria and children are most susceptible to malaria severity, this study would be relevant and generate interest in this setting, region and Sub-Saharan Africa. Specifically, there is a paucity of data that demonstrates the interplay of malaria densities with cellular immunity, cytokines and oxidative stress among Nigeria children. Hence, this work was instigated with the aim to determine the CD4th June 2019 at the University of Abuja Teaching Hospital (UATH), Gwagwalada and National Hospital Abuja (NHA), Nigeria. The UATH and NHA have 500- and 850-bed capacities, respectively. The UATH is located at 8.9531° N, 7.0614° E whileNHAis located at 9.0388° N, 7.4631° E. These are the two prominent tertiary hospitals situated at Nigeria’s Federal Capital Territory. The climate of Abuja is quite reminiscent of other tropical areas with 2 distinct seasons - the wet and dry season. The temperature ranges from 30 °C to 37 °C during the day and 18 °C–25 °C at night, with the highest temperature usually experienced in the month of March. The mean total rainfall is approximately 1650 mm/annum with more than 60% of this rain falling between July to September [This study was conducted during a 4-month period - 28th February to 30This was a hospital-based case-control study.et al [The minimum sample size for this study was determined from a previous case-control study by Efunshile et al  which reParticipants were enrolled using stratified randomization. Stratified randomization strata were constructed based on values of malaria density and a randomization scheme was performed separately within each of the 4 strata to avoid imbalance in participants’ selection. They comprised of one hundred and sixty-five children within 5–12 years categorized into four groups, viz;Forty-five children with laboratory-confirmed with mild Plasmodium falciparum malaria.Forty children with laboratory-confirmed with moderate Plasmodium falciparum malaria.Forty children with laboratory-confirmed with severe Plasmodium falciparum malaria.Forty children without malaria history for the past 1 year. This was based on routine hospital check-up records, as well as information from parents attesting that children were neither sick nor on any treatment within the past 12 months. These children were entirely from the affluent who reside in the mainstream city of the federal capital (Abuja) of Nigeria.All participants were identified and selected by nurses, pediatricians, and family physicians of these hospitals.Uncomplicated with mild fever.Malaria density count <10,000 parasites/μL.No anemia.No abnormal blood chemistry.No urinary proteinemia.High body temperature (39 °C) for ≥3 days.Mild anemia (9–11 mg/dL).No abnormal blood chemistry.No urinary proteinemia.Malaria density count between 10,000 parasites/μL and 100,000 parasites/μL.Proteinuria (+).Severe anemia (8 mg/dL).Hyperbilirubinemia (Bilirubin >3 mg/dL).High serum potassium (5.5 mmol/L).Malaria density count of >100,000 parasites/μL.Enrolment was based on participants who had no prior history of any immunodeficiency infections or diseases such as HIV/AIDS, diabetes mellitus, tuberculosis, typhoid, HCV and HBV. All participants were not on anti-malarial chemotherapy prior to sampling.Ethical approval for this study as mandated was obtained from 2 sources; the Human Ethical Research Committees of UATH  and NHA , while written consent was provided to individual parents/guardians of children. Participants’ confidentiality and privacy were entirely protected, no third party had access to participants’ data. All data were analyzed anonymously and saved in a personalized google cloud account. After analysis, all data were permanently deleted from the internet and computers.Age and sex of children were collected from their parents/guardians through a structured questionnaire. Whereas body mass indices (i.e. weight/squared height) were assessed by nurses. Data cleaning was done to identify and correct errors during collection to minimize their effects on the results. We screened and edited abnormal or faulty data such as outliers and were corrected by logarithmic transformation.+ T cell counts and complete blood counts and differentials. The remaining was dispensed into plain container (with no anticoagulant). Of which, serum was harvested by centrifugation at 10000 g for 10 min. Thereafter, it was used for cytokine and oxidation biomarkers measurement. Samples were analyzed within 1 h of collection.Five milliliters (5 mL) blood samples were collected from all children. Out of this, 2 mL was dispensed into an Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulant container which was used for CD4Malaria parasites were detected and identified microscopically using Giemsa stained thick and thin blood films, respectively, as described by Cheesbrough .Thick films were used to detect malaria parasites in the blood samples. Three drops of blood were placed in the center of a clean grease-free microscope slide to cover an area of 15 mm in diameter. The smears were allowed to air-dry and then flooded with 10% Giemsa stain which was retained for 10 min before the stains were washed off with water. Parasite species and morphology were determined by microscopic examination of thin films which were also prepared with the 10% Giemsa stain. Microscopic examinations of the stained slides were done using an oil immersion objective lens (×100 magnification).The parasite density in all samples was determined as previously described by Cheesbrough . CertifiParasite densities (parasite/μL of whole blood) were then calculated as follows:The number of malaria counted/WBC counted × WBC count μL of the participant.9/L of blood; all set by WHO to be used conveniently in facilities that lack the tools to determine patients’ absolute full blood count value [Furthermore, parasite densities for all participants were calculated using assumed WBC counts of 8.0 × 10nt value .The number of parasites seen per field were counted and the density scored as <10,000 parasites/μL (mild); between 10,000 parasites/μL and 100,000 parasites/μL of whole blood (moderate) and >100,000 parasites/μL (severe) .Serum IL-6 and IL-10 concentrations were measured by sandwich ELISA based on the protocol described by kit manufacturer . Accordingly, IL-10  and IL-6  were investigated.+ and CD8+ T cell lymphocytes, a dual-platform method  was utilized. A three-color flow cytometric analysis panel was done using a BD FACS™ brand flow cytometer . Before data acquisition, instrument parameters were checked and optimized using CaliBRITE beads (Becton Dickinson).To generate absolute counts of CD4Serum Ascorbic acid , Malondialdehyde  and α-Tocopherol  levels were estimated using competitive ELISA kits. While serum ferritin levels were measured using a sandwich ELISA kit . These analytes were assayed based on kits manufacturer’s instructions.Full blood counts and leucocyte differentials were analyzed using Mindray BC-5000  automated hematology analyzer following the manufacturer’s operational guidelines. All samples were analyzed within 30 min of collection. Daily quality control for all laboratory procedures such as flow cytometry and ELISA including blood smear microscopy staining and automated hematology analyses daily quality control ensured.P values < 0.05 were considered statistically significant at a confidence interval of 95%.All data were inputted into Microsoft Excel then exported to Medcalc software V.19.0.7 to analyze continuous variables as mean ± SD. We employed One-way ANOVA to define the difference between mean ± SD across study groups. Pearson’s correlation test was used to determine the association between leucocytes differentials and the various malaria densities. Dunnett’s Multiple Posttest was used to determine where differences existed. 