Febrile illnesses are among the most important reasons for medical consultation in sub-Saharan Africa and are frequently treated with antimicrobials due to the unavailability of appropriate diagnostic tools. This practice leads to antimicrobial resistance, with increasing mortality and morbidity as result. One of the few accessible diagnostic tools available in low resource settings is malaria rapid diagnostic tests (mRDTs) which contributed to reducing the over-prescription of anti-malarials, but cannot guide antibiotic prescriptions. To circumvent this problem, we explored whether combined testing with mRDT and c-reactive protein (CRP) could improve the diagnosis of febrile illnesses and subsequent prescription of antibiotics.Clinical specimens  collected from 396 febrile children (axillary temperature of ≥ 37.5 °C) were analyzed with rapid diagnostic tests  and microbiology culture to establish the possible cause of fever. Actual antimicrobial prescriptions given to the children were compared with those that could be given based on combined CRP-malaria testing.Pf-HRP-2. CRP test was positive in 84.3% (334/396) of the children, but bacterial infections were confirmed in 12.4% (49/396) of them. A possible cause of fever could not be established in 20.5% (81/396) of cases. Based on the diagnostic practice in place, 265 of the children with a positive mRDT-Pf-HRP-2 received anti-malarial treatment. Furthermore, 89.5% (111/124) of negative mRDT results received antibiotic treatment and 37.1% (46/124) received antimalarial treatment. Of these 124 cases, 80 had positive CRP tests and 44 negative CRP tests. If the results of CRP testing are considered, 44 CRP/mRDT negative children would not get antibiotic treatment, resulting in a 35.5% reduction in antibiotic prescriptions. However, 2 cases with a bacterial infection would be denied appropriate treatment.In total, 68.7% (272/396) of malaria cases were diagnosed by mRDT-PfHRP2 with CRP testing is particularly useful in children for whom both tests are negative as it results in a reduction of antibiotics prescriptions. However, there is a risk to miss potential severe bacterial infections and a close follow-up of these cases is strongly recommended.Combining mRDT- In Burkina Faso, like in many other sub-Sahara African countries, febrile illnesses in children are amongst the foremost reasons for medical consultation –3. In reIn Nanoro, a rural malaria-endemic area, many cases of fever have been reported in children under 5 years of age with many of them being malaria infection , 6. NextIn order to tackle antimicrobial resistance, there is a pressing need to develop better diagnostic strategies to improve the management of non-malaria fever in remote areas lacking suitable laboratory infrastructure. Next to malaria, it is highly relevant to develop diagnostic strategies for bacterial and viral infections as the later do not require treatment with antibiotics. C-reactive protein (CRP) is a relatively accurate and sensitive marker for inflammation and infection, and could be an indication of a (possible) bacterial infection –15. The Plasmodium falciparum specific histidine-rich protein 2 (mRDT-PfHRP2). RDT-positive cases are treated with appropriate anti-malaria drugs as recommended by the National Malaria Control Program (NMCP) guidelines ) by standard microbiological and biochemical methods  [Fresh stool samples collected in sterile containers by study nurses were subject to culture, microscopy, and Rota/adenovirus RDT testing. Firstly, we cultured the stool on Sodium Selenite broth and on appropriate agar followed by an incubation at 35–37 °C for 18–24 h. Subsequently, we identified suspected colonies  –31. Seco5 colonies forming units (CFU)/mL were considered significant bacteriuria [Urine samples were collected in sterile containers and tested using urine dipsticks . All samples tested positive for leukocytes and nitrite were cultured on appropriate agar and incubated for 18–24 h at 35–37 °C. Samples that resulted pure bacterial growth of > 10teriuria  and we iPfHRP2, the internal control lines confirmed the correct performance of these tests. In addition, we perform monthly internal quality controls of reagents and equipment using American Type Culture Collection (ATCC) standard reference strains. It is important to mention that the CRUN laboratory is subjected to external quality control organized by world health organization (WHO) and national institute for communicable diseases . Finally, at CRUN laboratory, microscopists are regularly submitted to an external quality control program on quarterly basis and only certified microscopists are allowed to perform malaria slide reading [To ensure the quality of the samples’ analysis, all experiments were conducted according to the standard laboratory operating procedures (SOPs) in place at CRUN laboratory. Furthermore, there was strict adherence to manufacturers’ instructions. For the semi-quantitative CRP rapid test and mRDT- reading .Data were double entered from study CRFs into OpenClinica software. The data were subsequently analyzed using STATA® statistical software version13 StataCorp LLC, College Station, TX, USA. Qualitative variables description was performed using the percentages. The median and mean were used for quantitative variables distribution. The baseline parasite density was described using geometric means.The characteristics of study participants are summarized in Table Results of microbiology laboratory analyses are also presented in Table Most of the children who were tested RDT positive for malaria also had a CRP positive test 93.4%; 254/272). But of those who were tested negative for malaria by RDT, 35.5% (44/124) also had a negative CRP test , and whose actual cause of fever remained unknown, the CRP test was positive in 63.0% (51/81) of the cases and negative in 37.0% (30/81) of the children , we also diagnosed a total of 30 bacterial infections but all are not treated with antibiotics. Among those, 12 bBSI and 18 bGE were diagnosed of which 10 bGEs were caused by EPEC that is not treated in accordance with the Burkinabe treatment guidelines. Furthermore, we also diagnosed 52 non-bacterial infections in mRDT-PfHRP2 positive cases.A more detailed distribution over the number of positive/negative mRDT-PfHRP2 (n = 19), there were 14 cases who were tested CRP positive and 5 were tested CRP negative. There were 22 cases of confirmed non-bacterial infection of whom 14 were tested mRDT-PfHRP2 negative but CRP positive and 8 who were tested negative for both RDTs.In febrile children with a confirmed bacterial infection and who were tested negative with mRDT-PfHRP2 and for whom the cause of fever could not be explained by the identification of an infective agent, there were 51 cases (63.0%) with a positive CRP test and 30 cases (37.0%) with a negative CRP test.Of the 81 cases who were tested negative with mRDT-PfHRP2 should have been treated with anti-malarials. Indeed, 265 (97.4%) of the children in this group did actual receive anti-malarials, but 7 children (2.6%) did not but the reason for this was not recorded in the CRFs  should not get anti-malarial treatment, but there were 46 children  who still received anti-malarials. In addition, 88  mRDT-PfHRP2 negative children were treated with 1 antibiotic, 22 children (17.7%) in this group were treated with 2 antibiotics and 1 child even received treatment with 3 antibiotics, irrespective of knowing the actual cause of fever.Children who were tested negative with mRDT-Based on the microbiology analyses we can also categorize what treatments were provided to the febrile children who actually had a bacterial infection. In the group of children with a confirmed bBSI (n = 23), there were 15 (65.2%) who received anti-malarials and 8 (34.8%) who did not. Importantly, 7 children (30.4%) with a confirmed bBSI did not receive antibiotic treatment, whereas 13 (56.5%) received 1 antibiotic and 3 (13.0%) were prescribed 2 antibiotics.Nineteen children  with a confirmed bGE received anti-malarials. In terms of antibiotics prescriptions, there were 11 children (44.0%) with a bGE who did not receive antibiotics, 12 (48.0%) were prescribed one antibiotic and two children (8.0%) with bGE received two antibiotics.One child with a bacterial urinary tract infection did not receive antimalarials, but did receive two antibiotics.PfHRP2 are treated with anti-malarials and those with a negative malaria RDT with antibiotics. Consequently, we diagnosed 272 malaria cases by mRDT, who should receive anti-malarial treatment as recommended by the NMCP guidelines of Burkina Faso. Of note is that within this positive mRDT-PfHRP2 group, 20 cases (7.4%) had also bacterial infection confirmed by microbiology analyses and that needed antibiotic treatment , who actually needed antibiotic treatment, but would on the basis of the negative result of the semi-quantitative CRP test not receive it.Within the group of 124 children with a negative mRDT (and who were given antibiotics for relief), there were 64.5% (80/124) had a positive CRP test and 35.5% (44/124) had a negative CRP test (see Table PfHRP2 could improve the diagnosis of febrile illness in rural Burkina Faso, and consequently would lead to a more correct prescription of antimicrobials. Our present