Plasmodium falciparum on Bubaque, the most populous island of the country’s remote Bijagos archipelago. A cross-sectional survey was performed at the start of the rainy season in August 2017. Participants were recruited using systematic random sampling in a two-stage stratified cluster design. Malaria parasitemia was detected using rapid diagnostic tests (RDTs) and quantitative PCR (qPCR). Data on housing, education, larval source management, socioeconomic status, anemia, and malaria preventive measures were collected. Multivariable logistic regression models were constructed to identify associations with P. falciparum infection. Four hundred four persons  were enrolled in the study. The prevalence of P. falciparum parasitemia was 5.8% by RDT (95% CI: 3.55–9.33) and 16.9% by qPCR (95% CI: 13.09–21.71). The prevalence of anemia was 74.3% (95% CI: 69.04–78.85) as defined by the WHO criteria. All sampled houses were found to have open eaves; 99.5% of the surveyed population reported sleeping under a bednet (95% CI: 97.8–99.9). Although reported LLIN use is high, there remains an appreciable prevalence of malaria, suggesting that transmission is ongoing and further tools are required to reduce the burden of the disease.Distribution of long-lasting insecticide-treated nets (LLINs), passive detection and treatment with artemisinin-based combination therapy (ACT), and intermittent preventive treatment in pregnancy (IPTp) are the mainstay malaria control measures of Guinea-Bissau’s national control programme. This study aimed to estimate the prevalence of  Plasmodium falciparum, with an estimated 111 million cases.1 Within the subregion, Guinea-Bissau reported an estimated 132,600 cases and 600 deaths from malaria that year. There is little published information on the epidemiology of P. falciparum infections in the country using molecular diagnostic techniques. Snounou et al.’s seminal 1993 study was an early PCR-based malaria survey during the rainy season in the village of Bor, west of Bissau, the country’s capital city. Of 79 asymptomatic persons  surveyed, 69 persons were reportedly infected with Plasmodium species, including 49 with P. falciparum, a positivity rate of 87%. A study by Santoguina et al.2 in 2009 of 2,348 asymptomatic persons  at three sites in the Gambia and Guinea-Bissau (West of Bissau), respectively, found an overall parasite prevalence of 25.5% (95% CIs: 23.7–27.3%). All three sites had comparable prevalence of P. falciparum infections.In 2016, the West African subregion had the highest number of cases of malaria due to P. falciparum in the country’s offshore Bolama Bijagos region has not previously been reported. The region’s approximate population of 30,000 lives on 20 different islands in the archipelago. The region has its own hospital, which includes a diagnostic laboratory, on the island of Bubaque. However, the remoteness of the islands from the mainland, and the distance between villages, impedes the collection of accurate epidemiological data. Other African islands have previously reported significant success in reducing malaria prevalence. Scaling up vector control programs on the islands of the Comoros,3 Zanzibar,4 and São Tomé and Príncipe5 have been credited for this. Mass drug administration with antimalarial drugs also offers the possibility of reducing prevalence on islands significantly and durably.6 To assess the susceptibility of the archipelago to such strategies, we conducted the first cross-sectional population-based malaria survey in the Bolama Bijagos region. The island of Bubaque was the chosen site for this malaria survey. It is situated approximately 50 km to the southwest of Bissau. The island has a tropical climate and a rainy season from June to October. A once to twice weekly shuttle boat connects the island to Bissau. A qualitative study by Durrans et al.7 demonstrated that travel patterns between islands and the mainland are complex and fluctuate according to seasons.The epidemiology of P. falciparum parasitemia as measured by rapid diagnostic test (RDT) and quantitative PCR (qPCR). Asymptomatic parasitemic individuals are the largest reservoir of P. falciparum, and estimating the size of this group in the population was the primary objective of this study.8The island survey allowed us to collect detailed clinical and epidemiological data including reported use of long-lasting insecticide-treated nets (LLINs), uptake of intermittent preventive treatment in pregnancy (IPTp), prevalence and severity of anemia, and prevalence of A census of the island was carried out in July 2017 to provide a sampling frame of households and an updated estimate of the island population. A cross-sectional population-based survey was carried out in August of that year.A malariometric cross-sectional survey was conducted using RDTs and qPCR on a representative group of asymptomatic individuals. A stratified cluster sampling approach was used to account for the stratification of individuals by village and clustering by households (primary sampling units). Probability proportional to size sampling was used in the island’s 14 villages. Households were selected by simple random selection using household census Global Positioning System coordinates. All asymptomatic household members aged 6 months and older were invited to join the study.Participants were interviewed using a standardized structured questionnaire. For participants younger than 7 years, the interview was completed with the main caregiver. Answers were recorded on tablets using Open Data Kit  and uploaded to a secure and encrypted server. Demographic information such as age, gender, education level, pregnancy status, and bednet use was recorded. Further household information was collected as guided by the Household Survey Indicators for Malaria Control and the Malaria Indicator Survey. This included questions on the level of education, material possessions, housing quality, treatment-seeking behavior, and vector control measures.Blood from each participant was obtained by finger prick sampling using a standard procedure onto Whatman 3MM filter paper and used for RDT  and point-of-care hemoglobin measurement . Approximately 20 μL of whole blood was collected onto filter paper from each participant, air dried, stored at −20°C, and shipped within a fortnight of collection to the molecular laboratory at the London School of Hygiene and Tropical Medicine, London, United Kingdom.Plasmodium species using a CareStart Malaria histidine-rich protein 2 (HRP2)/parasite lactate dehydrogenase (pLDH) (Pan) Combo RDT . The presence of HRP2 and lactose dehydrogenase in the blood identifies P. falciparum.Approximately 5 μL of whole blood was obtained from participants to detect Plasmodium falciparum species–specific qPCR was performed in duplicates, using 5 µL of samples DNA, forward (5′-GTAATT GGA ATG ATA GGA ATT TAC AAG GT-3′) and reverse (5′-TCA artemisinin-based combination therapy (ACT) ACG AAC GTT TTA ACT GCA AC-3′) primers, CYS5-FAM probe, and TaqMan™ Multiplex Master Mix . Quantitative PCR was performed in 96-well plate on the AB 7500 FAST system . The first WHO international standard for nucleic-acid amplification test (NAT) analysis was used as qPCR control in a form of a standard curve.9DNA was extracted from dried blood samples collected onto a filter paper. Blood spots were punched with a sterile hole puncher. DNA was extracted using a PureLink Pro 96 well Genomic DNA kit following the manufacturer’s instructions . t-test, Pearson’s chi-squared test, and Fisher’s exact test were used as appropriate. A generalized linear model forward univariable logistic regression was used to identify associations between P. falciparum parasitemia and questionnaire variables. Final multivariable logistic regression models were built using variables of interest identified a priori (age and education) and those which were significantly linked to the outcome in the sequential analyses to adjust for potential confounders. P-values less than 0.05 were considered statistically significant.Rapid diagnostic tests, qPCR, and survey questionnaire results were checked for completeness and analyzed in Stata v. 15 . Survey module settings were adjusted for the stratified cluster design with finite population corrections. The Shapiro–Wilk test was used as the test of normality. Student’s Ethical approval for the study was obtained from the Comite Nacional de Eticas de Saude in Guinea Bissau and from the London School of Hygiene and Tropical Medicine Ethics Committee. Written informed consent or adult caregiver consent in lieu when younger than 18 years was required. Written assent for participants aged 7–17 years was additionally required.Supplemental Table 3, respectively. The level of formal education achieved—primary, secondary, and higher—was comparable across genders ; 48.3% of households had a female person as their most educated member; such households had a similar proportion of P. falciparum cases as households in which the most educated person was male (P = 0.99). The mean hemoglobin level across all surveyed individuals was 110.6 g/dL (95% CI: 108.3–112.8). More than a quarter of participants had a normal hemoglobin level for their gender and age as defined by the WHO criteria  .10 Women were more likely to be anemic than men, according to the WHO age-adjusted criteria, with an average of 107 g/dL (95% CI: 105–109) versus 113 g/dL (95% CI: 111–117), respectively (P = 0.0006).Four hundred four individuals from 58 households took part in the survey . Charact genders , but malP. falciparum parasitemia with 25.6% compared with 14.9% for participants younger than 5 years, 18.7% for 16–30-year olds, and 15.9% for those older than 30 years (P < 0.001) .The overall estimated prevalence was 5.8% (95% CI: 3.5–9.3) by RDT (26/404) and 16.9% (95% CI: 13.1–21.7) by qPCR (75/404). Village prevalence rates ranged from 0% to 25% by RDT and 0–37% by qPCR . Partici30 years . Using qSupplemental Table 5); 99.5%  of participants reported sleeping under a mosquito net the previous night. All surveyed houses had open eaves, and none used indoor residual spraying (IRS); 15% of household heads scored three of three for questions on mosquito larva knowledge and management (95% CI: 7.7–27.1); however, this was not associated with a reduced probability of someone in the household testing positive . Regarding diagnosis and treatment, 14 children younger than 5 years were reported as having had a fever in the previous 2 weeks; 63.5% (10/14) of such children were tested either by microscopy or RDT by a local healthcare worker (95% CI: 32–95). Antimalarials were given to an estimated 56.9% (9/14) of these children (95% CI: 25.9–87.8), all first line (artemether with lumefantrine).An estimated 68.4% (20/30) of women pregnant in the previous 2 years reported having received a full course of IPTp (sulfadoxine with pyrimethamine) during their