2, then 18.8 ± 3.3 kg/mm2, 19.9 ± 2.1 kg/mm2, and 19.3 ± 4.3 kg/mm2 for those with moderate, severe and the controls, respectively  .+ T cell count of children without malaria, with mild, moderate and severe malaria was 710 ± 111 cells/mm3, 628 ± 105 cells/mm3, 592 ± 82 cells/mm3 and 390 ± 50 cells/mm3, respectively. The mean CD8+ T cell count of children without malaria, with mild, moderate and severe malaria was 428 ± 42, 325 ± 39, 282 ± 25 and 113 ± 18, respectively. Using one-way ANOVA and Dunnett’s Multiple Posthoc to determine difference between control and the three groups, there was significant decline in CD4+ T and CD8+ T cells count and within increase in severity of malaria densities (p<0.0001) (<0.0001) .The mean serum IL-10 levels of children without malaria, with mild, moderate and severe malaria was 321 ± 52 pg/mL, 815 ± 105 pg/mL, 842 ± 121 pg/mL and 935 ± 115 pg/mL, respectively. The mean serum IL-6 levels of children without malaria, with mild, moderate and severe malaria was 30.3 ± 8.7 pg/mL, 83.7 ± 13.5 pg/mL, 98.5 ± 15 pg/mL and 101 ± 18.4 pg/mL, respectively. After using one-way ANOVA and Dunnett’s Multiple Posthoc to determine difference between control and the three groups, serum IL-10 and IL-6 concentrations significantly elevated with increase in malaria density (p<0.0001) .+ T cell counts and induction of anemia through CD8+ T cell-dependent plasmodial clearance which invariably leads to rise in oxidative stress. To the best of our knowledge, this is the first comprehensive study on the T cell response, leucocyte profiles and oxidative stress level of children infected with malaria in Northern Nigeria.Varying reports have elucidated the roles of pro-and anti-inflammatory cytokines in malaria and disease outcomes. Essentially, severe malaria has repeatedly been reported to cause decline in CD4Our findings revealed no significant difference in Body Mass Index (BMI) in all categories (p = 0.513), which concurred, with a research carried out among Ghanaian children and no statistically significant variation was observed in BMI (p = 0.300) . This maet al reported no specific diagnostic indications given by the white blood cell count during malarial parasitemia [There was a positive but non-significant correlation between total White Blood Cells (WBC) and those with mild and no malaria. This is in agreement with a study carried out among Ghanaian children and found no statistical significance between those with low malaria parasitemia and those without parasitemia . Likewisasitemia . In anotasitemia . This miet al [There was positive but no significant association between neutrophils and monocytes in all grades of malaria. This does not agree with the report of significant monocytosis (p<0.05) observed in parasitemic patients in Uganda  and Beniet al .et al [There was a negative and significant correlation between children with severe malaria and lymphocyte count. In agreement with this finding was the significantly lower lymphocyte count reported in malarial patients in the study of Muwonge et al . In thiset al . Howeveret al .There was a significant decline in serum ascorbate with an increased in the severity of malaria densities. It is in agreement with prior study in Indian children, where a decreased serum ascorbate level was observed with an increase in malaria parasitaemia when compared with the mean ascorbate level of the healthy control group . Howeveret al on serum MDA levels in malarial children corroborated with ours. They reported a significantly higher serum MDA level in malaria parasitized children [et al [In this study, serum ferritin and Malondialdehyde (MDA) concentrations were significantly elevated with an increase in malaria density. In contrast, with the prior study in Edo state, South-south region, Nigeria, where no significant association was found between serum ferritin level and the presence of malaria parasite . This machildren . In the n [et al .+ T and CD8+ T cells count as the severity of malaria parasitemia increases (p < 0.0001). This aligns with various studies on T cell response to malaria in children, where majority reported declined CD4+ and CD8+ T cell counts in malaria-infected children [+ T cells are associated with severe malaria parasitemia.Findings from our study revealed a significant decline in CD4children –35. In achildren . This deOur study revealed serum IL-10 and IL-6 concentrations significantly elevated with increased in malaria density (p<0.0001). These agree with the study in Ugandan children, which reported significantly higher IL-6 and IL-10 only during acute malarial . SimilarPlasmodium falciparum, and very high levels of these cytokines have been implicated in the pathogenesis of severe malaria. These findings are consistent with the concept that high levels of proinflammatory cytokines, despite high levels of the anti-inflammatory cytokine IL-10, could contribute to the pathogenesis of severe malaria in children [Proinflammatory cytokines are involved in clearance of children –46.+ T cells especially, the memory T cells. This is one of the complications encountered when mounting a successful vaccination against malaria. Malaria is also known to expand CD8+ T cells and Treg cells and is implicated in severe malaria such as cerebral malaria because; T cell senescence and depletion markers are associated with CD8+ T cells. These together with the significant changes in the cytokine levels (IL-6 and IL-10) point to the debilitating effect of malaria on the immune system. This confirms that malaria leads to major immune dysfunction and affects how the body deals with subsequent malaria re-infection.Malaria has been confirmed to deplete CD4+ and CD8+ T cell counts, upregulation of IL-6 and high serum levels of oxidative stress biomarkers. Taken together, Ferritin, IL-6 and IL-10 could play significant roles in the inflammatory processes, severe anemia and pathologies associated with malaria.Based on these findings, severe malaria was significantly associated with declined CD4It is recommended that future studies should investigate the direct impacts of vitamin supplements on proinflammation, other cytokines/chemokines and cellular immune function during pediatric malaria. In addition, there is a need to investigate how immunity to malaria is lost despite repeated malaria re-infections in Nigerian Children.