study indicates that this approach has proven to be of limited value in our study area. Most cases with malaria infection, also had a high CRP level, which confirms the results of other studies [Plasmodium infection, the gold standard for malaria diagnosis, should be reconsidered in areas with high malaria prevalence and with the risk of significant numbers of false-positive mRDTs, in particular due to persisting HRP2 antigen [Our study aimed to assess whether the combination of semi-quantitative RDT-CRP testing with mRDT- studies , 34, 35. studies . Therefo antigen , 11, 36. antigen . However antigen .The strength of combined mRDT and CRP testing is highlighted in our study in case both tests are negative. This was the case in 44 children of whom the majority of the children, did indeed not need either anti-malarial and/or antibiotics treatment. However, if this diagnostic approach is followed, two children will be denied essential antimicrobial treatment. In this respect, health policymakers, will have to consider whether not treating this particular small group of children outweighs the savings in prescribing antimicrobials in general or not.In our study area, as was also previously reported malaria remains one of the most important causes of febrile illness , 39. HowAnother limitation of our work is that the current study design did not allow for statistical testing of sensitivity and specificity of the combined malaria and CRP testing. This is due to the fact that a majority of those recruited suffered from malaria and therefore will have inflammation ongoing. Perhaps performing a similar survey of the RDTs in a less malaria endemic region could indicate if the combined approach could be more useful.According to the NMCP guidelines in Burkina Faso, a febrile child who is tested positive by mRDT should receive appropriate anti-malarial treatment. In our study, health workers tend to adhere well to this guideline and this is in line with previous observations done in the same study area , 40. HowAlthough, in the present study, additional CRP testing did not provide a perfect solution to reduce over-prescription of antimicrobials. However, there is sufficient evidence that point of care CRP tests can contribute to control or reduce the unnecessary use of antibiotics and decreases the spread of antimicrobial resistance due to their ability to discriminate bacterial from non-bacterial infections in febrile patients , 41. In PfHRP2 and CRP RDT is only useful in case both tests are negative and a malaria infection can be excluded. This approach could result in a reduction in antibiotic prescriptions in the group of febrile cases, although there is a risk of not treating children with an actual bacterial infection.In high malaria-endemic areas, the combined testing of febrile cases with mRDT-


Malaria infection during pregnancy is a significant public health problem that puts pregnant women at risk. Interruption of transmission of asymptomatic malaria among a population remained a challenge and the host serves as a reservoir for the malaria parasite; and is also recognized as a major barrier to malaria elimination. This study aimed to assess the prevalence of asymptomatic malaria and associated factors among pregnant women in the Boset District, East Shoa Zone, Oromia, Ethiopia.Plasmodium infections. Epi info version 7 was used to code, enter, and clean data before being uploaded to SPSS version 25.0 for analysis. Bivariable and multivariable binary logistic regression were employed to find the associated factors. Variables in the multivariable model with a p-value < 0.05 were considered significantly associated with asymptomatic malaria.A community-based cross-sectional study was conducted to assess the prevalence of asymptomatic malaria and associated factors in pregnant women from February to March 2022. Using multistage sample techniques, 328 asymptomatic pregnant women were enrolled. Data were collected using a structured questionnaire. A rapid test and Giemsa-stained blood smear microscopy were used to diagnose Plasmodium species by microscopy and rapid diagnostic tests, respectively. Asymptomatic malaria during pregnancy was found to be significantly associated with not using an insecticide-treated bed net , lack of consultation and health education about malaria prevention during Antenatal care attendance , and living close stagnant water .Of the total 328 pregnant women who participated in this study, 9(2.74%) and 10(3.05%) were confirmed to be infected with The current study showed that asymptomatic malaria is prevalent in pregnant women. Not using insecticide-treated bed nets, inadequate health education during antenatal care, and living close to stagnant water are significantly associated with malaria infection. Thus, using insecticide-treated bed nets, health education, and avoiding stagnant water from residential areas could play significant roles in preventing asymptomatic malaria among pregnant women in the study area. Plasmodium, which are transmitted to humans by blood-feeding female Anopheles mosquitoes. Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae are the four human Plasmodium species  and 10(3.05%) [95% CI ] by using microscopy and RDTs, respectively. The density of parasitaemia, as determined from the thick blood smear, ranged from 790 to 5600 parasites /μl. The geometric mean of parasite density among the 7 parasitaemic pregnant women was 3528.6/μl [95% CI (3310–3750)]. The majority of the study participants who were diagnosed with malaria cases had moderate parasitaemia 5(71.4%), while 2(28.6%) had mild parasitaemia ] higher chance of having asymptomatic Plasmodium infection than those who did. Pregnant women who lived near stagnant water had 4.43 times  greater odds of having asymptomatic Plasmodium infection than those who did not live near stagnant water. In addition, pregnant women who did not receive malaria prevention education during their ANC follow-up had a 4.05-fold higher risk of malaria infection than their counterparts  ] and 3.05% [95% CI = ] using Giemsa-stained blood smear microscopy and RDT, respectively. These findings are lower when compared to the results reported previously from many similar studies conducted in different countries. For instance, the reported prevalence from the Republic of Congo was 7% [This study assessed the prevalence of asymptomatic malaria infection and associated factors among pregnant women. The prevalence rates of asymptomatic o was 7% ; the ruro was 7% ; the gloo was 7% ; the stuo was 7% ; a studyo was 7% , and theo was 7% . The lowP. falciparum. This result was almost in line with the study conducted in Ethiopia that reported 65–75% of malaria infections are caused by P. falciparum [P. falciparum in this study is a clear implication that there is a need for aggressive prevention and control of the disease, especially among pregnant women. Moreover, P. falciparum causes the most severe form of the disease and devastating complications not only to the mother, but also to the fetus. However, the proportion of malaria cases caused by P. falciparum in the study was lower than that of the WHO malaria 2017 report, which stated that P. falciparum was responsible for almost 99% of malaria cases [Nevertheless, the prevalence of asymptomatic malaria infection documented in this study is comparable to the findings of the studies conducted in Bangladesh, which was reported as 2.3% among pregnant women , and in lciparum . Howeverlciparum . This hiIn addition, the findings revealed not using ITN effectively increased the odds of developing malaria infection during pregnancy. This finding is in agreement with the findings of previous studies conducted elsewhere. For instance, the studies from Malawi , ArbaminFurthermore, the pregnant women who lived near stagnant water were more likely to have an asymptomatic malaria infection than their counterparts. This finding is also in agreement with the findings of research that were undertaken in the West Arsi Zone, Ethiopia , and theThe main limitations of the study include the inability to identify a direct temporal association between asymptomatic malaria infection and its likely risk factors because of being a cross-sectional study design; in the inability to diagnose all subjects using real-time PCR due to resources constraints; and fact that the data were collected during the season with low transmission probability which could have underestimated the prevalence of asymptomatic malaria among the study population.Plasmodium falciparum is most prevalent species in the area. Inadequate use of ITNs, living close to stagnant water, and inadequate consultation and health education about malaria prevention methods during ANC attendance were found to be factors associated with the occurrences of asymptomatic malaria infection among pregnant women.This study showed that asymptomatic malaria is prevalent among pregnant women in the study area. Thus, using insecticide-treated bed nets effectively, adequate consultation and health education on various malaria preventive measures, and avoiding stagnant water from residential areas could play significant roles in preventing asymptomatic malaria among pregnant women in Ethiopia in general and in the study area in particular.