pregnancy (95% CI: 46.3–84.5)  using qPCR. The survey was completed early in the rainy season (August), and so prevalence numbers may have been lower than if assessed a few months later (November) as observed in a neighboring country with similar malaria endemicity.11 Marked differences in estimated prevalence between villages were observed from 0% to 37% by qPCR. Such differences may reflect the limited number of samples obtained in smaller villages. Local circumstances and practices may also be at play to increase parasite exposure. Some of the highest prevalence numbers were found in village populations which resettled in temporary shelters next to their crop fields during the rainy season to facilitate the harvest. Despite high LLIN usage, such migratory practices may increase parasite exposure.The primary objective of this cross-sectional survey was to obtain a rainy season estimate of the prevalence of 2 Previous studies in the 1980s and 1990s, using light microscopy as the diagnostic tool, reported prevalence estimates ranging from 21%12  to 79% in children (2–9 years of age) in northwestern Guinea-Bissau,13 and 28% in the west of country.14 A study focusing on children aged 3–6 years in the capital city of Bissau in 1994 reported a prevalence of 59%,15 and another in 1999 reported a prevalence of 26% in children aged 1–2 years16; a study of children and adults in 2008 found a prevalence of just 3% at the end of the rainy season.17 The heterogeneity of eligibility criteria, seasonal timing, and diagnostic procedure across surveys make direct comparisons of results difficult. This problem is well documented in many sub-Saharan countries and is not limited to Guinea-Bissau.18 However, the trend across these surveys suggests a stable or slightly decreasing prevalence over the past 20 years in Guinea-Bissau.Elsewhere in Guinea-Bissau, the most recent survey, conducted in the coastal area of Caio in 2009, reported a prevalence of 4% with microscopy (95% CI: 2.6–5.4) and 23.7% with qPCR (95% CI: 20.9–26.7) .P. falciparum parasitemia by qPCR. In the rural region of Caio, Satoguina et al.2 also showed that qPCR-determined parasitemia was most common in adolescence, specifically among 11–15-year-olds, and then decreased in adulthood. Malaria parasitemia and malnutrition, with anemia as a cofactor, are among the major causes of childhood morbidity and mortality in sub-Saharan Africa.20 Thorne et al.21 surveyed 872 children in a cross-sectional study of younger than 5 year olds on three islands of the Bijagos archipelago and reported that 80.2% of children were anemic in 2013. This converges with the current study which found that 79.9% of children in that age-group are anemic. The interplay among anemia, malaria, other parasitic infection such as soil-transmitted helminths, and malnutrition is complex.25 Early antibody response in children may also play a role resulting in severe malaria outcomes.26 Socioeconomic status (SES) was not found to be associated with parasitemia. Previous studies consistently associate lower SES with high infection rates.28 The ubiquity of open eaves in island houses and the absence of stark wealth inequality among households are possible mitigating factors. Education, and maternal education in particular, has been reported as being a protective factor against child malaria infection.31 In this study, however, no significant association was found among the level of household education, the gender of the most educated household member, and malaria parasitemia. This study showed that all children younger than 5 years reported having slept under a mosquito net the previous night. This compares favorably to the results of Malaria Indicator Surveys conducted in other sub-Saharan countries between 2008 and 2015.32 Of note, self-reporting of LLIN usage can be unreliable, and on-site checks for usage are preferred when possible.The secondary objective of this study was to investigate associations between demographic, nutritional, educational, and socioeconomic indicators, and malaria parasitemia on Bubaque. In this study, 6–15-year-olds were found to have the highest prevalence of 34 In this study, nearly all persons reported sleeping under a mosquito net the previous night. Under the national malaria prevention program, LLIN distributions occur every 2–3 years. The benefits of more frequent distributions on parasite prevalence are uncertain. In a 2003 cross-sectional study of the capital city of Bissau, Rodrigues et al.17 reported that bednets were used regularly by 79% of respondents and increased to 92% by 2005. By comparison, the WHO1 estimates that, in sub-Saharan African countries as a whole, just 53% of the populations at risk slept under an LLIN in 2015 (95% CI: 50–57). A recent entomology study has reported that Anopheles gambiae was the main malaria vector on Bubaque and that adult mosquitoes showed full susceptibility to permethrin but moderate resistance to α-cypermethrin.35 A moderate frequency of the West African kdr pyrethroid conferring resistance allele was also observed. Expansion of such resistance traits in the mosquito population could compromise current control methods that rely on LLINs.The efficacy of LLINs in preventing malaria infection and mortality has been well documented in sub-Saharan Africa.36 and the introduction of malaria control measures, such as good case management using both RDTs and ACT, may have contributed to decreased prevalence.37 Community-wide free distributions of LLINs, as observed before the survey, is another effective means in reducing malaria prevalence.40 Aside from IPTp, other control measures including chemoprevention and IRS have not been widely used in Bubaque or in other regions of Guinea-Bissau. As prevalence of the parasite and the size of its human reservoir of infection decrease, it becomes increasingly challenging to diagnose subclinical parasitemia to interrupt forward transmission. The need for highly sensitive molecular diagnostics is therefore ever increasing.Stable climatic conditionsP. falciparum infections were identified with a combination of RDT and qPCR. In this first survey in the Bijagos Archipelago of Guinea-Bissau, a wide disparity in burden of subclinical P. falciparum infections was found at the village level on the island of Bubaque, with a moderate prevalence overall. Such levels of subclinical parasitemia were found despite regular LLIN distributions and high reported use by participants. This suggests that novel methods may be required, in addition to the existing malaria control tool kit, to reduce and ultimately stop transmission. Further studies are needed to better understand the fine scale transmission of the parasite among villages and islands.Asymptomatic Supplemental materials


Plasmodium species  circulating in whole blood samples were used. A screening was performed in the group of 515 patients, 162 seemingly healthy children (aged 1–15) and 353 adults, all inhabiting the villages in the Dzanga Sangha region (southwest CAR) between August and September 2021. As much as 51.2% of asymptomatic children and 12.2% of adults had a positive result in malaria rapid diagnostic tests (mRDTs). Our findings demonstrated a very high prevalence of asymptomatic malaria infections in the child population. Limited access to diagnostics, treatment and prevention of malaria during the global COVID-19 pandemic and less medical assistance from developed countries may be one of the factors contributing to the increase in the prevalence of disease in Africa.According to the latest World Health Organization malaria report, 95% of 241 million global malaria cases and 96% of 627,000 malaria deaths that were recorded in 2020 occurred in Africa. Compared to 2019, 14 million more cases and 69,000 more malaria deaths were recorded, mainly because of disruptions to medical services during the COVID-19 pandemic. The aim of this study was to assess the prevalence of asymptomatic malaria cases in children and adults living in the Dzanga Sangha region in the Central African Republic (CAR) during the COVID-19 pandemic. Rapid immunochromatographic assays for the qualitative detection of  Plasmodium: Plasmodium falciparum, Plasmodium vivax, P. ovale , P. malariae and P. knowlesi (simian species found in Southeast Asia). Thanks to recent developments in molecular diagnostics, it is possible to identify further enzootic species of Plasmodium in humans: P. simium, P. cynomolgi, P. coatneyi and P. inui; however, their prevalence and clinical picture have not been conclusively established [Malaria is a vector-borne parasitic disease, which in humans, is primarily caused by one of the five species of ablished ,2.According to the latest World Health Organization (WHO) report on malaria  , there wP. falciparum is responsible for 99.7% of all malaria infections in Africa [P. falciparum [Some experts estimate that because of the effects of the ongoing COVID-19 pandemic and a possible collapse of the public health care systems in many developing countries, the rates of morbidity and mortality of malaria in Africa are likely to increase rapidly . Accordin Africa . In 2020n Africa . Howevern Africa . The Cenlciparum . In CAR,lciparum . Becauselciparum . The present study aimed to assess the prevalence of asymptomatic malaria cases in children and adults living in the Dzanga Sangha region in the Central African Republic during the COVID-19 pandemic.The study was conducted between August and September 2021 and involved a group of 515 patients, children (aged 1–15 years) and adults (aged 16–76) of both sexes (settled Bantu and semi-nomadic BaAka Pygmies) living in the Monasao village (population of approx. 4000 people) and neighbouring villages in the Dzanga Sangha region  in the southwest CAR. Adults and children were recruited in the villages, then directed to the local health care facility in Monasao, where malaria rapid diagnostic tests (mRDTs) were used . The only inclusion criteria were the absence of clinical signs and symptoms of malaria (body temperature ≤37.5 °C). The exclusion criteria were: the presence of malaria signs and symptoms, anti-malarial treatment received in the past 28 days and difficulties in the venepuncture procedure. On behalf of the paediatric patients, the consent to participate in the study was given by their parents. The parents had been informed of the study’s purpose and methods. The interviews were conducted in their native language, either in Sango (a language spoken by the Bantu) or Mbenzele (a language used by the BaAka Pygmies). Medical personnel responsible for carrying out the study procedures measured the participants’ body weight and body temperature . Next, the medical staff performed RDTs for malaria as well as haemoglobin measurements to identify any possible cases of anaemia.Plasmodium species  in whole blood samples were used. Asymptomatic malaria was defined as the presence of Plasmodium species on mRDT with no clinical signs of the disease . The mRDT sensitivity  is >98.7% and its specificity  is >99.0% (according to the manufacturer’s specifications) [Immunochromatographic mRDT  detecting TM analyzer  by examining a venous blood sample. The definition of anaemia was based on the WHO criteria [Haemoglobin level was measured using a portable DiaSpectcriteria , where awww.statsoft.com (accessed on 10 October 2021). All data were presented as mean (standard deviation), range, median and 95% confidence interval (CI). Groups of data were compared using t-Student, U Mann-Whitney or Kruskal-Wallis test. Values of p < 0.05 or lower were considered significant.Statistical analysis was carried out using StatSoft, Inc. (2014) STATISTICA  version 12.0.  