Hemorrhagic disease (HD) caused by a group of elephant endotheliotropic herpesviruses (EEHV) is one of the leading causes of death for young elephants in human care. These viruses are widespread and typically persist latently in adult elephants with no negative effects; however, in juvenile Asian and more recently young African elephants, the onset of disease can be rapid and the mortality rate high. Measuring biomarkers associated with the immune response could be beneficial to understanding underlying disease processes, as well as the management of infection and HD. The goal of this study was to measure acute phase proteins and cytokines in serum collected from elephants infected with EEHV (13 Asian and 1 African) and compare concentrations according to presence, severity and outcome of disease. Serum amyloid A (SAA) and haptoglobin (HP) were higher in elephants with EEHV viremia than those without; concentrations increased with increasing viral load, and were higher in fatal cases compared to those that survived. In Asian elephants, SAA was also higher during EEHV1 viremia compared to EEHV5. Cytokine concentrations were typically low, and no statistical differences existed between groups. However, in individuals with detectable levels, longitudinal profiles indicated changes in tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) that may reflect an immune response to EEHV infection. However, the overall low concentrations detected using previously validated assays do not support the presence of a ‘cytokine storm’ and suggest more work is needed to understand if sub-optimal immune responses could be involved in disease progression. These results highlight the potential benefit of measuring circulating biomarker concentrations, such as APPs and cytokines, to improve our understanding of EEHV viremia and HD, assist with monitoring the progression of disease and determining the impact of interventions. CytoThis study will add to existing knowledge by analyzing serum samples from Asian and African elephants during EEHV viremia to compare concentrations of APPs SAA and HP, and cytokines TNF-α and IL-2, which are pro-inflammatory and associated with T-cell growth and differentiation, respectively. We investigated how these measures differ during viremia and non-viremia, among different types of EEHV, with increasing viral load, and between fatal and surviving cases of HD.Serum and whole blood samples submitted to the National Elephant Herpesvirus Laboratory (NEHL) at the National Zoological Park, Washington D.C. for routine EEHV testing were utilized in this study. Blood samples were collected according to approved phlebotomy protocols at each institution, typically from an ear vein through behavioral conditioning, or as part of a medical intervention. Whole blood was collected into EDTA anticoagulant tubes, refrigerated and shipped overnight to the NEHL. A second blood sample collected into serum tubes was allowed to clot at room temperature, before serum was separated, frozen at -20 to -80°C and shipped overnight to the NEHL or the Smithsonian Conservation Biology Institute. The presence of EEHV in whole blood was determined by conventional PCR (cPCR) and/or quantified by real-time PCR (qPCR) according to methodology already described by Latimer et al. , StantonThis study included serum samples from 14 elephants (13 Asian and 1 African), aged 1 year 2 months to 12 years and 1 months at the time of collection . For somSAA and HP concentrations were measured using a RX Daytona automated clinical chemistry analyzer . Commercially available reagents, calibrators, and two-level controls were used for each assay , which have previously been validated for elephants ,46,47. TCytokines were measured by enzyme immunoassay as described in Edwards et al. . In brie2, 103, 104, 105 and 106 or above) were included as categorical fixed effects. Samples from the one African elephant with EEHV3B were excluded from the comparison of EEHV type. In addition, including only samples from cases of higher EEHV viremia (> 5000 vge/ml), concentrations of each biomarker were compared between cases that resulted in either survival or fatality. Animal ID and EEHV type were included as random effects, and data were modelled either using all samples between the start and end of the case of viremia, or using the peak concentration for each biomarker within each case of viremia. All results are presented as the mean prediction ± standard error from the GLMM to account for repeated sampling over time within individuals.Serum samples were categorized as viremia positive or negative, by EEHV type, and for viral load based on qPCR testing of whole blood collected on the same day. Data were then analyzed using generalized linear mixed models constructed using the package ‘lmer’  in R 5656, usingOf 353 serum samples analyzed, 233 were categorized as positive by either cPCR or qPCR of whole blood collected on the same day. These contributed to 46 separate cases of viremia, where viremia was detected in at least one consecutive sample. Six cases were categorized as positive through cPCR (3 EEHV1 and 3 EEHV5) although viral load was not quantified, 17 were transient low concentrations , six were intermediate , and 17 were higher level viremia , although this relationship was driven by a single elevated IL-2 value in an Asian elephant calf with a swollen front limb that was not associated with EEHV viremia. Of the 17 cases of higher-level viremia  and HP (P = 0.009) were increased in EEHV-positive samples  compared to those that did not contain detectable EEHV ; TNF-α and IL-2 did not differ between positive and negative samples . SAA wasLongitudinal profiles of serum biomarkers during several cases of EEHV1, EEHV5 and EEHV3B viremia, are presented in Figs This study measured APP and cytokine biomarkers associated with the immune response during EEHV infection in elephants and found several that could be useful as indicators of disease severity or prognosis. The APPs SAA and HP were both higher in serum from elephants with viremia than those without, and increasing concentrations were associated with increasing viral load. SAA was also higher in cases of EEHV1 compared to EEHV5, and both APPs were higher during fatal cases than in individuals that survived a bout of viremia exceeding 5000 vge/ml. Although concentrations for three  of the five cytokines validated for elephants  were und3 vge/ml or more, and increased with increasing viremia. Visible clinical signs of EEHV HD are often absent until viremia is quite advanced; in the cases reported here viral loads of 19,000 vge/ml EEHV1 and 90,000 vge/ml EEHV5 were detected by qPCR without accompanying clinical signs of illness. Although clinical signs may not always be apparent, increased APP concentrations indicate that inflammatory processes are underway, and that supportive care should be initiated. We found concentrations of SAA to be greater in EEHV1 infection compared to EEHV5; indeed, APP responses were reduced or even absent in some cases of EEHV5 viremia exceeding 10,000 vge/ml. Historically, EEHV5 has only caused one known death [APPs are useful indicators of inflammation across species , and arewn death  comparedwn death , suggestIn general, APPs are non-specific, so elevated concentrations may occur in the absence of EEHV viremia. Indeed, some of the samples utilized for this study had been submitted for analysis in response to non-specific clinical signs  that upon testing were not associated with EEHV. We recently published reference intervals for several biomarkers in African and Asian elephants and found SAA to be elevated in 83% of active clinical cases, including both infectious and traumatic etiologies, and in 50% of deaths studied . The lacunpublished), suggesting that assay sensitivity may not be limiting the ability to detect immune responses. One potential explanation for overall low concentrations is that a reduced immune response could be involved with the pathogenicity of this disease. Other herpesviruses have been documented to exert a suppressive effect on the immune system, often via functional impairment of immune cells [Cytokines including TNF- α, IFN- γ, IL-1β, IL-2, IL-6 and IL-10 have been linked to the pathogenesis of other viral hemorrhagic diseases, such as dengue –77, and ne cells , includine cells , reducinSrivorakul and colleagues  recently


Timely health care among children with suspected malaria, and intermittent preventive treatment (IPTp) in pregnancy avert related morbidity and mortality in endemic regions especially in sub-Saharan Africa. Malaria burden has steadily been declining in endemic countries due to progress made in scaling up of such important interventions.The study assessed malaria health seeking practices for children under five years of age, and IPTp in Wakiso district, Uganda.A structured questionnaire was used to collect data from 727 households. Chi-square and Fisher's exact tests were performed in STATA to ascertain factors associated with the place where treatment for children with suspected malaria was first sought (government versus private facility) and uptake of IPTp.Among caretakers of children with suspected malaria, 69.8% sought care on the day of onset of symptoms. The place where treatment was first sought for the children (government versus private) was associated with participants' (household head or other adult) age (p < 0.001), education level (p < 0.001) and household income (p = 0.011). Among women who had a child in the five years preceding the study, 179 (63.0%) had obtained two or more IPTp doses during their last pregnancy. Uptake of two or more IPTp doses was associated with the women's education level (p = 0.006), having heard messages about malaria through mass media (p = 0.008), knowing the recommended number of IPTp doses (p < 0.001), and knowing the drug used in IPTp (p < 0.001).There is need to improve malaria health seeking practices among children and pregnant women particularly IPTp through programmes aimed at increasing awareness among the population. Malaria remains a major cause of morbidity and mortality in endemic countries particularly in sub-Saharan Africa including Uganda. Children under five years of age and pregnant women are high risk groups regarding malaria infection due to their low immunity. For that reason, global malaria prevention efforts such as access to long-lasting insecticidal nets (LLINs) and Indoor Residual Spraying (IRS) have put much emphasis on these groups to reduce the occurrence of malaria among themAlthough iCCM and IPTp policies have been implemented in many sub-Saharan African countries including Uganda for many years, there is still low uptakeMalaria is still endemic in most parts of Uganda where it is responsible for about 65% of maternal mortality, and 60% of spontaneous abortions among pregnant womenThe study was cross-sectional and collected quantitative data. The data was collected using a structured questionnaire focusing on: socio-demographic characteristics; malaria health seeking practices for children ; general household health seeking practices ; and knowledge and practices on IPTp . The household questionnaire was administered once for each household involved in the study. The study participants were household heads or other responsible adults such as spouses that were found at home during data collection. However, questions concerning uptake of IPT were asked to only female participants. For this reason, the questionnaire used in the study had a section that was specific for only females.The study was carried out in Ssisa sub-county, Wakiso district, Uganda located in the central region of the country. Wakiso district has 7 health sub-districts with a total of 229 HCFs where 73% are private and serve over 70% of the population. Ssisa sub-county is made up of 11 rural and peri-urban parishes of Bulwanyi, Bweya, Kitende, Nakawuka, Namulanda, Nankonge, Ngongolo, Nkungulutale, Nsaggu, Ssisa and Wamala which were all involved in the study. The population in the sub-county is engaged in various economic and social activities such as agriculture, animal husbandry, petty trading, stone quarrying, brick making and sand mining. Ssisa sub-county has a population of 94,238 and average household size of 3.820. A total of 727 randomly selected households from 29 villages were involved in the study as described in our earlier publicationData were entered in SPSS version 10  and analysed in STATA version 13.0 . Univariate and bivariate statistical tests were employed. Dependent variables of the study were: the place where treatment was sought by households that had a child below five years who was suspected of having malaria in the two weeks preceding the study (either private or government HCFs); and uptake of two or more IPTp doses during their last pregnancy among women who had a child within the preceding five years. Chi-square and Fisher's exact tests were performed to ascertain the independent variables that were associated with the dependent variables. The independent variables included individual  and household characteristics .Ethical approval to conduct the study was obtained from Makerere University School of Public Health Higher Degrees, Research and Ethics Committee (11353). The research was also approved and registered at the Uganda National Council for Science and Technology (SS 3294). Participation in the study was voluntary, and participants provided written informed consent after explaining to them the purpose of the research including the anticipated risks and potential benefits before taking part.Among the participants, 67.8% were female, 60.3% above 30 years of age, and 44.0% had attained secondary education and above. Regarding average monthly household income, over half of the participants (53.7%) earned less than 40 US dollars, while 65.1% had at least one child under five years of age in their household, with 32.7% having two or more children .The majority of participants (91.9 %) said children suspected of having malaria should be taken for treatment on the same day the illness begins. Among the participants, 29.3% had children under five years of age in their household who were suspected of having malaria in the two weeks preceding the survey. Among these households with suspected sick children, treatment was sought mainly from government HCFs (33.7%), private HCFs (30.7%), and CHWs (22.6%). The places where treatment for the children was first sought was mainly private HCFs (28.1%), followed by government HCFs (25.6%), CHWs (3.0%), and others (13.0%). The place where treatment was first sought for the children (government versus private HCF) was associated with participants' age (p < 0.001), education level (p < 0.001) and household income (p = 0.011) . Many ofThe nearest HCFs from participants' households were private clinics (39.3%), government HCFs (30.9%), and private hospitals (20.1%). Other facilities mentioned by participants were pharmacies/drug shops (4.4%). Many of the nearest HCFs (62.3%) were less than one kilometre from the participants' homes. The other facilities were between one to three kilometres (17.2%) or more than three kilometres (10.9%). The most likely ways in which the participants would go to the nearest HCF from their households were walking (65.7%), use commercial motorcycle (28.2%), public transport (3%), private car (1.5%), or bicycle (1.2%). The majority of participants (71.7%) used the nearest HCF from their household the last time a household member was suspected to have malaria. Among participants whose households did not use the nearest facility, the reasons given were suspicion of no drugs being available (33.3%), services being costly (29.8%), poor services offered (18.2%), services needed not offered (8.6%), or poor health worker attitudes (1.5%). A good number of participants (68.4%) were aware of the existence of CHWs who distribute malaria medicines in their villages. However, among participants who were aware of the existence of CHWs, only 17.3% thought they had malaria medicines at the time. In 37.3% of households, school going children had missed school in the previous 6 months due to malaria, with a mean number of days missed of 15.3 (standard deviation ± 29.9).Over half of the participants (54.2%) were aware of sulfadoxine-pyrimethamine (SP) as the drug used for IPTp. Other drugs participants mentioned were chloroquine (39.3%) and Coartem (6.5%), while 39.3% did not know. Females were significantly more knowledgeable about IPTp compared to males (p < 0.001). Among the females involved in the study (n = 493), 87% had children. The majority of females with children (81.9%) underwent IPTp when they were last pregnant. The main reasons given for those who did not undertake IPTp during their last pregnancy were: not being aware of the need (51.3%); no HCF being available (24.4%), and did not want (16.7%). Among those who took drugs for IPTp, 73.6% took SP, 4.8% took chloroquine, while 16.2% did not know what they took. The majority of women (94.6%) got IPTp the last time they were pregnant during ANC visits while for the rest, it was during another visit to a health facility. Among women who had a child in the five years preceding the study, 179 (63.0%) had obtained two or more IPTp doses during their last pregnancy. Uptake of two or more IPTp doses was associated with higher education level (p = 0.006), having heard messages about malaria through mass media (p = 0.008), as well as knowing the recommended number of IPTp doses (p < 0.001) and drug used in IPTp (p < 0.001) concerning the women .This study explored health seeking practices among under five children on malaria related care as well as uptake of IPTp in Wakiso district, Uganda. Findings indicate that nearly 70% of caretakers of children suspected with malaria sought health care on the day of onset of symptoms, and most of them traveled 3 kilometres or less to the nearest HCF from their home. The choice of facility (government versus private) for seeking malaria related care for children was influenced by the participant's age, education level and household income. Uptake of IPTp among women who had a child within five years preceding the study was generally low (63%) and influenced by level of education, having heard messages about malaria on mass media, as well as knowledge of the drug used during IPTp and recommended number of doses. The findings from our study emphasize the need for improving malaria health seeking practices including IPTp for better health outcomes among the population.Early diagnosis and appropriate treatment of malaria is critical to minimize severe cases and associated mortality as well as interrupt transmissionA large proportion of participants would need to travel less than one kilometre (62.3%) or between one to three kilometres (17.2%) to the nearest HCF from their homes. A short distance to a HCF implies that physical access for health care is less of a challenge hence increasing the likelihood of utilizing health services. Evidence suggests that long distances to HCFs impedes utilisation of essential services as has been previously observed in UgandaRegarding uptake of IPTp, 63.0% of women who had given birth in the five years preceding the study had obtained two or more IPTp doses during their last pregnancy. This level of utilisation of IPTp was lower than the national target of 80% for most African countries including UgandaOur study established that education was associated with uptake of IPTp among women who had a child five years preceding the study. Having attained higher education is likely to be associated with awareness of malaria prevention programmes. Indeed, individuals with higher education are more likely to be able to understand the benefits of IPTp for maternal and child survival as well as access ANC servicesThis study had a number of strengths such as involving both women and men in IPTp issues; explored health seeking practices for children, women and the general household; and also involved several parishes that had rural and peri-urban populations. Involving men in IPTp is particularly of key public health importance as male involvement has been shown to contribute to better maternal health outcomes including in developing countries This study provides useful insights into malaria health seeking practices including delays in taking children suspected of having the disease to health facilities, and low coverage of IPTp. There is need to improve health malaria seeking practices among childrn and pregnant women to reduce morbidity and mortality related to the disease. Programmes should therefore be designed to increase awareness on the importance of early seeking for health care for children and IPTp, as well as address other health system challenges affecting health seeking behaviour among the community.