P-values < 0⋅05. In the present study, the minimum week for a cure was 4 weeks, the maximum was 16 weeks and the overall time to cure severe acute malnutrition as measured by MUAC is judged to be 10 at 95 % CI (9⋅65–10⋅35). Families with six or more members are 2⋅16 times more at risk, children from homes with the lowest wealth index are at 1⋅4 times more risk, and children from food insecure families were 2⋅61 times more likely to require long-term treatment for MUAC. In the present study, the time to cure severe acute malnutrition by MUAC is determined as 10 weeks. Moreover, family size, low wealth index, and household food insecurity were risks to delay in cure time MUAC.Mid-upper arm circumference (MUAC) is simple to use and inexpensive in Ethiopia; both MUAC and target weight are employed, although the time to cure for MUAC is not indicated. The present study is aimed to determine cure time of MUAC for children in outpatient therapeutic program. A prospective cohort study was conducted among 414 severe acute malnourished under-five children admitted to selected health twenty-two posts from 1 February to 30 July 2021, in Oromia, Ethiopia. Data were coded, entered to Ep-data version 4.2 software, and transferred to SPSS for windows version 25 software for analysis. The Multivariate Cox Proportional Hazards model was used to fit independent determinants of time to cure. All tests were two-sided and statistical implications at  In addition, the management of severe acute malnutrition is critical for child survival and is a key cost-effective component of the scaling up nutrition outline for addressing undernutrition. In nations with a high incidence of undernutrition, timely, correct screening at the community level is vital to identify children with wasting. Moreover, the MUAC is used as a substitution to evaluate wasting in children. However, its legitimacy thrives in controversies.In the resource, limited sceneries where malnutrition is common, accurate measurement of weight and height may be a challenge. Mid-upper arm circumference (MUAC) is easier to quantify and interpret and it is similar in boys and girls and is relatively constant from 6 months to 5 years which leads to avoiding the obligation to determine the exact ageZ-score or (WHZ) only.A greater number of drawbacks are recognised in the usage of the existing, as evidence to answer the question about the relevance of strategy to identify the children at risk of acute malnutrition. There is doubt among practitioners and difference of opinion among practitioners and academic experts, concerning the strength of the indication to support this recommendation and the penalties for children identified with SAM using weight-for-height Z-score or (WLZ) have different associations with body composition, and length influences these associations differently, and MUAC acts more like a composite index of poor growth indexing.The MUAC and WHZ or weight-for-length .Weight gain is routinely monitored to assess hydration and growth during the treatment of children with complex severe acute malnutrition. However, changes in weight and MUAC gain speeds over time are barely described.Another challenge for MUAC or MUAC grounded on a solitary cut-off value for all the children less than 5 years of age has been used for numerous years as another nutritional status index for children through scarcities or refugee catastrophes, and as an extra screening tool in non-emergencies. However, it has newly been interrogated whether MUAC is age and sex-autonomous. A Study in Senegal suggests that MUAC is superior to WHZ in classifying high-risk children in the community and using both WHZ less than −3 and MUAC less than 115 mm increases specificity but decreases compassion to classify high-risk children. There is no advantage for programmes in combining WHZ and MUAC to identify high-risk childrensd, MUAC less than 110 mm, and with medical difficulties like loss of appetite and oedema but this is more meticulous more than 99 % over the age range 6–60 months and discharge only 15–20 % of weight gain.A problem that is encountered in admission standards for severe acute undernutrition is based on children with a weight-for-height below −3 . In addition, a weight-for-height Z-score ≥ −2 suggests that short children with stumpy MUAC fail to gain extreme fat during supplementation, and the practice of length as a criterion for gaging MUAC to determine treatment eligibility was outdated in policy and practice.Moreover, another study suggests that a cut-off of <11⋅5 cm is not enough to detect SAM children so it needs to increase to ensure early diagnosis of children and to avoid miss of children from referral for management of malnutrition. Therefore, to decrease the risk and death of undernutrition-sensitive cut-off point MUAC is needed.The anthropometric gauge that is used to identify severe acute malnutrition should also be used to trace whether a child has grasped nutritional retrieval, i.e. if MUAC is used to identify that a child has severe acute malnutrition. Likewise, if weight-for-height is used to categorise that a child has severe acute malnutrition, then weight-for-height should be used to measure and check nutritional recapture.However, the World Health Organization on its 2013 severe acute management guideline suggests that there is no difference in weight-for-height and MUAC; for a programme utilising MUAC weight gain is 15–20 % afterward oedema vanishes for the programme utilising weight-for-height: weight-for-height > –11 standard deviations or weight gain is 15–20 % after oedema dissolvesAs MUAC is easy to practice for classifying and follow up severe acute malnutrition among under-five children. However, the time to cure MUAC is not identified and the present study aimed to identify the time to cure for MUAC and factors affecting the survival of under-five children from severe acute malnutrition for MUAC in Ethiopia.. The study period was from 1 February to 30 July 2021.The study was conducted in Habro Woreda, West Hararghe Zone, Oromia, Ethiopia. Habro Woreda is found in the West Hararghe Zone of Oromia Regional State. The capital city of Habro Woreda is called Gelemso which is located about 326 km East of Addis Ababa and 76 km away from Chiro Zonal Town of West Hararghe. The total under-five population of the Woreda was 44, 243, 6–59 months was 40 392, and households of the Woreda were 56 099. The Woreda has 32 rural kebeles  and 5 urban kebeles . It also has thirty-two health posts and seven health centres and one general hospital, all of the health facilities, except the hospital are providing OTP services. The main income of the population depends on agricultureProspective cohort study.Under-five children were admitted for severe acute malnutrition in twenty-two health posts during the study period., a 5 % margin of error, 95 % CI and a power of 80 %. Then, it was considered by medcalc©version 119.1.1.3 survival analysis (log-rank test) at http://www.medcal.org. Finally, by bearing in mind 10 % for failure to follow up, the total study sample size was 414.The sample size was determined based on the expectations with a recovery rate of 93⋅3 %, a hazard ratio of 0⋅38In the West Hararghe Zone of Oromia Regional State, Habro Woreda, there are 32 rural kebeles  and 5 urban kebeles  and 32 health posts providing OTP service. From them twenty-two rural health posts were randomly selected, namely, Health post, Chafe 12 HP, Bareedaa HP, Wenne kallo HP, Medda Jaalalaa HP, Booraa HP, Maqrac HP, Ifaa Gemechu HP, Busoytu HP, Melkaa balloo HP, Odaa Mudaa HP, Daraaraa HP, Kallachaa HP, Gerbi Teka HP, Ibsaa HP, Kufa kaas HP, Dikkichaa HP, Bedada HP, Haro Carcar HP, Lallisaa HP, Lagabeeraa HP, odaa Ananii HP and Gadisa HP.Data were collected from the children's admitted for SAM management in health posts; a prospective cohort study was conducted for 6 months from February to July in randomly selected health posts. Data collection tools include socio-economic data , time . One supervisor and twenty-two data collectors were trained and involved in data collection. Data collectors received a one-day training on data collection tools and interpersonal reliability on Anthropometric measurement was checked by the technical error of measurement (TEM) after that organised to gather data once the main investigator was convinced about their competency. The primary investigator of the study and the supervisors critically followed the data collection procedure to minimise missing information and discrepancies.Z-score < −2. It often indicates recent and severe weight loss, although it can also persist for a long time.Weight-for-height : It is diagnosed by weight-for-height below −3 sd of the WHO standards, by MUAC <11⋅5 cm, and by clinical signs like bilateral oedema.If the child has severe acute malnutrition.It is also a form of severe undernutrition, the child's muscles were wasted, but wasting may not be apparent due to generalised oedema or swelling because of excess fluid in the tissuesZ-scores and having no oedema for at least 2 weeks, or MUAC is ≥125 mm and no oedema for at least 2 weeks.Weight-for-height/length is ≥ –2 .Household food insecurity access scale (HFIAS) gave the highest prevalence estimates of food insecurity, as might be expected given its inclusion of less severe manifestations of insecurity, including psychological anxiety and food consumption preferencesData were implicated, entered into Ep-data version 4.2 software, and transferred to SPSS for windows version 25 software for analysis. The occurrence of missing values, possible outliers and multicollinearity was checked by exploratory analysis. The wealth index was calculated using principal component analysis (PCA), based on PCA expectations. Accordingly, the households were considered into three wealth terciles for further analysis.Both bi-variate and multivariable Cox regression analyses were done. Kaplan Meier hazard curve with the log-rank test was fitted to identify the occurrence of a difference in recovery rate among the categorical variables. Mantel-cox and generalised Wilcoxon tests of equality of survival distributions are significant and one minus survival function line is parallel for those candidate variables of multivariable Cox regression .Fig. 1.For the different levels, under-five children with SAM cases followed in weeks from admission to the occurrence of the event (cured). Person time was calculated and the incidence was determined. In the present study, person time was reported in child week. Child week is the total follow-up times of each child from admission to the occurrence of the events (cured or censored).P ≤ 0⋅25 in the bi-variate Cox regression were selected for the multivariable Cox regression analysis. All statistical tests were measured significantly at P-values of < 0⋅05.Those variables with In the present study, 414 under-five children's participated of them 257 (62 %) were females and the reaming were males. When we come to the age of the majority of under-five children were below 24 