The research project was approved by the Committee on Bioethics at the Military Institute of Medicine, Warsaw, Poland (Decision No. 22/WIM/2020) under the Declaration of Helsinki (1996) and the rules elaborated by the European Union “Good clinical practice for trials on medicinal products in the European Community. The rules governing medicinal products in the European Community” (1999) ratified by the Committee of Ethics in Poland (March 1993). The tests performed in the CAR were carried out with the written consent of each participant and under the supervision of priest Wojciech Lula , with considerable help from the medical staff working at the healthcare centre.p = 0.0006). The mean body weight of the children with mRDT (+) was 17.0 kg. Children with mRDT (+) had significantly lower body weight (p < 0.0001). The mean body temperature of the children with mRDT (+) as well as those with mRDT (–) was 36.6 °C. The mean Hb g% of the children with mRDT (+) was 10.4 g/dL. Children with mRDT (+) had significantly lower Hb g% (p < 0.000001).The study involved 162 children  and 353 Plasmodium infections was much lower among asymptomatic adults than it was among children . The mean Hb g% of the adults with mRDT (+) was 11.5. Adults with mRDT (+) had significantly lower Hb g% (p = 0.0036).The prevalence of Plasmodium, including P. falciparum (99.0%), P. vivax (6.3%) and Pan   . More thMalaria remains a primary health issue in Africa as it accounts for 9% of all illnesses reported from this continent . In EuroAnopheles mosquitoes [The aim of this study was to assess the prevalence of asymptomatic malaria cases in children and adults living in the Dzanga Sangha region in the Central African Republic (CAR) during the COVID-19 pandemic. The WHO has indicated that the actual rates of malaria cases in the CAR could be underestimated, and in fact, they may be several times higher than the official reports suggest. This is mainly due to the fact that a significant number of malaria cases in the region are asymptomatic . Childresquitoes . The lonsquitoes ,16. Convsquitoes .The results of this study conducted during the COVID-19 pandemic (August–September 2021) revealed that as much as 51.2% of the involved paediatric group  had asymptomatic malaria. A study conducted before the pandemic (early 2020) in the same group of children (500 children aged 1–15) identified asymptomatic cases of malaria in 35.2% of the examined group. The results demonstrate very high malaria prevalence rates in the child population living in Central Africa. Our current findings are similar to those reported by Maziarz et al.  in northPlasmodium infections was much lower among asymptomatic adults (n = 353) than it was among children. The study found that only 12.2% of adults were infected with malaria. A lower prevalence rate in this group could be associated with protective antiparasitic immunity. In areas of high malaria transmission, the frequency of clinical events and average parasite densities are likely to decrease with age in line with increasing acquired immunity [Plasmodium infections are likely to become less severe and manifest with milder signs and symptoms. They may also be asymptomatic or take the form of infection with low subpatent parasite densities below the limit of detection (50–100 parasites/µL) by mRDT, depending on the gradual development of protective immunity and different degree of antigenic diversity. Age seems to be an important factor affecting the longevity of the humoral response, as adults seem to make better long-lived antibody responses to Plasmodium antigens (antibodies protect from reinfection) than children [The present study revealed that the prevalence of immunity . Subsequchildren . Disruption in the provision of diagnosis, prevention and treatment remains a major obstacle in the fight to eliminate malaria and other tropical diseases from Africa. The same may be true for the efforts undertaken to combat the COVID-19 pandemic. The WHO vaccination strategy assumed that a minimum of 40% of each country’s population would receive full primary immunisation against SARS-CoV-2 by the end of 2021 . To datePlasmodium carriers), possible long-term positivity of HRP2-based mRDTs even after complete elimination of P. falciparum from patients with effective treatment.Limitations of the study: no information on cases of malaria among pregnant women in the study group, lack of microscopy data and PCR confirmation of malaria infections  to the COVID-19 control strategies.The current prevalence of asymptomatic malaria in children living in the southwest of the Central African Republic is very high. In contrast, the prevalence of asymptomatic malaria in adults studied at the same time and place was found to be several times lower than in the paediatric population, which might be attributable to their acquired protective immunity against


Plasmodium falciparum and Schistosoma haematobium infections on the haematological indices in SAC and the confounding influence of malnutrition on the outcomes.School-aged children (SAC) are a high-risk demographic group for infectious diseases and malnutrition. The objective of this study was to assess the burden and the effect of S. haematobium by urine filtration technique. Multiple linear regression model was used to examine influence of independent variables on haematological parameters.This cross-sectional study was conducted in SAC 4–14 years old living in Ikata, Bafia and Mile 14-Likoko in Muyuka, Cameroon. Anthropometric measures of malnutrition were obtained and blood samples collected were used for detection of malaria parasites by Giemsa-stained blood films using light microscopy and complete blood count analysis using an automated haematology analyser. Urine samples collected were used to detect micro haematuria with the aid of reagent strips and the eggs of Plasmodium or S. haematobium and co-infection with both parasites was 16.2, 16.3 and 8.3%, respectively. Overall, malaria parasite (MP), urogenital schistosomiasis, malnutrition, anaemia, haematuria, microcytosis and thrombocytopenia was prevalent in 24.4, 24.6, 25.9, 74.4, 12.2, 45.4 and 11.1% of SAC, respectively. A significant linear decline (P = 0.023) in prevalence of P. falciparum infection with the severity of stunting was observed. Factors that significantly influenced haematological parameters included haemoglobin: age, stunting and MP; haematocrit: age and MP; white blood cell count: age; red blood cell count; age and MP; lymphocyte counts: stunting; mean cell volume: age; mean cell haemoglobin: age and stunting; mean cell haemoglobin concentration: sex, stunting and red cell distribution width-coefficient of variation: sex, age and stunting.Out of the 606 SAC examined, the prevalence of single infections with Plasmodium and S. haematobium infections are common while anaemia is a severe public health problem in Muyuka, Cameroon. The interaction between haematological parameters with malaria parasites as well as linear growth index was negative and other interactions indicate systemic inflammation. While findings provide contextual intervention targets to ensure the judicious use of the limited resources, there is need for regular monitoring and proper treatment to improve the health of the underserved population.Malnutrition, The online version contains supplementary material available at 10.1186/s12879-021-06201-9. Plasmodium falciparum and urogenital schistosomiasis (UGS) caused by the trematode helminth Schistosoma haematobium impose tremendous public health burdens in tropical and subtropical countries. Both diseases have been associated with poverty and factors such as low socio-economic status, poor sanitation, limited access to safe water, poor education and poor awareness, which also play a key role in their transmission  × 100, where n0 = anaemic children without malaria/UGS/stunting and n1 = anaemic children with malaria/UGS/stunting, whereby n0 + n1 = n, m0 = non anaemic children without malaria/UGS/stunting, and m1 = non anaemic children with malaria/UGS/stunting, whereby m0 + m1 = m. Geometric means were computed for those positive only and the log transformed counts were used in the analysis. Geometric mean parasite density (GMPD) of P. falciparum and geometric mean egg count (GMEC) of S. haematobium by age, sex and nutritional status and severity were compared by the Student’s t-test, and Mann Whitney U test where appropriate and the mean haematological parameters were compared by analysis of variance (ANOVA). Potential confounders of haematological values to be entered into a multiple linear regression (MLR) model were identified after exploratory analysis. Any potential confounder with a moderate (P < 0.2) relation with both the dependent variable and the confounder of interest was included in the later MLR models. The 95% confidence interval (CI) was reported and P-values < 0.05 were considered suggestive of statistical significance. All data was analysed using IBM-Statistical Package for Social Science (SPSS) version 21 .Descriptive measures such as the mean and standard deviation (SD), geometric means, frequencies, and proportions were used to summarize data. Differences in proportions between populations were compared using Chi  principles as stated in the Declaration of Helsinki. The population was sensitized in their various communities at the beginning of the study. Written informed consent was obtained from all parents/caregivers whose child/children participated in the study after explaining the purpose and benefits of their participation. Participation was totally voluntary, and a participant could decide to halt their participation in the study at any time without any penalty. Participants who had malaria and or urogenital schistosomiasis were given first line treatment as recommended by the national treatment guideline policy for uncomplicated malaria and helminths.The characteristics of the 606 SAC examined is presented in Table P. falciparum or S. haematobium and co-infection with both parasites was 16.2, 16.3 and 8.3%, respectively, with no significant variation with sex and