Plasmodium species infection rates and microscopic examination compliance with nested PCR results. Sensitivity and specificity analyses were performed in conjunction with predictive values. Also, phylogenetic characterization of species of malaria parasites was assessed. Plasmodium genus-specific nested PCR identified 565 positive cases including 536/611 (87.8%) confirmed cases from the microscopy-positive group and 29/199 (14.6%) diagnosed malaria cases from the microscopy-negative group. Our findings revealed a disease prevalence (69.8%) higher than that reported (25.5–55.1%) for the country. The diagnostic test had 94.9% sensitivity and 69.4% specificity, i.e., it missed 120 of the people who had malaria and about one-third of the people tested positive for the disease, which they did not have, respectively. In conjunction, the test showed 87.7% positive predictive value and 85.4% negative predictive value, which, from a clinical perspective, indicates the chance that a person with a positive diagnostic test truly has the disease and the probability that a person with a negative test does not have the disease, respectively. Further species-specific nested PCR followed by analysis of gene sequences confirmed species of malaria parasites and indicated infection rates for Plasmodium falciparum (Pf), 95.5% (540/565); P. ovale (Po), 0.5% (3/565); and P. malariae (Pm), 0.4% (2/565). In addition, 20 cases were coinfection cases of Pf-Po (15/565) and Pf-Pm (5/565). This study publicly reports, for the first time, a molecular survey of malaria cases in Togo and reveals the presence of other malaria parasites (Po and Pm) other than Pf. These findings might provide answers to some basic questions on the malaria scenario and, knowledge gained could help with intervention deployment for effective malaria control in Togo.Malaria particularly burdens people in poor and neglected settings across the tropics of Africa. Meanwhile, a large proportion of the Togo population have poor understanding of malaria epidemiology and parasites. This study carried out a molecular survey of malaria cases in southern Togo during 2017–2019. We estimated  Malaria is a major debilitating infectious disease that affects millions of people and impedes public health and socioeconomic development in Africa . In tropPlasmodium that are responsible for disease transmission. Studies on disease burden erroneously made believe that malaria cases are only related to Plasmodium falciparum (Pf) infection in Togo , Plasmodium ovale (Po), Plasmodium malariae (Pm), and Plasmodium knowlesi (Pk)] (Plasmodium species (Routine diagnosis using the standard method for the detection of hnicians . Polymerhnicians . A rangesi (Pk)] . In addi species .Plasmodium species from southern Togo.In order to advance knowledge of diagnosis efficacy and species of malaria parasites in Togo, this study carried out a molecular investigation on malaria clinical samples in comparison with light microscopic examination and assessed the phylogeny of This study was conducted in the two southern regions (Maritime and Plateaux) of Togo  where maLomé is the capital of Togo and lies on the Gulf of Guinea (Atlantic coast) in the extreme southwestern corner of the country. Its urban population was estimated at 837,437 inhabitants as of the 2010 census . Lomé haEthical approval for malaria samples collection was assented by the Togolese Ministry of Health’s Bioethics Committee following the institutional ethical guidelines by the Ethics Committee at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, as reported previously . InformeCentre Médico-Social de Doumasséssé and Clinique Chirurgicale Martin Luther-King, Lomé; Centre Médico-Social Notre Dame de la Consolation d’Agbonou Campement, Atakpamé; and Hôpital Bethesda d’Agou Nyogbo and Centre Hospitalier Préfectoral d’Agou Gare, Agou in September 2017 to December 2019. Individuals diagnosed by microscopic examination were referred to our study and samples were collected from those who consented to the study. The end of specimen collection was not determined by any factor other than the defined period of the study.Lomé, Agou-Gadzépé, and Atakpamé inhabitants usually access regional hospitals, polyclinics, and community health centers experienced in the management of malaria cases for treatment and medical attention. Blood samples were collected from Sample collection involved febrile and suspected malaria cases including asymptomatic individuals, aged between 3 months and 70 years old. Clinical malaria patients were subjects who presented themselves to health facilities with fever or a history of fever  in the last 24 h and were confirmed for Pf infection by microscopy . Suspected malaria cases were persons who complained of fever of less than 2 days or having a history of fever, diagnosed either negative or positive by microscopy, and received treatment with antimalarial drugs.Plasmodium species or case of coinfection of Plasmodium species was recorded from microscopy. All participants that were positive for malaria after diagnosis by parasitological blood smear test were treated with antimalarial drugs according to the national malaria programme guidelines.Finger prick/venipuncture blood samples were obtained from individuals who received parasitological diagnosis at the health facilities, using the Giemsa-stained thick blood smear microscopic examination under × 1,000 magnification. Asexual stages of malaria parasites were counted per 200 leukocytes and, parasite density was calculated as the number of parasites per microliter by assuming a fixed leukocyte count of 8,000 cells/μl of blood. The thick film was considered positive when malaria parasites were present and negative if no parasites were seen after 500 leukocytes were counted. A total of 810 blood samples were collected including 610 Pf confirmed cases and 199 microscopy-negative cases. Except one Pm case that was detected by microscopy, no infection case of other Fresh whole blood and serum samples were collected and sampled as dried blood spots (DBSs) and dried serum spots (DSSs) on Whatman FTA and 903 Protein Saving Cards , respectively, according to the instructions of the manufacturer. They were then packed in transparent zip-lock airtight plastic bags with silica gel desiccant to ensure quality of spots storage. Genomic DNA was extracted from the blood samples applied to FTA Cards using the QIAGEN DNeasy Blood and Tissue Kit  according to the instructions of the manufacturer and as reported by Plasmodium species ssrRNA genes with primers as reported previously , followed by a final extension at 72°C for 10 min. Two microliters of the first PCR product were used for the second-round amplification, with conditions and concentrations identical to those used for the first, except that rPLU3 and rPLU4 were used as inner primers  was used to perform nested PCR amplification of eviously . For fir primers  and amplt humans . The fir+ Imaging System . All positive results of Plasmodium-infected cases were confirmed again through direct sequencing of the second round nested-PCR products by the BGI Company (China), using nested primers. Sequencing reads were imported into the EditSeq module of Lasergene-Ver7.11 to construct ssrRNA gene sequences of the nested-PCR products, which were then blasted at https://blast.ncbi.nlm.nih.gov/. Partial sequences of 18S ribosomal RNA genes of the Togo isolates were aligned with those of other Plasmodium isolates obtained from blast analysis, using the ClustalW method  of MEGA-Ver7.02 to generate a phylogenetic tree based on the neighbor-joining method  were also calculated for the population that was tested in this study.Plasmodium genus-specific nested PCR identified 565 malaria-positive cases including 536 and 29 cases from the MPG and MNG groups, respectively. In other words, nested PCR confirmed 536 out of 611 (87%) cases that were true positive from the MPG and identified 29/199 (14.6%) malaria cases that were found negative  with Giemsa-stained thick blood smear microscopic examination, we obtained 611 positive malaria cases [referred to as microscopy-positive group (MPG)] including 610 cases of Pf and one case of Pm, while the remaining 199 cases were tested malaria negative and were referred to as the microscopy-negative group (MNG). The use of negative  in the Mnegative . ClinicaFurther species-specific nested PCR and species confirmation through direct sequencing of the nested-PCR products identified higher infection rate [95.5% (540/565)] for Pf than those for other species, which were 0.5% (3/565) for Po and 0.4% (2/565) for Pm. In addition, 20 cases were coinfection cases of Pf and Po [2.7% (15/565)] and Pf and Pm [0.9% (5/565)] . However18S rRNA locus. Phylogenetic analysis of the fragments of 18S SSu rRNA gene sequences of the sample cases tested in this study showed species specificity and clustered with homologous sequences of isolates of Plasmodium species from different countries ; MW492393 and MW492394 (Po); MW492392 and MW504627 (Pm)].Phylogenetic relationships of unique sequences that were amplified were constructed on the basis of similarities with published homologous sequences from different countries based on neighbor-joining analysis of the ountries . Gene seTogo, an integral part of tropical Africa where malaria is endemic, has its entire population at risk of malaria infection and disease transmission occurs all year round , 2021. SPlasmodium species, and