months 311 (75⋅1 %), on the other hand, more than half of the children were from the household with family members of six and above 250 (60⋅4 %), the rest were below six family members. When we come to the household wealth index from the participants 166 (40⋅1 %) households were under the lowest tercile of the rank in a similar way majority of households or family members of under-five children admitted with severe acute malnutrition were food insecure with 302 (72⋅9 %) .Table 1In the present study, children were followed for 16 weeks after admission for severe acute malnutrition in health posts. From the total follow-up, 354 (85⋅5 %) were cured during treatment and the remaining were not cured, transferred to the stabilisation centre and defaulters of the treatment were 30 (7⋅2 %), 22 (5⋅3 %) and 8 (1⋅9 %), respectively .Fig. 4.In the present study, the minimum week for the cure was 4 weeks, the maximum was 16 weeks and the overall time survival from severe acute malnutrition by MUAC is determined as 10 weeks with 10 weeks, 95 % CI (9⋅65–10⋅35). However, time to cure varies with different variables, for example; at the age of 6–11 months cure time becomes 11 weeks becomes 95 % CI (10⋅47–11⋅53) and at the age of 12–23 months, it becomes 9 weeks becomes 95 % CI (8⋅66–9⋅34). In addition, family members with six and more members have a median time for MUAC is 11 weeks with 95 % CI (10⋅51–11⋅49). In the same way, among food insecure food-insecure 11 weeks with 95 % CI (10⋅48–11⋅52), among the lowest wealth index 11 weeks with 95 % CI (10⋅41–11⋅59), and with the highest wealth index the cure time will fall to 9 weeks with 95 % CI (8⋅23–9⋅77) .Table 2Factors associated with cure time to MUAC in Proportional Hazards Model, after adjusting for related variables to control confounders and testing for Kaplan–Meier test to identify parallel line assumptions. Under-five children with severe acute malnutrition from family size 6 and above member were 2⋅16 times at higher risk for long duration in treatment  compared to lower family size. Children from low wealth index category family members have a 1⋅4 times hazard of long-term treatment for severe acute malnutrition to cure for MUAC . In addition, SAM children from food-insecure households were 2⋅61 times at higher risk during treatment by MUAC compared with food secured households  .Table 3In the present study, the minimum and maximum time to cure is determined as 4 and 16 weeks, respectively, and the time to cure severe acute malnutrition by MUAC is determined as 10 weeks. The following factors associated with time to cure were family size, wealth index and household food insecurity associated with cure time by MUAC from severe acute malnutrition. and another study from North Gondar Zone, reveals that the median time to recovery was 38⋅5 days or 5 weeks. However, in the present study, the median week for survival was determined as 10 weeks for MUAC and this is a higher time to stay in treatment. This may be because the above study durations were based on target weight gain from admission but the discharge criteria in this study were on target MUAC or 12⋅5 cm. This may suggest us children who are declared cured and discharged from severe acute malnutrition management programme by weight gain were may not cure of MUAC.A study conducted in Dire Dawa among different health institutions suggests the median recovery time was 8⋅7 weeks. This may be because children in a large family may face several problems that may include lack of care, competition for supplementary treatment food, Plumpy'nut or ‘Nefis Aden’, among severely malnourished children and other family members.When we come to the factors associated with time to cure; in the present study, family size is associated with time to cure. The present study is similar to the retrospective study conducted among under-five children admitted to the therapeutic feeding unit at Dubti referral hospital. Even though there are different ways that means study conducted in Afar is followed by target weight as discharge criteria but in the present study, we have followed MUAC <12⋅5 cm as discharge criteria for severely malnourished children. The similarity of the cure time among poor or low wealth index households in different studies may low income or being poor directly affect cure time.Another factor associated with time is the wealth index in a parallel way study conducted at Dubti referral hospital; Afar region reveals that household income in Ethiopian Bir (ETB) is associated with time to cure.On another hand in the present study household food insecurity, one of the associated factors with time to cure this may be because of food-insecure children from the food insecure household food-insecure with severe acute malnutrition more and it may result in long cure time from the SAM.MUAC bids an advantage on screening malnutrition and following up the progress of the intervention on malnutrition particularly in developing countries and hard to reach area MUAC is easy to care out. In addition to that MUAC is easy for utilisation and there is no need of age of a child, which minimises relaying on weight and age. Cure by MUAC is longer than that of weight and we have suggested that discharge of MUAC less than 12⋅5 cm were result in relapse or repeated admissionIn the present study, the median time to cure severe acute malnutrition by MUAC is determined as 10 weeks. Moreover, family size 6 and above members, low wealth index category and household food insecurity were identified as risks to stay at treatment for MUAC.From the present study, we recommend that it is better to use MUAC as diagnosing and discharge criteria for severe acute malnutrition among under-five children and better to increase treatment week to 10 and above or until MUAC becomes 12⋅5 cm for all under-five children irrespective of admission criteria.


P. vivax (Pv) infected individuals (Asym) feature different epidemiological, clinical and biochemical characteristics, as well as hematological parameters, potentially predictive of clinical immunity in comparison to symptomatic Pv infected individuals (Sym).Herein, we tested the hypothesis that Asymptomatic Between 2018 - 2021, we conducted 11 population screenings ) in 13 different riverine communities around Iquitos city, in the Peruvian Amazon, to identify Pv Sym and Asym individuals. A group of these individuals agreed to participate in a nested case - control study to evaluate biochemical and hematological parameters. Pv Asym individuals did not present common malaria symptoms , had a positive/negative microscopy result, a positive qPCR result, reported no history of antimalarial treatment during the last month, and were followed-up weekly until Day 21 (D21). Control individuals, had a negative malaria microscopy and qPCR result, no history of antimalarial treatment or malaria infections during the last three years, and no history of comorbidities or chronic infections.From the 2159 individuals screened during PS, data revealed a low but heterogeneous Pv prevalence across the communities (11.4%), where most infections were Asym (66.7%) and submicroscopic (82.9%). A total of 29 Asym, 49 Sym, and 30 control individuals participated in the nested case - control study (n=78). Ten of the individuals that were initially Asym at D0, experienced malaria symptoms during follow up and therefore, were included in the Sym group. 29 individuals remained Asym throughout all follow-ups. High levels of eosinophils were found in Asym individuals in comparison to Sym and controls.For the first-time, key epidemiological, hematological, and biochemical features are reported from Pv Asym infections from the Peruvian Amazon. These results should be considered for the design and reshaping of malaria control measures as the country moves toward malaria elimination. Plasmodium vivax (Pv) is the most geographically widespread human malaria parasite. However, compared to P. falciparum (Pf), global malaria efforts have been less successful at reducing the burden of Pv, owing to its unique biology and treatment complexity , highlight the persistence and high prevalence of Pv asymptomatic malaria infections as detected by molecular tools . TherefoTo better characterize asymptomatic infections in the Peruvian Amazon and to extend our understanding of its biological process, we carried out eleven cross-sectional studies in a total of thirteen communities from the Peruvian Amazon between 2018 and 2021 to search for malaria cases. Next, we carried out a nested case - control study between Pv symptomatic (Sym), asymptomatic (Asym), and control healthy individuals. We tested the hypothesis that Pv Asym infected individuals feature different epidemiological, clinical, and biochemical characteristics, and hematological parameters predictive of clinical immunity compared to Pv Sym infected individuals.Eleven population screenings (PS) or cross-sectional studies were conducted between September 2018 and November 2021 in a total of thirteen riverine communities, six of which are located near to Iquitos City: Tarapoto , Santa Rita , San José de Lupuna , Llanchama , and San Pedro  located at the basin of the Nanay river and Centro Fuerte community  located at the Momon river; and seven communities from the Mazan district: Libertad , Primero de Enero  and Puerto Alegre  located at the basin of the Mazan river; and Huaman Urco , Salvador , Urco Miraño  and Lago Yuracyacu  located at the basin of the Napo river  that agreed to participate in the study, after giving informed consent, filled out a comprehensive survey related to malaria history and symptoms, and were asked for finger-prick blood samples for malaria diagnosis by microscopy and qPCR . To prevent bias, the individuals were not given a symptoms list; instead, a self-explanation from each one’s symptoms was recorded. Microscopy and molecular diagnosis were performed within 24 hours after sample collection. Pv Sym individuals were classified as people presenting at least one malaria symptom , a Pv positive or negative microscopy result, and a Pv positive qPCR result at the time of PS (D0). Pv Asym individuals did not present malaria symptoms during the previous seven days before PS, had a Pv positive or negative microscopy result, and a Pv positive qPCR result at the time of PS (D0). Sym individuals detected with llection . We obta®, Padua, Italy). Whole blood samples in microtainers were centrifuged to separate red blood cells from plasma, and stored at −20°C until processing  or any of the follow-up days , and reported no history of antimalarial treatment during the last month , Pv Asym (n = 29), and control individuals (n = 30), were invited to donate 60 – 100 mL of blood for immunological, biochemical, and hematological testing in our laboratory facilities in Iquitos city. A complete medical evaluation was carried out by a physician prior to sample collection. Sym individuals detected with Plasmodium spp., infection by microscopy or qPCR at D0 or any day during follow-up, received immediate appropriate