age. Significantly higher  prevalence of single infection with S. haematobium (25.9%) and co-infection with P. falciparum and S. haematobium (13.4%) were observed in SAC from the Likoko locality while single P. falciparum infection was observed in Ikata (20.9%), respectively. The higher prevalence observed in the anaemics was not statistically significant when compared with their counterparts as shown in Table P. falciparum and S. haematobium was lower in children who presented with fever contrary to the significantly higher (P = 0.008) prevalence of co-infection observed in those with fever (13.8%). With regard to malnutrition and its forms, the prevalence of S. haematobium and co-infection was comparable among the different groups unlike that of P. falciparum. The prevalence of P. falciparum was significantly lower  in those malnourished (10.2%) or the stunted (8.6%) than those normal, respectively  in prevalence of P. falciparum infection with the severity of stunting with those normal having the highest prevalence (18.4%) while those with severe stunting had the least (6.5%). No significant trend in the prevalence of S. haematobium and co-infection was observed with the severity of stunting even though the prevalence was lowest in those with severe stunting  as revealed in Fig. Observation from the study demonstrated a significant linear decline  prevalence of moderate and severe stunting, respectively, as shown in Fig. The prevalence of moderate and severe stunting was 17.7 and 5.1%, respectively. Males and children 10–14 years old had significantly higher  than males (20 eggs/10 mL of urine) as shown in Table P. falciparum, the majority  had low parasite density. As shown in Fig. P. falciparum density was 12.2, 10.6 and 1.7%, respectively and this was lower in malnourished than well-nourished children. In addition, the prevalence of low and moderate P. falciparum density decreased with the severity of stunting. With respect to S. haematobium, the prevalence of low and high egg density was respectively, 16.3 and 8.3% and, a comparison with the nutritional status showed no significant differences with the severity of stunting  in children with co-infection (82.0%) when compared with those with single infection and those negative for both infections. Mild, moderate and severe anaemia was prevalent in 17.3, 54.8 and 2.1% of the population, respectively. Although no significant difference  in the severity of anaemia was observed, moderate anaemia was most common in children with co-infection (64%) when compared with their counterparts as shown in Fig. Anaemia was common in the study population 74.4%) even in SAC negative for both infections (70.2%). The prevalence of anaemia was significantly higher  mean Hb (10.5 (1.2) g/dL) concentration and mean RBC (4.1 (4.4) × 1012/L) counts in children infected with P. falciparum when compared with those negative, respectively , stunting (P = 0.004) and MP status (P = 0.013); Hct: age (P < 0.001) and MP status (P = 0.038); WBC: age (P = 0.034); RBC: age (P = 0.001) and MP status (P = 0.034); lymphocyte counts: stunting (P = 0.015); MCV: age (P < 0.001); MCH: age (P < 0.001) and stunting (P = 0.001); MCHC: sex (P = 0.046) and stunting (P = 0.004) and RDW-CV: sex (P < 0.001), age (P = 0.001) and stunting (P = 0.023) as shown in Additional file A multiple linear regression analysis involving the 606 participants, with the different haematological variables as the dependent variable and sex, age, HAZ (stunting index), MP status, UGS status and co infection as independent variables, revealed that co-infection did not significantly influence any haematological parameter  is lower than the 33.9% observed in a simultaneous study in SAC in non-schistosomiasis endemic area within the same Muyuka Health District [Plasmodium infection when compared with non-endemic foci warrants further investigation.Findings from the study revealed similar prevalence of both  earlier , 14. HowDistrict . These fDistrict  and NortDistrict . While tDistrict . NonethePlasmodium and S. haematobium with age. On the other hand, UGS was more common in females with haematuria being a common manifestation consistent with other studies [Plasmodium and S. haematobium infections [Observations from the study revealed no significant differences in the prevalence of both  studies , 34. Thi studies  in Niger studies  in Camer studies  in Nigerfections , 3.Plasmodium and S. haematobium co-infection in SAC (8.3%) is like that reported in Gabon [S. haematobium endemic areas due to the presence of an efficient Bulinus snail vector and the use of untreated waterways for domestic, farming and recreational use. Risk of co- infection with Plasmodium in these areas is also high as these areas are part of the holoendemic stratum with high and perennial malaria parasite transmission [The prevalence of in Gabon , higher in Gabon  and lowein Gabon . Ikata, smission . FurtherPlasmodium infection and higher GMPD/ μL of blood in those malnourished or stunted with significant linear decline in infections with the severity of stunting. Similarly, the lowest prevalence of S. haematobium and co-infections and higher GMEC was observed in those with severe stunting. Despite the previous work by Olney et al. [S. haematobium infection [Malnutrition prevalence, underweight and stunting varied significantly with sex and age with significantly higher occurrence of moderate and severe stunting in males and children 10–14 years. The prevalence of stunting is similar to that observed in SAC in Ghana , but higy et al. , which ry et al.  reportednfection . Whilst P. falciparum infection include haematological alterations of which anaemia is the most common [S. haematobium and co-infections. The perpetual presence of anaemia in apparently healthy SAC negative for both infections with significantly higher occurrence in children 4–9 years old and those with co-infections necessitates caveat in attributing the causality of anaemia to these factors. Although the high prevalence of anaemia in those 4–9 years old could be linked to the high parasite densities of both P. falciparum and S. haematobium and microcytosis observed, the attributable risk of anaemia associated with these infections in the population remains low. Notwithstanding, findings from the multivariate analysis asserts a negative interaction between haemoglobin level with malaria parasites and S. haematobium as well as the association between haemoglobin and linear growth index.The hallmarks of t common , 50 unliP. falciparum and S. haematobium was very high. This prevalence is similar with those in children in Ethiopia [While anaemia is one of the markers of morbidity associated with schistosomiasis even though it may be masked by anaemia resulting from malaria , the preEthiopia  with modEthiopia . NeverthEthiopia , 54 whilEthiopia .P. falciparum or S. haematobium or co-infection and those not infected. Infections with P. falciparum however, significantly lowered the Hb concentration and mean RBC counts. This effect is further bolstered by the significant association of P. falciparum with red cell indices including Hb, RBC and Hct in the multiple regression analysis. In line with findings by Kotepui et al. [P. falciparum infection with these red cell indices is not atypical and may be related to the enhanced destruction of infected and uninfected erythrocytes combined with decreased erythrocyte production leading to malaria related anaemia [Findings from the study revealed that the mean Hct, WBC, lymphocyte and platelet counts, MCV, MCH, MCHC and RDW-CV were comparable amongst those infected with i et al. , 56, the anaemia , 58.Observations from the study highlighted age as a significant factor in most of the haematological parameters evaluated while sex was found to be associated with MCHC and RDW-CV. Since haematological parameters are interrelated with each other as well as with sex and age, of significance is the intricate positive interaction between the anthropometric malnutrition proxies of height-for-age and haematological parameters including haemoglobin, lymphocyte, MCH, MCHC and RDW-CV. As noted in previous studies , 60, theSchistosoma mansoni while, microcytosis and thrombocytopenia is common in individuals with malaria [S. haematobium is, however, uncertain. Although the context of interpretation is limited to the few cases of thrombocytopenia observed, findings from the study demonstrated no statistically significant association between platelet counts and parasite densities. It is worthy to note that children in the 4–9 years old age group had the highest parasite densities of both Plasmodium and S. haematobium. On the other hand, the high prevalence of microcytosis in the population may be attributed to the high prevalence of anaemia observed which is partly accounted for by the presence of the parasitic infections and chronic nutritional deficiencies.Other haematological abnormalities of significance observed in the study population included thrombocytopenia, and microcytosis which were higher in SAC 4–9 years old. Furthermore, a higher occurrence of microcytosis was observed in males than females. Thrombocytopenia is commonly found in individuals living in areas endemic for  malaria –67. Its The findings of the study should be viewed in the context of the limitations of the cross-sectional nature of the design which did not provide the changes in the burden and morbidities following control measures that could have been captured in a longitudinal study. Hence, there is a need for further investigations to ascertain the causal relationship between enhanced control interventions and changes in demographic as well as clinical characteristics and burden in this at-risk group to formulate more appropriate public health interventions.Plasmodium falciparum and S. haematobium infections are of public health concern while, malnutrition, microcytosis and thrombocytopenia are common, and anaemia is a severe public health problem in Muyuka, Cameroon. The interaction between haematological parameters with malaria parasites as well as linear growth index was negative even though the attributable risk of anaemia associated with these conditions remains low in the population. Other positive interactions between the linear growth index and haematological parameters indicate the occurrence of chronic malnutrition in addition to anaemia as well systemic inflammation. While findings provide contextual intervention targets to ensure the judicious use of the limited resources there is need for regular monitoring and proper treatment to improve the health of the underserved population.Additional file 1. Multiple linear regression analysis examining the influence of independent variables on some haematological parameters. Although not statistically significant UGS had a negative influence on Hb, Hct, WBC, RBC and lymphocyte counts, MCV, MCH and MCHC.