a number of studies demonstrated that PCR is more reliable than microscopy in detecting malaria parasites in infected individuals, especially in those with low parasite density  is the essential cause of malaria burden in tropical Africa (Plasmodium species detected (95.5%) among the sample cases, aligning onto the evidence that Pf is always highly endemic in West African countries because the region usually experiences long periods of rainy reason which enhances perennial or semiperennial Anopheles breeding sites. We, also detected low infection rates of both ovale and malariae cases . The detection of these two “bashful” malaria parasites in such a Pf-endemic area is consistent with previous studies from West Africa, which reported that Po and Pm are often associated with Pf infections, especially in areas where falciparum is highly endemic  low incidence of the disease, (ii) lack or rarity of severe cases as they are regarded less important to Pf in terms of public health issues, or (iii) practical difficulties in microscopic identification as Po and Pm share morphological characteristics resembling those of Pf  . Thus, iAlthough considered “mild,” Po and Pm may constitute an important cause of morbidity and high risk of mortality. For example, fever episodes related to parasitemia peaks of Pm (in Liberia and The Gambia) or Po , in both children and adults have been reported . Also, PThis study publicly reports, for the first time, a molecular survey of malaria infections in Togo and also reveals the presence of other species of malaria parasites—Po and Pm, which were not previously reported. In addition, findings from this study indicated errors in microscopic examination including error in species diagnosis and undiagnosed cases. For example, in this study population, one-third of the people were microscopically tested positive for the disease they did not have. Thus, overlaps of different species of malaria could further aggravate the disease burden in Togo if appropriate actions are not taken, especially in situations where diagnosis of monospecies of malaria cases seems already “problematic.” Collectively, this preliminary study advanced our understanding of the malaria epidemiology in southern Togo and provided information to improve disease control/surveillance policy. However, large-scale studies across the whole country are required in the future to assess malariae and ovale malaria epidemiology.https://www.ncbi.nlm.nih.gov/, MW490726, MW492389, MW492390, MW492391, MW504628, MW492393, MW492394, MW492392, and MW504627.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Permission was obtained from all malaria subjects before collecting the specimens. Blood collection was made under a study protocol reviewed and approved by the Togo Ministry of Health’s Bioethics Committee (Authorization N°019/2019/MSHP/CBRS), following institutional ethical guidelines by the Ethics Committee at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, as reported previously .KK and J-HC conceptualized the study. KK, KA, KN, and KKK collected and analyzed the specimens. KK, S-WF, and KKK conducted the experiments. KK interpreted the data and wrote the manuscript. EMA, X-KG, and X-NZ revised the manuscript critically for intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plasmodium falciparum infection has revealed many previously undetected (by microscopy) yet transmissible low-density infections. The proportion of these infections is typically highest in low transmission settings, but drivers of submicroscopic infection remain unclear. Here, we updated a previous systematic review of asexual P falciparum prevalence by microscopy PCR in the same population. We aimed to explore potential drivers of submicroscopic infection and to identify the locations where submicroscopic infections are most common.Adoption of molecular techniques to detect P falciparum prevalence by both microscopy and PCR. Surveys of pregnant women, surveys in which participants had been chosen based on symptoms or treatment, or surveys that did not involve a population from a defined location were excluded. Both the number of individuals tested and the number of individuals who tested positive by microscopy or PCR, or both, for P falciparum infection were extracted. Bayesian regression modelling was used to explore determinants of the size of the submicroscopic reservoir including geographical location, seasonality, age, methodology, and current or historical patterns of transmission.In this systematic review and meta-analysis we searched PubMed and Web of Science from Jan 1, 2010, until Oct 11, 2020, for cross-sectional studies reporting data on asexual Of 4893 identified studies, we retained 121 after screening and removal of duplicates. 45 studies from a previous systematic review were included giving 166 studies containing 551 cross-sectional survey microscopy and PCR prevalence pairs. Our results show that submicroscopic infections predominate in low-transmission settings across all regions, but also reveal marked geographical variation, with the proportion of infections that are submicroscopic being highest in South American surveys and lowest in west African surveys. Although current transmission levels partly explain these results, we find that historical transmission intensity also represents a crucial determinant of the size of the submicroscopic reservoir, as does the demographic structure of the infected population (with submicroscopic infection more likely to occur in adults than in children) and the PCR or microscopy methodology used. We also observed a small yet significant influence of seasonality, with fewer submicroscopic infections observed in the wet season than the dry season. Integrating these results with estimates of infectivity in relation to parasite density suggests the contribution of submicroscopic infections to transmission across different settings is likely to be highly variable.Significant variation in the prevalence of submicroscopic infection exists even across settings characterised by similar current levels of transmission. These differences in submicroscopic epidemiology potentially warrant different approaches to targeting this infected subgroup across different settings to eliminate malaria.Bill & Melinda Gates Foundation, The Royal Society, and the UK Medical Research Council. Plasmodium parasite. Routinely, malaria detection is undertaken using microscopy of blood films or rapid diagnostic tests, although in recent years there has been an increase in the use of more sensitive molecular methods in research contexts. These techniques The ability to accurately detect malaria infection during population surveys is a cornerstone of effective surveillance and control of the These infections are also relevant to public health because of their potential to be transmittable, despite being undetectable by conventional diagnostics. Although typically characterised by lower parasite densities and infectivity than microscopically detectable infections,Evidence before this studyPlasmodium falciparum infections missed by microscopy can be substantial , that submicroscopic infection is more frequent in adults than in children (a phenomenon attributed to increased levels of immunity in adults and the associated suppression of parasite densities by this immune response), and that this missed fraction  is typically highest in areas with low transmission intensity.Cross-sectional surveys of malaria prevalence in endemic populations have revealed the widespread presence of infections with parasite densities lower than the threshold of detection by microscopy. These submicroscopic infections can infect mosquitoes and contribute to onwards transmission; therefore, understanding when and where these infections are most common is vital to the control of malaria transmission. A previous systematic review of these surveys highlighted that the fraction of Added value of this studyThe results presented here support previous research linking submicroscopic infection to older individuals and to areas where current transmission levels are low. However, the increased availability of data used in our study allows a richer and more complex characterisation of variation in submicroscopic epidemiology between settings. Our findings highlight marked geographical variation in the size of the submicroscopic reservoir, which cannot be explained by current transmission levels and instead appear primarily to be driven by the historical patterns of transmission in a setting. We also identify both seasonality and variation in diagnostic quality as drivers of submicroscopic infection prevalence across the population. Integrating these results with estimates of infectivity in relation to parasite density suggests the contribution of these submicroscopic infections to transmission across different settings is likely to be highly variable.Implications of all the available evidenceOur work highlights material differences in submicroscopic malaria epidemiology across settings and suggests the absence of a one-size-fits-all solution for malaria control efforts targeting this infected subgroup. These differences likely warrant different and setting-specific approaches to targeting the submicroscopic reservoir if malaria is to be most effectively controlled in the approach to elimination.Plasmodium falciparum infections compared with PCR-based methods in cross-sectional surveysDespite this potential