treatment at the health centers, following the Peruvian Ministry of Health Guidelines, after sample collection. All the Asym individuals received counselling and were directed to seek healthcare in a malaria reference center in case symptoms appeared during the follow-up period, samples were collected before individuals received antimalaria treatment. After sample collection, all the participants that had a positive malaria screening by qPCR were referred to the nearest health center for antimalaria treatment following the Peruvian Ministry of Health Guidelines. Pv non complicated malaria in adults were treated with chloroquine 10mg/Kg on days 1 and 2, and 5mg/Kg on day 3, plus primaquine 0.5 mg/Kg/day for seven days; while, Pf non complicated malaria in adults was treated with mefloquine 12.5mg/Kg/day for 2 days, plus artesunate 4 mg/kg/day for 3 days, plus primaquine 0.75mg/Kg/day for 1 day  in the thick smear, assuming a concentration of 8000 WBCs/μL. Slides were read on-site and then again by a microscopy expert in our laboratory at Iquitos city. A slide was declared negative if no malaria parasites were found after examining 200 fields ® Blood DNA Mini Kit  was used to extract genomic DNA from 40 μL of red blood cells with EDTA or 0.72 cm2 of filter paper impregnated with whole blood following the manufacturer’s instructions with slight modifications  supplemented with SDS 10% w/v). Extracted DNA was stored at 4°C for immediate use and at −20°C for later analyses.The E.Z.N.A.Plasmodium spp. screening was performed using a qPCR that targets the cytochrome oxidase III (COX-III) mitochondrial gene (216 bp) for the detection of Plasmodium spp DNA using SYBR Green. Briefly, 5 μL of extracted DNA was mixed with 0.2 μM of primers. COXIIIF: 5’-CGGTAGATAGGGAACAAACT-3’ and COXIIIR: 5’-CTTTGCCTGGAGGTTACG-3’, 12.5 μL PerfeCTa® SYBR® Green FastMix® and 6.5μL PCR-grade water per reaction. Reactions were carried out in an Real-Time PCR CFX96TM Bio-Rad  using the following qPCR conditions: initial denaturation at 95°C for 3 min, followed by amplification for 45 cycles of 15 s at 95°C, 15 s at 60°C, and 15 s at 68°C, and a final extension of 3 min at 70°C. Amplification was immediately followed by a melting program consisting of a stepwise temperature increase from 70°C to 90°C with an increase of 0.5°C/s up for a total of 5 s. Positive, negative, and blank controls were added to each plate. The same positive control was added to each plate to ensure inter-assay comparability. Analysis of the differences in melting curves provided an accurate differentiation between positive and negative samples.Plasmodium spp identified during screening were used to detect the small rRNA subunit (18S) of Pv and Pf species using previously described probes with small modifications , hemoglobin, hematocrit, platelets, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), neutrophils, lymphocytes, monocytes, eosinophils, and basophils. We also evaluated the following biochemical clinical analytes: alanine transaminase (ALT), aspartate aminotransferase (AST), urea, creatinine, alkaline phosphatase (ALP), bilirubin, direct bilirubin, indirect bilirubin, gamma-glutamyl transpeptidase, glucose, proteins, potassium, and sodium. These parameters were measured for all enrolled individuals in the nested case -control study. The automated Pentra 60C Hematology Analyzer  and Cobas c311 Automated Analyzer , were used for hematological and biochemical assays, respectively. All the 26 analytes were measured using recommended commercial kits from the same brand of the Automated Analyzers. International references values were applied for assessment. For all procedures, the standards of laboratory quality control were followed.post-hoc when necessary.Data were analyzed using R statistical software (Version 4.0.0) and RStudio software (Version 1.2.5042) and models packages “rstatix”, “stats”, “epiR” and “UpSetR”. For all analyses, p values < 0.05 were considered statistically significant. For the cross-sectional study, a Fisher’s exact test was used to evaluate the differences in the proportion of malaria cases between PS in communities with more than one PS. Two independent univariable logistic regression analysis was performed to determine associated factors to develop an Asym malaria infection. The first analysis included sociodemographic information such season, sex, age group, diagnostic status, travel history and occupation. The second analysis was focused on the household characteristics. The multivariable logistic regression analysis only included factors from the univariable analysis with results of likelihood ratio test . The two final predictive models contained all potential variables that were significantly associated with Asym malaria infection. For the nested case-control study, a Kruskal-Wallis test was used to compare results between clinical status groups , and a Dunn test as Geographical analysis was done using QGis (v. 3.10.10) and R packages’ ‘sp’, ‘spdep’, and ‘sf’. First, Heatmaps were constructed with the number of Pv Sym and Pv Asym malaria cases confirmed by qPCR per house in each community employing a kernel density estimation (KDE) on QGis. Later, two indexes were calculated to assess spatial autocorrelation of the number of Asym cases in each site: Global Moran’s I and Local Moran’s I. If a village had more than one visit, indexes were calculated for each one of those. As our dataset was composed of coordinates, we defined neighbors by the distance between them. To avoid isolates, we calculated the threshold distance at which any point that had at least one neighbor inside its own village. From here, we performed 99,999 Markov chain Monte Carlo (MCMC) permutations to assess the significance of our Global Moran’s Index. Finally, to assess local spatial autocorrelation, we computed the Local Moran’s I using the same minimal distance for each village and correcting for false discoveries (FDR). This process allows us to classify houses in four categories: High - High, houses with a high number of Asym cases surrounded by points with a high number of them; Low - Low, houses with a low number of Asym cases surrounded by houses with a low number of them, and High - Low and Low - High respectively.The studies involving human participants were reviewed and approved by the Ethical Committee of the Universidad Peruana Cayetano Heredia . The participants provided written informed consent at the beginning of the study during PS (SIDISI code 101518 approved on 10/27/2017) and to participate in the nested case - control study (SIDISI code 101497 approved on 10/26/2017).A total of 2159 individuals (≥18 years old) and 688 households were surveyed across 11 cross-sectional studies carried out between 2018 and 2021, in 13 communities in four districts, with different living conditions from the Peruvian Amazon , had at least one bedroom , and no more than two beds  per house. The predominant material for walls and floors was wood 91.9% (632/688) and 57.6% (396/688), respectively; and roofs were mainly made of calamine or tin . The availability of potable water was scarce in most communities , and the primary water source for drinking was obtained from adjunct rivers or rain . The proportion of households that possessed more than one net treated with insecticide was high , and a low proportion of residents  reported their houses were sprayed with insecticide ; therefore, qPCR was used as the reference method for confirmation of Pv infections for all analyses  in Jun/Jul 2019; however, six months later (Jan/Feb 2020), this prevalence significantly decreased to 11.5% (15/130)  despite being wet season  in Nov/Dec 2018 to 22.7% (25/110) in Jun/Jul 2019 and 19.9% (29/146) in Jan/Feb 2020 , reflecting the persistence of malaria transmission in this specific area in comparison to other communities  of the individuals experienced headaches, and only 23.2% (19/82) of individuals reported fever; 18.3% (15/82) reported dizziness, and 17.1% (14/82) reported muscle pain. Others also reported general discomfort  and chills , while some individuals also reported loss of appetite, abdominal pain, fatigue, stomach ache, diarrhea, and eye pain. Among the Pv Asym individuals, 86% (141/164) did not report any symptoms commonly associated with malaria; however, some developed symptoms including, but not limited to, muscle pain , dizziness , general discomfort , and eye pain , only 78 individuals agreed to be followed up until D21 and at the last day of follow-up donated blood for immunological, hematological, and biochemical assays. Here, 44.9% (35/78) were classified as Pv Sym at D0, however, 17.9% (14/78) of Asym individuals classified as such at D0, experienced malaria symptoms during the follow-up weeks (28.6% (4/14) at D7, 21.4% (3/14) at D14, 21.4% (3/14) at D21), and 28.6% (4/14) on the final day of sample collection, and were, therefore, added to the symptomatic group. 6.1%, (3/49) from this group reach microscopy-detectable parasitaemia. 37% (29/78) individuals remained Asym throughout all follow-ups, within this group, five infections (17.2% (5/29)) became detectable by microscopy (40% (2/5) at D7 and 60% (3/5) at D14)  compared to Pv Sym infections  , where most infections were Asym (66.7%) and submicroscopic (82.9%).The results from PS in Santa Rita, San Jose de Lupuna and San Pedro communities, located at the basin of the Nanay river, showed a median prevalence of Pv infections by qPCR of 21.3% (CI 95% [11.5 – 25.4]) and 1.25% (CI 95% [0 – 10.6]) by microscopy, where the highest prevalence was found on June-July 2019 and the lowest in January-February 2020. Notably, the mean ratios of Asym versus Sym Pv episodes during these collections showed and increment from 2019 (~3.0) to 2020 (~4.5). Furthermore, analysis of these results revealed a shift from 2018 to 2020 in Santa Rita where Pv prevalence found by qPCR only reduced by 15%, whereas Pv prevalence found by microscopy decreased by half. The assessment of Pv infections at one time point in Tarapoto and Llanchama communities, also located near to Napo River basin, and Centro Fuerte community (near to the Momon river) showed similar findings of low Pv prevalence and high proportions of Pv Asym individuals. The assessment of malaria prevalence in communities from Mazan district, such as Libertad, Primero de Enero, and Urco Miraño, along with their historical records , also shPlasmodium ssp. recurrences in individuals from Western Thailand . Previous experiences of studies carried out by our research group in the same settings in 2015, found an estimated geometric mean of Pv parasitemia of 90 par/μL (95% CI [42–190]) where 50% of them had less than 10 par/μL . Age treDuring the case-control study, 44.9% (35/78) individuals were classified as Pv Sym at D0, 17.9% (10/78) of Asym individuals from D0, experienced malaria symptoms during the follow-up weeks and were, therefore, added to the symptomatic group (49/78), and 37% (29/78) remained Asym throughout all follow-ups. Moreover, 17.2% (5/29) of Asym infections