Globally, approximately 15% of all babies are born with low birth weight (< 2.5 kg) and ≥ 90% of them are born in low- and middle-income countries. Malaria infection in pregnancy remains a public health concern as it can affect both the mother and the newborn. Notably, it increases the risk of newborns with low birth weight. The World Health Organization (WHO) recommends intermittent preventive treatment with ≥ 3 doses of sulfadoxine-pyrimethamine (SP) during pregnancy in areas with moderate to high malaria transmission in Africa. The aim of this topical review is to give an overview of the impact of malaria infection during pregnancy on low birth weight, with focus on East Africa where malaria is endemic.Eleven studies were selected according to a predefined set of criteria.Three studies showed a significant reduction in the prevalence of low birth weight with intermittent preventive treatment with SP, whereas four studies found no significant impact of such treatment on low birth weight. The number of SP doses and compliance to this treatment may in part explain these discrepancies. Pregnant women with frequent symptomatic malaria infection had significantly higher risk of placental malaria.The WHO recommendation of ≥ 3 doses of intermittent preventive treatment with SP during pregnancy seem effective in preventing low birth weight, but treatment compliance is a challenge. Malaria prophylaxis is important during pregnancy, especially in endemic areas of malaria, such as East Africa. Plasmodium falciparum) during pregnancy remains a major public health problem, especially in sub-Saharan Africa [Malaria in pregnancy affects more than 25 million pregnant women every year, both in high and low malaria-endemic areas . Pregnann Africa , 4.. [There are several adverse outcomes associated with malaria, which affect both the mother and the newborn, including stillbirth, preterm birth, maternal and neonatal mortality, congenital malaria, maternal anaemia and low birth weight  . Thirty-.  estimate. .According to a systematic review from 2019, 20.5 million infants worldwide were born with LBW, which represents 14.6% of all births. Of these LBW infants, about 90% were born in low- and middle-income countries (LMICs), and in sub-Saharan Africa the prevalence of LBW was 24% [P. falciparum tends to sequester on the placenta and thus the density of blood parasites available is below the detection limit of microscopy and, therefore, can only be detected by sensitive molecular tools, thus being a submicroscopic parasitaemia [In pregnancy, sitaemia , 9. Placsitaemia .There is a paucity in compiling data from studies of malaria and LBW in East Africa, a part of sub-Saharan Africa endemic for falciparum malaria. Such information might be important, not only for the pregnant woman herself and the antenatal care staff, but also for other stake-holders and policy-makers. To address this knowledge gap, provide a topical review is here provided of the relationship between malaria during pregnancy and LBW with focus on East-Africa.In this topical review a list of relevant MeSH words was generated and used to search for relevant articles from the database PubMed. The search was performed on 25 January, 2021. The search covered research conducted in East African countries, which according to PubMed consists of Burundi, Djibouti, Eritrea, Ethiopia, Kenya, Rwanda, Somalia, South Sudan, Sudan, Tanzania and Uganda. The search covered peer-reviewed research published between January 2000 and January 2021, and the articles from PubMed were imported into an EndNote library. Search terms included “Infant, Low Birth Weight”, “Malaria”, and “Africa, Eastern”. Studies were included in this review if they focused on (i) infants with LBW; (ii) malaria infection in pregnancy; (iii) were conducted in East Africa, and (iv) included an abstract and the free full text in English was available. Reviews or meta-analysis were not included in the selected literature-search, however we included such reports if relevant in our discussion. The inclusion of studies were not restricted to any particular study design, hence e.g. randomized trials and observational studies were eligible for inclusion. Studies that did not examine associations between infants with LBW and malaria infection during pregnancy in East Africa, were excluded. Studies that did not have LBW as a primary outcome were also excluded.The literature search initially retrieved 42 articles Fig. . Taking Table . [Braun et al.  did a cr. [In a non-randomized community trial in Uganda, Mbonye et al.  investig. [Mikomangwa et al.  did a fa. [Mosha et al.  investig. [In a hospital-based cross-sectional study in Rufiji, Tanzania, Ndeserua et al.  investig. [Ndyomugyenyi et al.  investig. [In a hospital-based study in Western Kenya, Van Eijk et al.  investigTable . [Kalinjuma et al.  did a co. [P. falciparum infection and LBW in Central Sudan, an area with unstable malaria transmission. Cases were women who had LBW deliveries and the controls were women without LBW deliveries. The prevalence of submicroscopic malaria infection was higher in the cases compared with the controls: 27.6% versus 7% (p < 0.001). By using multivariate analysis, this study showed that malaria infection in the placenta was not significantly associated with LBW, while submicroscopic P. falciparum infection (p = 0.001) or a combination of submicroscopic infections and histologically determined infections (p = 0.012), were significantly associated with LBW.A case–control study by Mohammed et al.  investigKapisi et al.  did a co. [Dong et al.  investigP. falciparum and measures of placental malaria [Eleven original articles comprising 14,148 pregnant women were included in this topical review. Notably, with the current stringent search strategy, studies from only four of the 11 countries located in East Africa were retrieved. Seven studies investigated the effect of IPTp-SP , whereas malaria . Only th malaria , 13, 17.. [P. falciparum infection may [Kapisi et al.  divided . , 18, 19.. . In conttion may , 19. How. [. [. [Most studies in this review investigated the effect of one to three doses of IPTp-SP –17. A sy.  investig. . Mikoman. [.  found th. [. [. .P. falciparum SP resistance on the effectiveness for malaria in pregnancy in Africa [A systematic review and meta-analysis from 2015 of randomized and quasi-randomized trials investigated anti-malarial drugs for preventing malaria during pregnancy and the risk of LBW . This ren Africa . The resn Africa .. [The study from Central Sudan found that among women who had LBW deliveries, the prevalence of submicroscopic malaria infection was significantly higher compared with the controls . A limit.  could noP.falciparum over the past decade, especially in Africa. A review by Rijken et al. [Plasmodium vivax, but prevention of malaria during pregnancy is not emphasized in national guidelines in the Asia–Pacific region as it is in sub-Saharan Africa. In line with this, the WHO recommendation for interventions for the control of malaria during pregnancy are mostly based on findings from sub-Saharan Africa. Based on this, several studies should examine malaria during pregnancy and make strategies to prevent malaria infection during the pregnancy for the Asia–Pacific region as well.There has been a significant progress in reducing the prevalence of n et al.  investigThe aim was to summarize data collected during the past 20 years (i.e. in the period after the WHO recommended to use IPTp-SP) on the relationship between pregnancy and malaria with focus on LBW infants in Eastern Africa in the form of a topical review to provide and facilitate such information to relevant stake-holders . There are some limitation to this approach, e.g. there are several challenges in measuring the prevalence of LBW because many infants in LMICs are not weighed at birth, some are misclassified and the quality of data-reporting varies . In addiThis topical review reports that among pregnant women in sub-Saharan Africa, three or more doses IPTp-SP were associated with a decreased risk of LBW and an increased birth weight compared to the standard 2-dose regimen. These studies provide support for the current WHO recommendation to provide at least three doses of IPTp-SP during pregnancy. Some studies concluded that SP may no longer protect against the risk of LBW in those areas of high-level resistance in Africa. Future studies should thus monitor the SP-resistance to guide the use of IPTp-SP. Women who did not use mosquito prevention methods such as bed nets had higher risk of submicroscopic placental malaria. Based on these results, one should continue to advice pregnant women in East Africa  should use bed nets or other mosquito prevention methods to decrease the risk of submicroscopic malaria.