relevance to malaria transmission, our understanding of the factors influencing the size of the submicroscopic reservoir remains far from complete. Previous reviews have found that microscopy misses on average, half of all Resolving these gaps in our understanding of submicroscopic epidemiology has material consequences for the future of malaria control. Given that submicroscopic infections can contribute to malaria transmission, understanding when and where they are most prevalent is vital to the control of the disease. Despite mixed reports surrounding more recent progress,Here we update previous reviews on submicroscopic malaria infection prevalence,P falciparum prevalence by microscopy and PCR in the same population were included. Surveys of pregnant women, in which participants were chosen on the basis of symptoms or treatment, or which did not involve a population from a defined location were excluded. Submicroscopic infections were defined as where infection was detectable by PCR but not by microscopy. Specificity of microscopy compared to PCR is high  and characteristics of the survey location and timing . Where available, information on the age range of survey participants were also extracted.From each study, we extracted information on the number of individuals tested by PCR and microscopy, as well as the number of tested individuals positive for Data were analysed using an ANOVA based approach to assess differences in the mean prevalence ratio  and the factors underlying these differences. Data were weighted according to the cross-sectional survey sample size and controlling for the PCR prevalence recorded in the survey. Tukey's honest significant difference (HSD) test was used to post-hoc examine pairs of factors for significant differences in the mean prevalence ratio. These analyses were done using R statistical software, version 4.0.2.In line with a previous review,i is the log odds of microscopy prevalence in survey i, PCRi is the log odds of PCR prevalence, and δi is the log odds ratio of microscopy to PCR prevalence. δi is defined as:where LM0 is a coefficient shared across all surveys describing how log odds of microscopy prevalence varies with log odds of PCR prevalence, and δiʹ is a survey-specific intercept coefficient describing the effect of PCR prevalence on microscopy prevalence. This model structure allows δi to vary between surveys, with β0 controlling the extent of this variation. This model was fitted within a Bayesian Markov chain Monte Carlo based framework, implemented in JAGS.GitHub.where PCR is the mean survey PCR prevalence, βSurveys done in Africa were geolocated and prevalence estimates  from the Malaria Atlas ProjectWhere MAP estimates were unavailable (dates earlier than 2000), it was assumed that the year 2000 was reflective of historical transmission intensity because the substantial increase in international financing for malaria control occurred from 2000 onwards (approximately a twentyfold increase between 2000 and 2015).We integrated the results of the regression modelling described earlier  with estimates of infectivity for submicroscopic and microsopically detectable infections to estimate the potential contribution of submicroscopic infections to transmission across different settings. Estimates of comparative infectivity of microscopically-detectable infections versus submicroscopic infections (hereafter referred to as the infectivity ratio) are variable, ranging from a 2× to a 20× difference.–ve) is set to 1, and the infectivity ratio  is a multiplicative factor reflecting the fact that microscopically detectable infections  are more infectious. The equation's denominator reflects total onwards transmission occurring within the population, the numerator the amount of transmission attributable to submicroscopic infections. These analyses assume that submicroscopic and microscopically infected populations do not differ in other factors that are likely to influence transmission .where the relative infectiousness of submicroscopic infections  revealed marked geographical variation in the prevalence ratio , being lower in South American surveys than all other regions  and higher in west African surveys than all other regions  might be observed in areas with similarly historically low transmission in Africa. Our results indicated that both regional historical prevalence (in the year 2000) and current prevalence (both averaged over the administrative unit 1 level) were significant predictors of the prevalence ratio when controlling for survey PCR prevalence , suggesting that historical transmission levels, in addition to current transmission levels, are an important determinant of the submicroscopic reservoir size.We next classified each survey in our review from Africa into three transmission archetypes on the basis of the historical and current levels of transmission at the administrative unit 1 level  and fittWe also collated surveys carried out in specific age groups and defined three age-based categories: young children (0–5 years) giving 49 prevalence survey pairs, older children (6–15 years) giving 62 prevalence survey pairs, and adults (>15 years) giving 53 prevalence survey pairs. The prevalence ratio varied significantly between age groups , and was significantly lower in adults (indicating a greater proportion of submicrosopic infections) than in young children  and older children  and increased immunity  have been linked to lower parasite densities.Another potential limitation is the strong geographical bias in our transmission archetype stratification, which precludes exact determination of the extent to which variation in submicroscopic malaria is driven by geography compared to transmission patterns. The majority of surveys assigned to the historically low and currently low archetype are from east Africa, while the majority of surveys in the historically high and currently high archetype are from west Africa. The observed results across transmission archetypes could, therefore, be reflecting geographical variation rather than variation driven by past transmission history. However, the fact that the proportion of infections that are submicroscopic in historically high and currently low settings  was consistently lower than that observed for historically low and currently low settings, together with the results observed for South America, provide tentative support for an effect of transmission history on the size of the submicroscopic reservoir, independent of variation due to geographical location.P falciparumP falciparum strains), whereas low-transmission settings might select for non-virulent parasites with lower rates of replication better able to persist and avoid causing symptomatic infection .Several hypotheses could explain these results, including various haemoglobinopathies and human genetic traits that have been linked to lower average parasite densities.Our analyses revealed a significant influence of seasonal effects on submicroscopic carriage, with submicroscopic infections more common in the dry season. This is in keeping with previous work showing that parasite densities rise slightly during the rainy season .Our work suggests that the contribution of submicroscopic infections to onwards transmission is likely to be highly variable across settings. However, this analysis is based on the detection of asexual parasites and does not provide direct insight into gametocyte densities. Additionally, due to data constraints, we did not consider a range of relevant factors, such as age profile of the infected population and related skin surface area effects (whereby adults have larger skin surface areas available for biting by mosquitoes compared with children), and adjusting for these would likely increase the contribution to transmission from older children and adults (who are more likely to have submicroscopic infection). The relationship between asexual parasite and gametocyte density is highly non-linear and the distributions of parasite densities in the submicroscopic range can differ substantially between settings.Despite their potential relevance to maintenance of malaria transmission, our understanding of submicroscopic infections remains far from complete. Do submicroscopic infections represent a substantial source of transmission and threat to future progress? Do these infections need to be targeted to achieve malaria elimination? Although more work is required, our findings highlight important differences in submicroscopic epidemiology between settings and suggests the absence of a one-size-fits-all solution for malaria control targeting this infection subgroup. Such variation will probably warrant different approaches to malaria control if the infection is to be controlled most effectively in the effort towards elimination.GitHub.Complete details of the code used to analyse the collated data can be found on LCO reports grants from UK Royal Society during the conduct of the study; grants from the Bill & Melinda Gates Foundation and UK Research and Innovation, outside the submitted work. ACG reports grants from the Bill & Melinda Gates Foundation and the Medical Research Council (MRC) Centre, during the conduct of the study; grants from The Wellcome Trust, Medicines for Malaria Venture, Integrated Vector Control Consortium, PATH, National Institute for Health, and personal fees from The Global Fund, outside the submitted work. All other authors declare no competing interests.