detected by qPCR became detectable by microscopy but remained Asym and only 6.1% (3/49) became Sym at the last day of follow-up. Few longitudinal studies in Amazonia have reported data on the clinical evolution of Asym parasitemia . SimilarIn this study, Asym infection were prevalent during dry season. Indeed, the high prevalence of Pv Asym infections during the dry season has been frequently reported with median duration of Pv infections that can last up to 9 months, with oscillating parasitemia between ultra-low-density and high-density infections that can bridge the dry season and could become a potential source of transmission for the wet season . As AsymAsym individuals tended to be found in houses with wooden walls, and the possession of at least one not impregnated mosquito net had a protective effect on the house, as these families had 56% decrease odds to present an Asym family member. Our results are similar from a study conducted in 2009 in individuals that lived in the forest of central Vietnam, with resembling living conditions as the participants from our study . In mala9/L) as well increased levels of Protein Reactive C , categorizing them with mild anemia , howeverAdditional results from physical evaluations of the participants showed normal corporal mass index and no palpable livers or spleens were reported (data not shown). This finding reinforces results from a previous study that Pv Asym infections could go unnoticed by laboratory routine exams or clinical evaluation . We arguOne of the main limitations of this study was the absence of other pathogen screenings, the exploration of micronutrient deficiencies and the immunological and genetic backgrounds of the participants. This information was only registered by interview and could have been fundamental for the interpretation of hematological and biochemical clinical parameters between Pv Sym and Asym individuals. Futures studies will integrate these epidemiological data with results of immunological studies on PBMCs, plasma, and serum samples from the same individuals in order to better characterized the innate and acquired immunological response of Pv Asym individuals in the Peruvian Amazon. This information is relevant to augment the general knowledge of asymptomatic condition in infectious diseases.In this study, we compared different epidemiological, clinical and biochemical characteristics, as well as hematological parameters between Sym and Asym Pv infected individuals, and control individuals from the Peruvian Amazon, in the search of biomarkers of clinical immunity to Pv infection. Our data revealed a low but heterogeneous Pv prevalence across communities, where most infections were Asym and submicroscopic. During this study, most Sym individuals experienced headache, and not fever, as a common symptom. As previously reported, we observed fluctuation of low parasitemia in Pv Asym individuals during the follow-ups and only a small proportion of these individuals became Sym or microscopy detectable. Further, we identified that houses with wooden walls were associated to Asym infections and that unimpregnated mosquito nets had a protective effect against Asym infections. Finally, we showed that Pv Asym infections may go unnoticed by laboratory routine exams or clinical evaluation. A solid understanding of malaria Asym infections is important for the design of novel malaria control strategies as the country works towards to its elimination.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethical Committee of the Universidad Peruana Cayetano Heredia . The patients/participants provided their written informed consent to participate in this study.KT, DG, JV, and EV contributed to the conception and design of the study. EV, KG, JT, KT, AC, MG, and CR performed the methodology. SSGC, JG and CA performed the statistical analyses. Investigation: EV, SG, and KT. Resources: KT, DG JMV, and EV. Data Curation: SSGC, MG, EV, KT, JT, and JG. Writing – original draft preparation: EV, SSGC, JG, and KT. Writing – review and editing: EV, SSGC, JG, KG, JT, KT, AC, MG, CR, CA, DG, and JV. Visualization: SSGC, JG, and CA. Supervision: KT, DG, and JV. Project administration: KT, DG, and JV. Funding acquisition: KT, EV, DG, and JV. All authors contributed to manuscript revision and approved the submitted version.Funding for this study was provided by The International Centers of Excellence for Malaria Research (ICEMR) program (U19AI089681), NIH–USA; FOGARTY International Center (D43TW007120), NIH-USA. EV is supported by Proyecto Concytec – Banco Mundial “Mejoramiento y Ampliación de los Servicios del Sistema Nacional de Ciencia Tecnología e Innovación Tecnológica” 8682-PE, through its executing unit ProCiencia [08-2018-FONDECYT/BM-Programas de Doctorados en Áreas Estratégicas y Generales].We thank all residents and local authorities from the communities for their enthusiastic participation in the study, as well as all field workers for their dedication during the fieldwork. EV acknowledges the financial support of Proyecto Concytec – Banco Mundial “Mejoramiento y Ampliación de los Servicios del Sistema Nacional de Ciencia Tecnología e Innovación Tecnológica” 8682-PE, through its executing unit ProCiencia [08-2018-FONDECYT/BM-Programas de Doctorados en Áreas Estratégicas y Generales]. We thank Oscar Nolasco and Roberson Ramirez for their technical support for during qPCR experiments. We also thank logistic and administrative support of Rosa Alban, Yulissa Vasquez, and Yovana Maldonado from the Malaria Laboratory at UPCH.This study received funding from the World Bank Group. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.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 is largely documented and this parasite is the main target for malaria eradication, other Plasmodium species persist, and these require more attention in Africa. Information on the epidemiological situation of non-P. falciparum species infections is scarce in many countries, including in the Democratic Republic of the Congo (hereafter Republic of the Congo) where malaria is highly endemic. The aim of this study was to determine the prevalence and distribution of non-P. falciparum species infections in the region south of Brazzaville.Although Plasmodium infection in blood samples was detected by microscopic analysis and nested PCR .A cross-sectional survey was conducted in volunteers living in rural and urban settings during the dry and rainy seasons in 2021. Socio-demographic and clinical parameters were recorded. Plasmodium spp. infection was 31.2% and 63.7%, respectively. Microscopic Plasmodium malariae infection was found in 1.3% of participants, while sub-microscopic studies detected a prevalence of 14.9% for P. malariae and 5.3% for Plasmodium ovale. The rate of co-infection of P. malariae or P. ovale with P. falciparum was 8.3% and 2.6%, respectively. Higher rates of sub-microscopic infection were reported for the urban area without seasonal fluctuation. In contrast, non-P. falciparum species infection was more pronounced in the rural area, with the associated risk of the prevalence of sub-microscopic P. malariae infection increasing during the dry season.Of the 773 participants enrolled in the study, 93.7% were from the rural area, of whom 97% were afebrile. The prevalence of microscopic and sub-microscopic P. falciparum species in malaria control programs, surveillance measures and eradication strategies in the Republic of the Congo.There is a need to include non-The online version contains supplementary material available at 10.1186/s13071-022-05312-9. Anopheles mosquitoes. The WHO estimated that in 2020 there were 241 million malaria cases in 85 malaria endemic countries worldwide, an increase from 227 million in 2019, mainly in countries in the WHO African Region  as mono-infection), while P. malariae was prevalent at a level of 1.3% (10/773) individuals (including 0.5% [4/773] as mono-infection). The rate of P. falciparum/P. malariae co-infection was 0.8% (6/773). According to the districts, the rate of positive microscopic infection was significantly (P < 0.0001) higher in Goma Tsé-Tsé  than in Madibou . Plasmodium malariae was identified in 1.4% (8/573) of the participants living in Goma Tsé-Tsé District with 0.7% (4/573) of cases co-infected or not with P. falciparum. Plasmodium malariae was detected in 1% (2/200) of the participants living in Madibou as co-infection with P. falciparum. Although the prevalence of microscopic Plasmodium  spp. infection was low in participants aged < 5 years , parasite density in children in this age group was higher both for P. falciparum and P. malariae than in children aged between 6 and 14 years  of all samples , 14.9% (79/532) and 5.3% (28/532) of participants, respectively. Dual Plasmodium infection with P. falciparum/P. malariae accounted for 8.3% (44/532) of Plasmodium spp. infections, while dual infections with P. falciparum/P. ovale and P. malariae/P. ovale accounted for 2.6% (14/532) and 0.2% (1/532), respectively. Triple infections were detected in 1.7% of all participants.Sub-microscopic 7% 339/53 of all sPlasmodium spp. infection was significantly higher (P = 0.0099) in Madibou  than in Goma Tsé-Tsé . A similar trend was observed for the prevalence of mono-infection by P. falciparum (Madibou: 56% (89/160); Goma Tsé-Tsé: 41% [153/372]), while the reverse was observed for P. malariae infection  and P. ovale infection . The prevalence of sub-microscopic infection was high in all age groups , but no relationship was observed between P. malariae infection and the risk of anemia (Table P = 0.0003) for increased P. falciparum prevalence in Madibou. Plasmodium falciparum infection was associated with gender in Goma Tsé-Tsé , but no such association was found in Madibou. According to the age group, P. falciparum prevalence for all age group was higher in Goma Tsé-Tsé District than in Mabidou District, with participants aged 5 to 14 years having a higher risk of malaria infection. The presence of fever was significantly  associated with P. falciparum infection in the two districts. No association was observed between the socio-demographic characteristics of the study population and the microscopic prevalence of P. malariae in the two districts , and in the absence of bed net use. Participants aged between 15 and 19 years were at the highest risk  of sub-microscopic P. falciparum infection in Goma Tsé-Tsé District (Table P = 0.045) of sub-microscopic P. malariae infection during the dry season. No risk factor was identified for the presence of sub-microscopic P. ovale infection , suggesting a strict compliance of the inhabitants of these localities to the malaria control measures.Since publication of the systematic review on the malaria situation in the Republic of the Congo in 2016 , studiesnfection –32. HowePlasmodium spp. detected by microscopy was 31.2% in this study. Plasmodium falciparum remained the most prevalent species, in accordance with prevalences reported in many previous studies conducted in African malaria endemic areas [P. falciparum in the overall study population demonstrates the importance of malaria control strategies specifically against this species. The prevalence of malaria infection was significantly higher in villages of Goma Tsé-Tsé District than in Madibou District, which may be related to the different ecological situations in these two areas  [Plasmodium spp. in this study was higher than that reported in recent studies in Benin (23.7%) by our research group [The overall prevalence of ic areas –36. The . urban) . The prech group , but simch group .