Compared with individuals receiving artemether-lumefantrine, those receiving artesunate-amodiaquine had greater odds of being in the high-incidence group , while individuals receiving dihydroartemisinin-piperaquine had a lower odds of being in high incidence group . Individuals residing in the forested areas of Sokourani and Karamogobougou had significantly greater odds of being in the high-incidence group compared with individuals residing in the semi-urban area of Bougoula-Hameau 1 . This study highlights the importance of fine-mapping malaria risks even at sub-district levels for targeted and customized interventions.Throughout a phase IIIb/IV efficacy study of repeated treatment with four artemisinin-based combination therapies, significant heterogeneity was found in the number of clinical episodes experienced by individuals during the 2-year follow-up. Several factors, including host, parasite, and environmental factors, may contribute to the differential malaria incidence. We aimed to identify risk factors of malaria incidence in the context of a longitudinal study of the efficacy of different artemisinin-based combination therapy regimens in Bougoula-Hameau, a high-transmission setting in Mali. Risk factors including age, residence, and treatment regimen were compared among individuals experiencing eight or more clinical episodes of malaria (“high-incidence group”) and individuals experiencing up to three clinical episodes (“low-incidence group”). Consistent with the known association between age and malaria risk in high-transmission settings, individuals in the high incidence group were significantly younger than individuals in the low-risk group (mean age, 7.0 years versus 10.6 years, respectively;  Dihydroartemisinin–piperaquine is an alternative, safe, and efficacious first-line artemisinin-based combination therapy (ACT) in Mali.,pfmdr1 and pfcrt parasite genotypes predominated , the combination of artesunate–amodiaquine provided an approximate 2-fold longer protective effect than artemether–lumefantrine.pfmdr1 86Y and pfcrt 76T mutant parasites > 80%), artemether–lumefantrine provided up to a 1.5-fold longer post-treatment protective effect than artesunate–amodiaquine.Throughout the initial clinical and parasitological evaluation of the effect of repetitive administration of four ACTs on the incidence density of malaria during a 2-year follow-up  would differ according to host demographic factors , residence, and ACT received during the study.Plasmodium falciparum is hyperendemic with seasonal peaks during the rainy season. Parasitemia prevalence ranges from 40% to 50% during the dry season (January–May) and 70% to 85% during the rainy season (June–October).This study was conducted in Bougoula-Hameau, a peri-urban center of approximately 7,000 people located near the city of Sikasso in southern Mali. Bougoula-Hameau and the surrounding villages are located in the Sudanian zone dominated by savanna woodland, with tall grass dotted with trees. The climate is affected by the humid forest zone, with a rainy season of up to 6 months or more. The predominant ethnic groups are Senoufo, Samogo, Mossi, Fulani, and Bambara. The area is rural, with agriculture being the main economic activity. The region receives more rain  than any other Malian region, and is known for its fruit and vegetables. This was a secondary analysis of data collected during a phase IIIb/IV clinical trial of ACTs conducted by the WANECAM studyBased on our previous report,2 tests were used to evaluate unadjusted associations between categorical variables  and malaria incidence groups, as defined earlier. t-Tests were used to compare unadjusted associations between age (treated as a continuous variable) and malaria incidence. Logistic regression was used to estimate odds ratios (ORs) and 95% CIs of the effect of each variable of interest on malaria incidence classification while adjusting for other variables in the model. Statistical analysis was performed using SAS version 9.4 .χOf the 1,168 patients enrolled in this study, 68 experienced at eight or more episodes of malaria, whereas 602 patients experienced three or fewer episodes of malaria during the 2 years of follow-up, for a total of 670 patients included in the analysis.t-test, P < 0.0001). In addition, the distribution of location of residence and treatment arm was significantly different between the susceptible and resistant groups . There was no significant difference in the distribution of gender between the two incidence groups , so this variable was not included in the final multivariable logistic regression models.Unadjusted associations between malaria incidence and potential risk factors are presented in Table P = 0.0004). Individuals residing in the forested areas of Sokourani and Karamogobougou had significantly greater odds of being at high incidence compared with individuals residing in Bougoula-Hameau 1 . In addition, individuals receiving artesunate–amodiaquine during the study had a greater odds of being in the high-incidence group compared with individuals receiving artemether–lumefantrine , whereas individuals receiving dihydroartemisinin–piperaquine had lower odds of being in the high-incidence group compared with individuals receiving artemether–lumefantrine . Individuals treated with artesunate–pyronaridine did not show a significant difference in incidence compared with those treated with artemether–lumefantrine  during the 2 years of repeated administration of the same ACT.After adjusting for all other variables in the model , experiencing eight or more malaria clinical episodes during the 2-year follow-up, and 602 individuals who had a lower incidence , experiencing three or fewer malaria clinical episodes during the 2-year follow-up. Consistent with the known association between age and malaria risk in high-transmission settings,In our study, we observed that individuals residing in the two villages in a forested area near a permanent river were significantly more likely to be in the high-incidence group, despite these villages being within 5 to 7 km of the reference village and major population center at Bougoula-Hameau 1, because they have greater exposure. This observation highlights the heterogeneity of malaria transmission, even across small geographic areas, and the impact of microclimate differences , on the level of malaria transmission. Spatial heterogeneity in malaria risk was also observed farther north in Bandiagara, Mali, where Coulibaly et al.,The association between age and malaria risk in high-transmission settings such as Mali has been well documentedPlasmodium falciparum malaria).pfmdr1 86Y and 1246Y mutations and the pfcrt 76T mutation (associated with chloroquine resistance) have been shown to result in reduced sensitivity to amodiaquine, but increased sensitivity to lumefantrine.pfmdr1 and pfcrt mutations in the parasite population, as noted in our previous work, conducted in a similar and very close area of our study sites, with 68.1% (n = 191) and 75.3% (n = 85) of pfcrt 76T mutant alleles in 2012 and 2014, respectively, whereas the pfmdr1 86Y mutant allele frequency was 26.7% (n = 191) and 15.3% (n = 85) in 2012 and 2014, respectively.n = 60) and 18.3% (n = 60) of the pfcrt 76T and pfmdr1 86Y mutant alleles, respectively, in 2016.In our study, individuals receiving artesunate–amodiaquine had significantly greater odds of being in the high-incidence group compared with individuals receiving artemether–lumefantrine. Amodiaquine is metabolized primarily in the liver by CYP2C8 to its biologically active metabolite desethylamodiaquine,,P. falciparum infections carrying plasmepsin 2 duplications associated with piperaquine resistance.,Plasmodium spp. malaria incidence after repeated treatment.In contrast to artesunate–amodiaquine, in our study, individuals receiving dihydroartemisinin–piperaquine for the 2 years were less likely to be classified as high incidence compared with individuals receiving artemether–lumefantrine. Dihydroartemisinin–piperaquine efficacy in sub-Saharan Africa has been shown to be similar to that of artemether–lumefantrine.Individuals receiving artesunate–pyronaridine during the 2 years of repeated treatment did not show a significant difference in the odds of being classified as high incidence compared with those receiving artemether–lumefantrine, despite the half-life of pyronaridine being longer than that of lumefantrine, suggesting the impact of other post-treatment protective factors such as human genetic polymorphism or parasite genetic determinants.pfmdr1 and pfcrt), as the distribution of these mutations may affect the efficacy of ACT partner drugs being used in Mali and other high-burden malaria endemic areas. Our results identify a need for evaluating other human genetic factors that may influence the risk for malaria, such as hemoglobinopathies, erythrocyte surface proteins, and immunogenetic factors. Fine-mapping malaria risks, even at sub-district levels, may be required for targeted and customized interventions.Our study demonstrated that host factors , environmental factors , and treatment policies  can affect an individual’s risk of malaria. The data also highlight the importance of continued molecular monitoring of mutations within genes that have been shown to modulate susceptibility to multiple drugs (e.g., Supplemental materials


The R (v.4.0.2) software was used for analyses. Results: A total of 1126 participants consented for the study in the four sites. The diagnostic accuracy of the CareStart Pf mRDT was 0.93.6% (0.911–0.961) in Yaoundé, 0.930% (0.90–0.960) in Ngounso, 0.84% (0.794–0.891) in St Vincent Catholic Hospital Dschang and 0.407 (0.345–0.468) in Dschang district hospital. For SD Bioline Pf/PAN the accuracy was 0.759 (0.738–0.846) for St Vincent Catholic Hospital Dschang and 0.426 (0.372–0.496) for the Dschang district hospital. The accuracy was slightly lower in each case but not statistically different when PCR was considered as the reference. The likelihood ratios of the positive and negative tests were high in the high transmission settings of Yaoundé (10.99 (6.24–19.35)) and Ngounso (14.40 (7.89–26.28)) compared to the low transmission settings of Dschang (0.71 (0.37–1.37)) and St Vincent Catholic hospital (7.37 (4.32−12.59)). There was a high degree of agreement between the tests in Yaoundé (Cohen’s Kappa: 0.85 ± 0.05 (0.7–0.95)) and Ngounso ) and moderate agreement in St Vincent hospital Dschang (k: 0.58 ± 0.06 (0.44–0.71)) and poor agreement in the District Hospital Dschang (Cohen’s Kappa: −0.11 ± 0.05 (−0.21–0.01)). The diagnostic indicators of the SD Bioline Pf/PAN were slightly better than for CareStart Pf mRDT in St Vincent Catholic hospital Dschang, irrespective of the reference test. Conclusions: Publicly procured malaria rapid diagnostic tests in Cameroon have maintained high accuracy (91–94%) in the clinical diagnosis of malaria in high malaria transmission regions of Cameroon, although they failed to reach WHO standards. We observed an exception in the low transmission region of Dschang, West region, where the accuracy tended to be lower and variable between facilities located in this town. These results underscore the importance of the routine monitoring of the quality and performance of malaria RDTs in diverse settings in malaria endemic areas.Background: There was an increase in the number of malaria cases in Cameroon in 2018 that could reflect changes in provider practice, despite effective interventions. In this study, we assessed the diagnostic performance of two malaria rapid diagnostic tests (mRDTs) for diagnostic confirmation of suspected cases of malaria in public and private health facilities in two malaria transmission settings in Cameroon. Methods: We evaluated the diagnostic performance of CareStart pf and SD Bioline Pf/PAN mRDT and compared these parameters by RDT type and transmission setting. Nested PCR and blood film microscopy were used as references. The chi square test was used for independent sample comparisons, while the McNemar’s test was used