Atraumatic splenic rupture is a rare and life-threatening condition, if not diagnosed. We present two cases with a history of travel to endemic areas, who came to the emergency department with abdominal pain and were diagnosed to have spontaneous splenic rupture as a complication of severe malaria. Both patients were treated surgically by splenectomy. A high level of clinical suspicion is critical in every malaria patient presenting with abdominal pain, even if it is mild. Clinical imaging modalities like ultrasonography and computed tomography (CT) are crucial diagnostic tools in managing such patients. Malaria is a mosquito-borne infection caused by parasites from the plasmodium group. There are five parasite species that cause malaria in humans. Plasmodium falciparum  and Plasmodium vivax (P. vivax) are the most common organisms that can cause malaria in humans . MalariaCase 1A 38-year-old gentleman with no past medical history presented to the emergency department with complaints of left upper quadrant abdominal pain for four days. The pain was severe, constant, and associated with nausea and vomiting. He had recent travel history to Ghana, normal bowel and bladder habits, and denied any history of trauma or fall. His vitals were as follows: temperature: 36.5 degrees Celsius, heart rate: 84 beats per minute, respiratory rate: 20 per minute, blood pressure: 113/76 mmHg, and oxygen saturation: 98%. On abdominal examination, there was marked tenderness in the left hypochondrium. Other systemic examinations were within normal limits.Laboratory examinations revealed high c-reactive protein levels and low platelet count. Malaria smear test was positive showing early trophozoite state (ring forms), suggestive of falciparum malaria. Ultrasound abdomen was done and showed mildly enlarged spleen, with no obvious focal lesion. A trace amount of fluid was seen in the splenorenal space Figure . He was On the second day of admission, the patient was still having severe abdominal pain, despite taking regular analgesics. CT abdomen with contrast was performed and revealed perisplenic hyperdense collection or hematoma with suspicion of a distorted capsule at the inferior border, hemoperitoneum, and multiple mesenteric lymph nodes enlargement Figure .The patient was managed as a case of acute abdomen due to spontaneous rupture of the spleen and underwent emergency laparotomy and splenectomy. During the operation, splenic laceration, tear in the capsule, significant amount of clotted and non-clotted blood in both upper compartments, and moderate splenomegaly were detected. The patient was discharged on the sixth day after surgery. Influenza and pneumococcal vaccine were given on the day of surgery. On the fifth postoperative day, Haemophilus influenzae and meningococcal vaccines were given. The patient was followed up to 10 days postoperatively and was clinically stable.Case 2A 34-year-old gentleman, with no past medical history, presented to the emergency department with a history of left upper abdominal pain and fever for three days. He had a history of travel to Nepal recently. No history of nausea or vomiting was noted. His vitals were as follows: temperature 40.6 °C , heart rate: 129 beats per minute, blood pressure: 92/50 mmHg, respiratory rate: 20 per minute, and oxygen saturation: 99%. On general examination, he was dehydrated. Local examination revealed mild tenderness in the left hypochondrium.Intravenous normal saline was given, and laboratory workups showed high c-reactive protein. Malarial smear was positive for plasmodium vivax malaria. The patient was admitted to the critical care unit as a case of severe malaria. Ultrasound abdomen showed mild splenomegaly with hypoechoic linear echogenicity extending from the cortex and no vascularity on doppler examination that suggested splenic laceration Figure .Conservative treatment was given initially with intravenous fluids and anti-malarial drugs artemether-lumefantrine 20 mg/120 mg tablets, four tablets, twice daily for three days, and primaquine 30 mg, once daily for 12 days, which showed initial improvement, but later he had a drop in hemoglobin along with hypotension and severe dehydration. CT abdomen with contrast was performed, and it showed features of splenic laceration of grade III with a mild volume of hemoperitoneum and preserved splenic vessels, giving an impression of spontaneous splenic rupture Figure .The patient underwent splenectomy and postoperative days were uneventful. Influenza and pneumococcal vaccines were given on the day of surgery. Meningococcal and Haemophilus influenza vaccines were given on the fourth postoperative day and discharged home on Day 7 with follow-up in the surgical outpatient clinic.Malaria parasites have been around since the dawn of time. They most probably evolved in Africa along with humans, and mosquito fossils reveal that malaria vectors have been there for at least 30 million years. Humans are the sole vertebrate hosts for Plasmodium parasites and Anopheles mosquitoes are the vectors. This parasite specificity also suggests a long and adaptive association with humans .In malaria, the precise mechanism of spleen rupture is unknown. The following three mechanisms are implicated in the process of splenic rupture: i) Increased intrasplenic tension owing to cellular hyperplasia and engorgement, ii) spleen compression by abdominal muscle during physiological activities like sneezing, coughing, defecation, etc., iii) vascular occlusion due to reticuloendothelial hyperplasia, culminating in thrombosis and infarction . This caMost cases of spontaneous splenic rupture linked with Plasmodium vivax occur during acute malaria illness. A main predisposing factor appears to be a lack of prior immunity to malaria . AlthougConservative management with antimalarial drugs is the best option for patients who are hemodynamically stable. For children and adults with P. falciparum malaria, artemether-lumefantrine is the effective and safe treatment, whereas, for the dormant liver stage of P. vivax, primaquine is the treatment of choice -8. NonopThere is growing evidence that treatment of spontaneous rupture of the malarial spleen without splenectomy should be attempted in malaria-endemic areas . In our Spontaneous splenic rupture in complicated malaria is extremely rare. There should be a high index of suspicion for spontaneous splenic rupture in a patient who is diagnosed to have malaria and presented with left upper quadrant pain, even without a history of trauma. Ultrasound and computed tomography will aid in early diagnosis to avoid the fatal complications of malaria. Perisplenic hematoma with hemoperitoneum should be managed with abdominal exploration. And nonoperative management is the best option if the patient is hemodynamically stable. Prophylactic measures should be taken by travelers to endemic areas.