-P. falciparum species in the whole study population, only P. malariae was detected by microscopy at a rate of 1.3% . This prevalence was higher in the rural area of Goma Tsé-Tsé District (1.4%) compared to the urban area of Madibou District (1%), suggesting that this variation in prevalence was probably due to the socio-demographic characteristics of each study area. Suitable containers or conditions for water to pool and persist, deforestation and agriculture have all been associated with an increased risk of malaria transmission [P. malariae prevalence observed in this study was comparable to that reported in Benin, in which an increase of this prevalence was observed over the past 10 years [-Plasmodium falciparum parasites that must be taken into account in the WHO strategic plan for malaria eradication [Among the identified nonsmission . The P. 10 years . A potendication .P. falciparum infection in Madibou District. In the villages of Goma Tsé-Tsé District, where the prevalence of P. falciparum was high and almost stable throughout the year, men and children appeared to be at higher risk of getting malaria infection. All of these results suggest that malaria management strategies must be directly applicable to the area they are being used: in urban areas, they should take seasonality into account; in rural areas, they must be permanent, with a particular focus on the sensitization of men and adolescents. Fever was significantly associated with P. falciparum infection in the two districts of this study. We did not observe any association between socio-demographic factors or clinical outcomes with an increased prevalence of P. malariae, probably due to low number of events and, therefore, power issues. However, many studies have reported a significant association of P. malariae infection with severe anemia, probably due to prolonged erythrocyte destruction and bone marrow suppression with a minimal reduction in erythrocyte life-span at a low parasitemia level [P. malariae infection [P. malariae infection was reported to be 2% [The rainy season was associated with a higher prevalence of ia level , 39. Pulnfection . The pooto be 2% .Plasmodium spp. was observed, being more pronounced in older participants (> 15 years). These findings are similar to those reported in a previous study from Ghana (66%) [Plasmodium infection endemicity. Indeed, in Benin, the survey was conducted in villages, while in Ghana, the study covered a mixture of communities. In addition, the sub-microscopic prevalence in our study varied with respect to the study district, being lower (59.8%) in the rural area compared to the urban areas (73%). The high prevalence of sub-microscopic infections observed in the present study, as well as in other studies [, represents a major challenge to malaria control programs in the Republic of the Congo since the management of malaria patients is based on the microscopic detection of parasites. Our findings suggest that despite the best efforts of the national malaria program, malaria transmission will continue to be high because half of infected individuals remain afebrile and will, therefore, not be identified as having malaria and subsequently treated. Among the non-P. falciparum identified by PCR, P. malariae and P. ovale were detected in 14.9% and 5.3% of participants, respectively. In terms of co-infection, participants co-infected with P. falciparum/P. malariae (8.3%) were the most prevalent, followed by participants co-infected with P. falciparum/P. ovale (2.6%). It is notable that although non-P. falciparum parasites were hardly detected by microscopy, the high rates of sub-microscopic detection of these parasites translate into perennial transmission of these species in endemic areas, thereby constituting a major challenge to achieving malaria eradication. The presence of persons with P. ovale and P. malariae co-infected with P. falciparum in this study highlights the impact of those two latter parasites in asymptomatic and chronic malaria infection. This becomes more critical with triple infection, which was detected at a sub-microscopic prevalence of 1.7% in the present study. Plasmodium malariae and P. ovale are rarely associated with severe cases of malaria, but their persistence in the body remains a great challenge for malaria eradication. Although P. malariae does not cause malarial relapse from persistent liver-stage parasites, recrudescence of blood-stage parasites can occur after long periods without signs or symptoms, even after the infected person has left the endemic region [P. malariae infection can occur because of its extended pre-patent period when inadequate drug levels in the blood cannot eliminate newly emerging merozoites [Plasmodium ovale is responsible for malaria relapses, even after months or even years without the person showing symptoms, due to the presence of hypnozoites [P. falciparum species were more pronounced during the dry season. The reason for this association remains unclear. The small numbers of non-P. falciparum species detected in our study is a limitation to this study, especially in sub-analyses on demography or other clinical factors.Compared to microscopic malaria infection, a higher sub-microscopic infection (63.7%) of na (66%) , and higna (66%) . This di studies , 30, repc region , 18. Everozoites , 42. Planozoites , 12. In -P. falciparum species in malaria control programs, surveillance measures and eradication strategies in the Republic of the Congo. Therefore, a continuous systematic prevention promoting the use of bed nets and intermittent treatment of pregnant women, screening and treatment of Plasmodium-infected individuals living in high-transmission settings may strengthen malaria intervention measures.The results of this study highlight the need to include nonAdditional file 1: Table S1. List of primers used for the nested PCR.


Plasmodium vivax, Plasmodium ovale spp., and Plasmodium malariae. Biological features such as association with low-density infection and the formation of hypnozoites responsible for relapse make their elimination challenging. Studies on these parasites rely primarily on clinical samples due to the lack of long-term culture techniques. With improved methods to enrich parasite DNA from clinical samples, whole-genome sequencing of the neglected malaria parasites has gained increasing popularity. Population genomics of more than 2200 P. vivax global isolates has improved our knowledge of parasite biology and host-parasite interactions, identified vaccine targets and potential drug resistance markers, and provided a new way to track parasite migration and introduction and monitor the evolutionary response of local populations to elimination efforts. Here, we review advances in population genomics for neglected malaria parasites, discuss how the rich genomic information is being used to understand parasite biology and epidemiology, and explore opportunities for the applications of malaria genomic data in malaria elimination practice.Malaria elimination includes neglected human malaria parasites  Plasmodium. Five species are commonly accepted as human malaria parasites: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, and Plasmodium ovale wallikeri. The latter two are recognized as morphologically indistinguishable, sympatric, but non-recombining species only in the last decade , production of fewer merozoites per schizont (average of 8), and an extended erythrocytic cycle (72 h)  . Low-dene (72 h) . Gametocfections . Finallyrs later . These clciparum . Since nlciparum , a bettePlasmodium DNA  technologies have enabled whole-genome sequencing (WGS) of a large number of clinical isolates of malaria parasites, allowing the study of parasite populations at the genomic level – population genomics. However, there have been significant barriers to using the WGS technology within the neglected malaria species. The most notable is the reliance on clinical samples, which often have low parasitemia and contamination with human genomic DNA. Since the human genome (∼3000 Mb) is about 100 times larger than the malaria parasite genome (25–35 Mb), the problem associated with the human genome is obvious. Therefore, leukocyte depletion using filtration methods  is instrumental for obtaining high-quality parasite genomes . Additiodium DNA . SWGA prmalariae . Anothermalariae , is partmalariae . Here weP. falciparum 3D7 genome is the most complete reference Plasmodium genome  dramatically improved assembly quality, reflected in the substantially reduced number of unassigned contigs and better resolved subtelomeric regions where there were nearly four times as many hypervariable pir genes as identified from the Sal-I assembly  and one P. ovale curtisi (Nigeria) were historic samples isolated in 1974 and 1977, respectively, and passaged through chimpanzees. Subsequently, a high-quality genome assembly of a P. malariae strain from Uganda using long-read sequencing technology and draft genomes of P. ovale curtisi (from Ghana) and P. ovale wallikeri (from Cameroon) assembled from P. falciparum co-infections were reported  and P. malariae (∼34 Mb).In terms of accuracy and completeness, the m genome , and it m genome . Howeveromosomes . Shortlyuritania . De novoor genes . The 201assembly . The rec1 genome . With th in 2016 . The tworeported . ComparePlasmodium genome assemblies  are especially affected as they depend on discordant read alignment and read depth , both cosemblies . FortunaP. falciparum being the most well-annotated species, the most recent version of PlasmoDB (Update 57) included updates of 223 P. falciparum genes and less than 60 updated genes for any other human or primate Plasmodium species  gene was common in Malagasy P. vivax strains infecting Duffy-negative individuals  in the Greater Mekong subregion (GMS), where nearly all local populations are Duffy positive  defines the degree of multiple infections and the similarity of those infections . It is slciparum . As genolciparum . COI is WGS data . When coWGS data  and for WGS data . Single-WGS data , but mayFWS) statistic is one popular method  of monoclonal infection (FWS ≥ 0.95) than other endemic areas such as Thailand, Indonesia, and Ethiopia (52–71%), consistent with the decline of vivax malaria incidence in Malaysia followed by unstable transmission and outbreaks , microsatellite markers, and more recently, SNPs  , phylogeny, pairwise IBD, and admixture. It may also be more directly studied by comparing populations using