to test for the dependence of categorical data in paired sample testing. A  Most RDTs in the market are of the lateral flow format and may detect HRP-II only (Pf mRDT) or both HRP II and LDH combined in the same format (Pf/PAN mRDT). The LDH is produced by all common species of the malaria parasite. In the combined RDT format, monoclonal antibodies of the isoforms of each of the common species of the malaria parasite are used on the test strip. Therefore, Pf/PAN mRDTs detect both P. falciparum and other species (Pf/PAN). Cameroon adopted the use of RDTs ten years ago, and despite significant gains that have been associated with the implementation of this policy, several challenges have emerged. First, there is a perception among health care providers that mRDTs are not accurate in the diagnosis of malaria among suspected cases. Indeed, one study in Yaoundé, Cameroon noted a low sensitivity of HRP−2 based mRDTs when parasite density is low [P. falciparum histidine rich protein antigen in rapid malaria diagnostic tests as well as factors associated with mRDT transportation, storage, and adherence [Malaria remains a daunting public health challenge worldwide . Despitene 2021) . Other bne 2021) . The maly is low , althougy is low . Care pry is low ,8. Mfuh y is low , noted ty is low ,11,12. Odherence ,11,13,14The protocol of this study, associated informed consent forms, questionnaires, participant education and recruitment materials, and other study forms, were submitted to the IRB of the Cameroon Baptist Convention Health Services (CBCHS-IRB) with respect to ethical and scientific compliance with applicable research and human subject regulations. Written informed consent was be obtained for each study participant. An explanation of study procedures and implications of study participation (risks/benefits) including potential use of study information for scholarly publication was provided. Consent was obtained after basic eligibility had been established. Participants were provided with adequate time to consider the information provided about the study and to ask additional questions before deciding to participate. For non-mature minors, the study details were explained to at least one parent/guardian for parental permission and then to the minor for assent. For infants, the study details were explained to at least one parent/guardian for parental permission. Mature minors who were able to consent for themselves (students), were allowed to provide self-consent, but they were advised to speak with and involve their parents/guardians or other adults in the study participation process if they felt it was safe to do so. All participant data were stored securely and were only released after permission from study participants. An ethical clearance (reference IRB2019-40) was provided by the CBCHS-IRB before the study started. The study sites include four hospitals from two regions in Cameroon. The sites are described and listed below . Yaoundé: In Yaoundé, the study took place in the town of Mfou, situated 30 min by car from Yaoundé. This rural setting is located at 4°27′ N and 11°38′ E, in the Centre region of Cameroon. The climate is typically equatorial with two discontinuous dry and wet seasons. The annual average rainfall measures 1727 mm with an average temperature of 23 °C . Average annual temperature and rainfall are 27 °C and 539 mm, respectively. Ngounso: Located 2 km from the small town of Magba in the Noun division in the West region of Cameroon. It is situated at 5°57′ N and 11°13′ E. The climate is equatorial, characterised by a tropical transition of the Sudano-Guinean type, generally with a long rainy season from mid-March to mid-November and a short dry season from mid-November to mid-March . It is characterised by a rainy season extending from mid-March to mid-November and a dry season extending from mid-November to mid-March. This town forms part of the major western highlands of Cameroon. Dschang: Dschang is situated in the Menoua division of the West region of Cameroon. Its geographical coordinates are 5°27′ N and 10°4′ E. It has an equatorial monsoon climate with alternate mountains and valleys determined by the altitude averaging 1400 m  compared to Menoua (Dschang). The sites chosen represented the major transmission blocks in the southern part of Cameroon. Additionally, Dschang was selected because it falls within a low transmission transect in the Western Highlands of Cameroon and semi-urban with respect to Yaoundé. Ngounso is a rural locality with seasonal malaria transmission and situated at a lower altitude compared to Dschang.This diagnostic accuracy study was a prospective cross-sectional study in which consenting volunteers suspected of malaria were consecutively enrolled to test for malaria infection using the mRDTs, microscopy and PCR. α/2 is the critical value of the normal distribution at α/2 where α = 0.05 for a confidence interval at 95%, p = estimated sensitivity = 90% and d = precision = 5%. We, thus, estimated 138 malaria infected participants. We further adjusted for non-response rate (10%) as well as for those who may not fulfil the inclusion criteria (10%) among eligible patients. This adjusted the sample size to a minimum of 166 malaria positive patients. Given that the prevalence of malaria using microscopy obtained from health facility data was 60% in the same period (April) in 2018, we projected to enrol a minimum of 266 suspected cases of malaria in each of the study areas. Eligibility: All suspected cases of malaria infection, as defined by the national guideline for malaria case management, from individuals who provided written consent were enrolled into the study. In Cameroon, a suspected case is any individual who reports to the health facility with fever (axillary temperature > 37.5 °C or history of fever with 24 h) and any or more of the following symptoms: lethargic, chills, nausea, joint ache, general body weakness. Participants were identified among all those who consulted clinical staff and were sent to the laboratory for a malaria test. The clinical staff had no role in the study and consent to study procedures was obtained in the laboratory environment by a research assistant. The facilities were both outpatients and inpatients and were recruited in SU-Yaoundé, Dschang and CBCHS-IHC Ngounso between March 2019 and July 2020. This period corresponds to two rainy seasons in the locations and, therefore, periods of moderate to high malaria transmission. The study was interrupted during the dry season between December and February. Participants were sampled consecutively and administered consent and/or assent. Those who accepted were enrolled and then tested for malaria. The sample size for this study was determined using the following formula and considerations. We considered that the Pf test should be at least 90% sensitive in detecting malaria infection among suspected cases. We choose a sample size n, such that the confidence interval is maintained within 5% in an infinite population. By using the following formula:Index testing: Each participant agreed to provide about 0.5 mL of blood from finger prick, of which 200 µL was used for dried blood spots, 5 µL for each of two types of mRDTs and 20 µL for slide microscopy. The rest was spotted on a Whatman N0 3 filter paper for PCR testing.Plasmodium. falciparum, which is the major parasite species in most malaria infections in Cameroon.CareStart™ malaria Pf (HRP2) Ag RDT . This mRDT detects parasite histidine rich protein (HRP-2), specifically secreted by SD Bioline Pf/PAN mRDT . This is a combination test that detects two proteins viz. HRP-2 and Plasmodium lactate dehydrogenase (pLDH), which is shared by all known Plasmodium species in malaria infections.Two mRDTs were used for this assessment.Both types of lateral flow immunochromatographic tests rely on the capture of dye labelled antigen–antibody complex by a fixed monoclonal antibody to produce a visible line on a strip of nitrocellulose inside a cassette . The bloBoth mRDTs are routinely used in health facilities in Cameroon for malaria diagnosis. The Pf mRDT is recommended for use by community health workers to detect malaria infections among suspected cases in the community while the Pf/PAN mRDT is used in health centres and hospitals for parasitological confirmation of malaria among suspected cases. Prior to the deployment of the test system in Cameroon, community health workers as well as laboratory technicians and other health workers at emergency units were trained on the general protocol for the testing and interpreting of mRDTs test outcomes. The retraining of study technicians was performed before study implementation. We compared the diagnostic performance of different tests separately using the standard Giemsa-stained microscopy or PCR as reference (for Yaoundé and Ngounso). This molecular test was additionally chosen because it has documented high sensitivity and specificity in detecting sub-microscopic parasitaemia compared to microscopy, although it is not used routinely. We further reasoned with reducing parasite rates, in the near future, the PCR test may become a useful reference when the National Malaria Control Program re-orientates its strategy towards pre-elimination. Therefore, it was important to set a baseline.For microscopy, slides were prepared and read by a trained technician in each site. Approximately 6 µL of blood was placed on a clean grease-free slide, about 1 cm from the frosted end and a circle of about 1 cm in diameter of thick film was made after defribrinating the blood through spreading anti-clockwise for about two min. The slide was left to dry in open air away from flies for about one hour. It was later stained with 10% freshly prepared Giemsa for 15 min. The slide was thereafter washed by allowing slow running tap water to wash off the stain with the slide indented about 30 degrees to the running water. The slide was left to dry completely and observed under a binocular microscope in oil immersion. The criteria for stopping the reading was the detection or not of malaria parasites. Slides with low density parasitaemia were read for at least 10 min, whereas slides with high density parasitaemia were read for 2 min or less. This was sufficient to detect malaria parasites on stained slides. In the case of discordant readings, the result of a second study microscopist served as a tie breaker.P. falciparum msp-2 gene. Briefly, parasite DNA was extracted using the Chelex-100 method as previously described [For samples that underwent the PCR test, the test was based on amplification of the escribed  and amplTM hot start polymerase at 5 units/µL and 3 µL of DNA extract in a total reaction volume of 25 µL. The amplification conditions were as follows: pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, primer annealing at 42 ° C for 1 min, elongation at 65 °C for 2 min × 30 cycles and termination at 72 °C for 3 min. For nested PCR, each tube consisted of 20.25 µL of nuclease-free water, 2.5 µL of 10× Thermopol buffer, 0.5 µL of dNTPs, 0.25 µL of each primer (S1 and S4) at a concentration of 2.5 µM, 0, 25 µL of OneTaqTM hot start polymerase (5 units/µL) and 1 µL of amplicon from the first amplification, in a total volume of 25 µL. The following amplification conditions were applied: denaturation at 94 °C for 30 s, primer annealing at 50 °C for 1 min, elongation at 72 °C for 2 min × 30 cycles and termination at 72 °C for 3 min. Each tube consisted of 18.25 µL of nuclease-free water, 2.5 µL of 10× Thermopol buffer, 0.5 µL of dNTPs, 0.25 µL of each external primer (S2 and S3) at a concentration of 2.5 µM, 25 µL of OneTaqThe amplicons were analyzed using electrophoresis through 2% (wt/vol) agarose gels stained with ethidium bromide. The DNA was visualized under UV.The expected product size of 1200 bp was measured against a 100 bp DNA ladder  with a range of fragments from 1000–1517 bp. Samples that had the 1200 bp band were classified as positive and samples that did not have the 1200 band were classified as negative. P. falciparum