Wright’s fixation index (FST) and shared IBD  . As NGS cade ago , which c in 2016 , and mor in 2016 . TogetheCambodia , southerCambodia , MalaysiCambodia , MyanmarCambodia ], South Cambodia , Peru , South Asia (India), and Southeast Asia  or genetic relatedness  can be compared with geographical distances by the Mantel test to identify differentiation beyond what is expected from geographic isolation alone. In one instance, the identification of P. vivax parasites with significant IBD sharing from two sites 700 km apart in the Brazilian Amazon evidenced long-distance migration  may be used to identify genes under selection. This type of analysis allows the identification of genes showing elevated genetic diversity and positive D  or negative D values (negative selection or selection sweep) , AP2 transcription factors, and genes potentially related to drug resistance  . The abilciparum . However signals . XP-EHH sistance ; Table 1pressure . The div vectors .P. falciparum -resistant and –sensitive parental strains before and after CQ administration, the authors calculated an effect e-value for each marker and identified those on chromosome 1 with consistently high e-values across progeny pairs. Targeted sequencing on chromosome 1 and transcriptomic analysis suggested that CQ resistance may be mediated by changes in pvcrt transcription resulting from an upstream motif. While sample sizes for these analyses were small (4–10 linkage group selection pairs), and it is uncertain how mutations in primate-adapted strains may affect the results, they represent an important step forward in phenotypic analyses in P. vivax. In general, however, the lack of a long-term culture system and the need for non-human primates make it less feasible to apply phenotypic assays to genetic mapping in neglected malaria parasites. Therefore, we rely heavily on population genomics to infer underlying resistance mechanisms.Selection signals from population genomics are especially useful for neglected malaria parasites where resources are scarce and traditional means of genetics are difficult. Genetic linkage mapping using experimental crosses and genome-wide association studies (GWAS) have successfully mapped loci associated with drug resistance in lciparum . To datePvdhps and Pvdhfr, mediating resistance to the antifolate drugs sulfadoxine and pyrimethamine (SP), respectively  and 2 (pvmrp2)  .P. vivax populations from Oceania and the GMS, some of which harbor genes related to drug resistance  but was absent in Indonesia or Ethiopia  may also differ among populations and potentially lead to drug resistance. CNVs with a marked geographical difference in frequency were identified between sistance . Among tsistance , had ∼20Ethiopia . A shortarasites . DetectiP. vivax culture or convenient animal models for genetic manipulation precludes direct evaluations of their roles in drug resistance. The expression of these identified P. vivax genes in transgenic P. knowlesi (P. knowlesi (P. vivax genes. Incorporating population genomics into the study of neglected malaria parasites may provide insights into the nature of the selective pressure.Population genomic studies have identified various genes under selection, some of which may mediate drug resistance, but the lack of a long-term knowlesi , a closeknowlesi , have opP. falciparum barcode consisting of 24 SNPs (P. vivax populations appear to require more SNPs, likely due to higher genetic diversity (P. vivax genomes available at that time (P. vivax WGS data have become available from more geographic regions, a new 71-SNP barcode has been designed, allowing the geographic origins of parasites to be predicted at 91.4% accuracy (Even with the continuous reduction of WGS costs, it is still prohibitively expensive to perform large-scale population genomic studies of malaria parasites. Yet, the availability of WGS data of parasites from various endemic areas such as those generated from the MalariaGEN Consortium has informed researchers to design ‘genetic barcodes’, which offer a reductionist approach to inferring transmission networks and population structures by using a small set of SNPs . Barcode 24 SNPs  has prov 24 SNPs , althougiversity . A set ohat time . Though hat time . The addhat time . Recentlaccuracy . Though accuracy , while taccuracy , but it P. knowlesi, P. cynomolgi, and P. simium are increasingly recognized as additional targets for malaria elimination. Malaysia has been cleared of human malaria since 2018 but continues to report thousands of human cases of P. knowlesi (in vitro culture in human RBCs has opened new venues to study these parasite species in the lab, which may serve as model systems for functional genomics of their closely related human malaria species, such as P. vivax (P. knowlesi from a rhesus macaque was sequenced, revealing a 23.5 Mb nuclear genome (P. knowlesi genome of 24.77 Mb to consist of 14 contigs with 25 gaps (de novo assemblies of clinical field samples (P. cynomolgi was sequenced in 2012, resulting in three draft genomes (P. simium draft genome was only recently sequenced, containing over 2000 scaffolds (Zoonotic malaria species infecting humans such as knowlesi . As globknowlesi . Genomicknowlesi . The adaP. vivax . In 2008r genome . Subsequ 25 gaps . Recentl samples . P. cyno genomes ; the ass genomes . The P. caffolds . ImprovePlasmodium species due to the ability to amass a large amount of data with few samples and little a priori knowledge. Of the zoonotic species, population genomics studies have been undertaken primarily on P. knowlesi due to the large number of human infections occurring in Malaysia (P. knowlesi populations are structured not just geographically and between clinical and laboratory samples but also by primate hosts (P. simium, advancement in population genomics includes compelling evidence that the species diverged from P. vivax in a rare occurrence of reverse zoonosis (P. simium samples revealed high genomic similarity to new-world P. vivax samples, but not to old-world P. vivax samples (P. vivax spread to the Americas and diverged from its Asian cousins before adapting to local macaques. Finally, very little genomic research has been done with P. cynomolgi field populations. The development of genomic tools and resources for studying zoonotic malaria species may provide crucial findings on the parasite’s host adaptation and the biology of closely related human parasites.Population genomics techniques have a clear benefit in neglected Malaysia . Most note hosts . The orite hosts . A recente hosts  leaves tte hosts . In P. szoonosis . Analysi samples , supportWith technical improvements to enrich parasite genomes from very small amounts of DNA, the number of genomes of the neglected malaria parasites sequenced is rapidly growing, providing a new means to address many aspects of parasite biology. High-coverage WGS paves the way to precisely determine the relatedness of recurring parasites with parasites from the initial infection, setting up a platform for studying the biology of relapses . PopulatP. vivax (P. ovale spp. and P. malariae, researchers would benefit immensely from large numbers of genomic data collected from various populations to hasten our understanding of their epidemiology and biology. Insights into how they transmit and recur or relapse in different settings will become especially important as P. falciparum decreases and other species stand in the way of malaria elimination. In simian species, including P. knowlesi, P. cynomolgi, and P. simium, population genomics may give insights into their transmission and possible additional hosts, and may even help researchers assess the threat of human transmission. For P. vivax, increased sample acquisition on finer geographical scales remains an important objective, especially for tracking regional parasite movement. We still lack P. vivax genomes from west Africa, where Duffy negativity is most prevalent. Additional locations where there are relatively large numbers of cases but relatively few WGS samples include the areas between Bangladesh and Myanmar, and northeastern South America, specifically around Venezuela (Population genomics of neglected malaria parasites offers a new frontier of malaria research. While the malaria community has recently generated an impressive number of over 2200 whole-genome sequences for P. vivax , the stuP. vivax . In P. oenezuela . Furtherenezuela . ImproveAB and LC conceived, drafted, revised, and gave final approval to the article. Both authors contributed to the article and approved the submitted version.


Plasmodium of which five infect humans, namely Plasmodium falciparum, P. vivax, P. ovale, P, malariae, and P. knowlesi. It is a life threatening disease of tropical countries around the globe, and more than 600,000 deaths occurred in 2020  whilst the South-East Asia region accounts for 2.2% of fatal cases and the Eastern European countries for 2.5%. There is also malaria in the Western Pacific region and in the Americas with Brazil having the highest incidence rate .For diagnosis WHO recommends “prompt parasite-based diagnosis by microscopy or malaria rapid diagnostic test (RDT) in all patients suspected of malaria before antimalarial treatment is administered. Light microscopy entails visualization of the malaria parasites in a thick or thin smear of the patient's blood” WHO, . 1 MicroP. vivax detection as circulating antigen and is being used in discriminating RDTs  have been developed around 1995, successfully tested, and finally implemented worldwide  albeit with lower sensitivity, WHO has called an immediate action to address these problems , it hardly can be replaced by highly sensitive molecular tools such as PCR or LAMP (often referred to as Gold Standard), because of the technical requirements and costs. Usually, in elimination areas, index cases (with significant parasitemia) are identified by microscopy. Subsequently, family and close-by living individuals are treated without further confirmation  microscopy which has a limit of detection of 10–50 parasites/μl , and most importantly must allow a diagnostic test at affordable costs for developing countries. For the latter requirement, it might be that innovative funding schemes could support such development.In conclusion, microscopy as diagnostic test for malaria will remain despite other means of diagnosis such as RDTs, because of its ability to enumerate parasitemia and to identify all five species, as well as to detect the transmissible gametocytes. Ideally, to overcome human limitations, automatic microscopic devices with embedded artificial intelligence would provide the same or better quality of results (in particularly with constant improving) and could eventually become the reference for malaria diagnosis based on microscopic examination in endemic areas.The author confirms being the sole contributor of this work and has approved it for publication.Author HP-B acts as scientific advisor to the company Noul Co., Ltd.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.