only or in a mixed infection with other species; (2) two bands on pLDH and control lines. In this case, the test outcome was interpreted as positive for other species than P. falciparum but was not used for diagnostic calculations. Since these tests are qualitative tests, and despite the fact that the intensity of the bands might reflect parasite density, no cut-offs were set. A faint line on results window was interpreted as positive, based on manufacturer recommendations. Two trained assessors performed and read the result of each index test. Microscopy was also read by a trained hospital technician who was blinded to the index test. The presence of a trophozoite was noted as a positive test. The presence of a visible band after nested PCR and gel electrophoresis was considered a positive PCR result. Results of the RDT test was delivered to the patient in sites where mRDT was routinely practiced, while microscopy results were delivered to patients in the hospitals that had the technical platform to perform slide microscopy in routine practice. The PCR testing was retrospective and, therefore, the result was not provided to the patient.A positive test was defined as the presence of a red line on the test window in addition to a clearly visible control line. In the absence of a control line, the test was considered invalid and repeated. A negative test was defined as the presence of a red line on the control window only. Thus, for a Pf/PAN test, the result could be either (1) 3 bands on test (Pf line plus pLDH line) and control windows. In this case, the test was interpreted as the presence of Sensitivity = The proportion of suspected cases with true malaria infection who have a positive mRDT test result and calculated using the following formula: a/a + c.Specificity = The proportion of suspected cases without malaria who have a negative mRDT test result and calculated using the following formula: d/b + dOverall accuracy = The overall accuracy of each mRDT test was defined as the proportion of correct mRDT assessments as a function of all mRDT assessments in the study population and for each type of reference test. It was calculated using the following formula:The primary outcome of this study was the diagnostic accuracy and agreement of HRP2 analyte in HRP2 mRDT, the HRP2 and pLDH combo RDTs and microscopy and/or PCR in each site. The performance parameters (sensitivity and specificity) of the RDTs were compared to manufacturer supplied data. The secondary outcomes were a comparison of the performance of the HRP2 analyte in both mRDT test presentations and microscopy and/or PCR within the same hospital in the West, between sites of different malaria transmission intensities within the West region of Cameroon, and between the West and the Centre regions. We defined the diagnostic accuracy using the following modalities in 4.Predictive value of the positive test = This modality defined the probability of a malaria infection among study volunteers with a positive mRDT test result. It was calculated as follows: a/a + b5.Predictive value of the negative test = This was defined as the probability of malaria free participants with a negative mRDT test result, calculated using the following formula: d/c + d.6.The likelihood ratios (LR) of the positive and negative tests were calculated and post-test probabilities expressed as percentages estimated from Bayesian theorem. Thus,Diagnostic accuracy of CareStart™ malaria HRP2 and SD Bioline Pf/PAN mRDTs in different study sites.Performance of two band mRDT (HRP-2 only) against microscopy as gold standard.We determined the performance characteristics of the two band mRDT rapid tests that detect HRP-2 antigen in patient samples. The study enrolled a total population of 1126 participants in the four sites over the study period. In all the sites, there were more females than males marked in the sites in the West region compared to the site in SU-Yaoundé. In CBCHS-IHC Ngounso, West region. However, this difference was not significant. The majority of the study participants were aged 20–40 in the Western regional sites , followed by those in the age group 21–40. In SU-Yaoundé, individuals 20 years or under formed three quarters of the study participants followed by those 20 years and under .DiagnosAs shown in When PCR was taken as the reference, almost the same trend in sensitivity, specificity, likelihood ratios and test agreement were observed. However, we note the there was an increase in the likelihood ratios of the positive test outcomes in SU-Yaoundé and CBCHS-IHC Ngounso, and a slight reduction in St Vincent Catholic hospital and the District Hospital Dschang . The samSince Pf/PAN mRDT was evaluated in the two sites in the Dschang but not in Yaoundé, the performance of this test in these three sites is reported below.As shown in Given the results, we sought to look at how microscopy performed with PCR as the referent method. The following table presents the results obtained in the four study sites when we compared results of microscopy with those of the PCR test as the reference method.As shown in p < 0.05 in each case). We further compared the diagnostic parameters of Pf mRDTs (HRP-2) against Pf/PAN RDTs in each of the two facilities in Dschang when both diagnostic tests were performed on the same participant and on two independent samples, respectively. In each case, we used both microscopy and PCR as gold standards separately. Our analysis showed that Pf/PAN mRDTs performed better in terms of sensitivity and specificity irrespective of the good standard applied. In the same vein, irrespective of the index test used, the test performance was higher in StVC Dschang than in DH Dschang  for sensitivity and specificity comparisons. Likewise, when PCR was used as referent, the same significant differences were obtained (p < 0.001 for sensitivity and p < 0.02 for specificity).When the Pf mRDT and the combo tests were performed on the same samples in the StVC Dschang , no diffFurthermore, a pairwise comparison of test parameters taking into consideration all the study sites showed a consistent lower test performance at the DH Dschang, irrespective of the test and referent used .The goal of this study was to evaluate the diagnostic accuracy and performance characteristics of two malaria rapid diagnostic tests deployed for the diagnosis of malaria between and within health facilities located in semi-urban areas across different malaria transmission settings in Cameroon. The tests were the Pf mRDT , which is a two-band test that detects histidine-rich protein 2 secreted by Plasmodium falciparum only, and the three-band test Pf/PAN mRDT (SD Bioline Pf/PAN) that detects both HRP-2 and Plasmodium lactate dehydrogenase (pLDH), the latter secreted by all five main species of malaria parasites infecting humans. All the RDTs were of the lateral flow format and obtained from the same supplier as the study site.The results show that across different malaria transmission regions, the main malaria RDTs used in Cameroon have remained accurate since they were first deployed in 2011 with few exceptions ,17. ThesOur analysis shows that in Dschang, compared to the other study sites, the Pf only and Pf/PAN mRDTs have performed less than expected. In the district hospital of Dschang in particular, the diagnostic parameters were worse. The sensitivity at this site was 17% for the pf only mRDT and 23% for the Pf/PAN mRDT during the study period. The trend could be observed in the St Vincent Catholic hospital where the sensitivity of the pf only mRDT was 65% and 46% for the Pf/PAN mRDT test. Moreover, the predictive values of the positive and negative tests also indicate high rates of positivity and false negative diagnosis. Several reasons could account for this poor quality of malaria RDTs. First, the HRP-2-based RDTs are known to vary with age with decreasing false positivity in older age groups compared to younger age groups . This poThe occurrence of other species than are detected by conventional malaria RDTs in our settings can also affect the performance of such RDTs. In the DH of Dschang, previous reports have indicated the circulation of Plasmodium vivax in the population of febrile patients. An initial  and follThe ability of the mRDTs to test negative among non-diseased participants was equally appreciably higher in other sites than in Dschang. This is reflected in the predictive values of the positive and negative test results that were consistently poorer. The positive predictive value varies by season and age group and was shown to be higher in children than in adults in Burkina Faso during the malaria transmission season . AlthougThe likelihood ratio is a useful tool for assessing the effectiveness of a diagnostic test because they determine how much more probable it is to find a positive result in an infected person compared to an uninfected person. Our results further show that in regions of moderate to high transmission of malaria in Cameroon, both mRDTs have a strong ability to predict malaria infections among febrile cases suspected for malaria, as indicated by estimates of post-test probability of disease. The likelihood ratios of the positive test for both the pf only and the Pf/PAN mRDT were high in Ngounso and, Yaoundé and moderate in StVC Dschang. This translates to a post-test probability of >97% for Ngounso and Yaoundé and 91.3% for StVC Dschang. Previous studies have shown that when parasite densities are high, there are greater odds of obtaining a positive test outcome among febrile malaria suspects ,30. The The pattern of mRDT performance across different sites mirrors the altitude patterns compatible with malaria transmission, irrespective of the study population. In regions at a low altitude, such as Ngounso and Yaoundé (about 700 m above sea level), malaria transmission is shown to be high, and RDT performance is appreciably high, although it failed to satisfy the 95% threshold recommended by the WHO. In Dschang (about 1380 m above sea level), on the other hand, the mRDT performance was poorer, although this varied between facilities in the same town. The McNemar’s analysis of paired samples in our population in these two health facilities yielded similar results (with little differences) to the independent sample test, further lending validity to the consistency of the test outcomes after performance and the overall results.Our study had some limitations, among which we did not conduct a density-dependent analysis of mRDT performance across the sites. However, while this was an interesting option, the limitations in funding did not allow full expert quantification of malaria parasites for over a thousand patients within the timeframe. This is the first study conducted in Cameroon that compares the clinical performance across geographies to provide useful clinical data on RDT performance and guidance on a consideration of regional specificities in some cases in the interpretation of mRDT results. Our results are also useful in that it will help the national malaria control program in its recent quest to implement the national quality assurance program for an accurate and a timely malaria diagnosis in Cameroon. We intend to examine the molecular species contributing to malaria infections among samples from Dschang to further clarify the utility of RDTs in this region. Lastly, it would have been more interesting to use the genus primers for the PCR analysis of malaria positive samples, which we intend to undertake with new funding. Overall, our results may prove is useful to move the program to its ambitious goals of malaria pre-elimination within the current decade.The present diagnostic study shows that malaria rapid diagnostic tests in Cameroon have maintained a high accuracy (91–94%) in the clinical diagnosis of malaria across regions of different malaria transmission, although they failed to reach the WHO threshold. These results underscore the importance of routine monitoring of the quality and performance of malaria RDTs in diverse settings in malaria endemic areas.