The Republic of Djibouti is a malaria endemic country that was in pre-elimination phase in 2006–2012. From 2013, however, malaria has re-emerged in the country, and its prevalence has been increasing every year. Given the co-circulation of several infectious agents in the country, the assessment of malaria infection based on microscopy or histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDT) has shown its limitations. This study, therefore, aimed to assess the prevalence of malaria among febrile patients in Djibouti city using more robust molecular tools.All suspected malaria cases reported to be microscopy-positive were randomly sampled (n = 1113) and included in four health structures in Djibouti city over a 4-year period (2018–2021), mainly during the malaria transmission season (January–May). Socio-demographic information was collected, and RDT was performed in most of the included patients. The diagnosis was confirmed by species-specific nested polymerase chain reaction (PCR). Data were analysed using Fisher’s exact test and kappa statistics.Plasmodium falciparum, 88 (11.2%) Plasmodium vivax, and 44 (5.6%) P. falciparum/P. vivax mixed infections. In 2020, P. falciparum infections were confirmed by PCR in 50% (144/288) of negative RDTs. After the change of RDT in 2021, this percentage decreased to 17%. False negative RDT results were found more frequently (P < 0.05) in four districts of Djibouti city . Malaria occurred less frequently in regular bed net users than in non-users .In total, 1113 patients with suspected malaria and available blood samples were included. PCR confirmed that 788/1113 (70.8%) were positive for malaria. Among PCR-positive samples, 656 (83.2%) were due to P. falciparum hrp2 gene deletion, which leads to false negative cases of P. falciparum.The present study confirmed the high prevalence of falciparum malaria and, to a lesser extent, vivax malaria. Nevertheless, 29% of suspected malaria cases were misdiagnosed by microscopy and/or RDT. There is a need to strengthen the capacity for diagnosis by microscopy and to evaluate the possible role of The online version contains supplementary material available at 10.1186/s12936-023-04546-x. In 2020, the World Health Organization (WHO) reported 241 million cases and 627,000 deaths due to malaria worldwide despite the fact that considerable efforts have been made to control and/or eliminate malaria , 57.7–64.4%) than females , with a sex-ratio of 1.6 (262 missing data on sex). The mean (± standard deviation [SD]) and median ages were 30.4 years (± 18.0 years) and 28 years, respectively, ranging from 2 months to 95 years.A total of 1113 patients with samples available for molecular diagnosis were included during the malaria transmission season in 2018–2021 . The samples were collected from Health Centre 1 (n = 615), Health Centre 2 (n = 325), Einguella Health Centre (n = 29), and Djibouti City General Hospital (n = 144). The proportion of males  was significantly higher . Between 2020 and 2021, the prevalence in the period January-April decreased significantly  from 75.9% (259/341) to 68.1% (344/505).PCR was successfully performed and interpretable in all 1113 (100%) samples, consisting of 729 dried blood spots and 384 RDTs, available for molecular diagnosis. The overall prevalence of malaria based on PCR-confirmed cases was 70.8% (788/1113) in 2018–2021. This prevalence was 81.2% (65/80) in 2018 between September and December and 64.2% (120/187) over the whole of 2019. In the period September-December only, the prevalence in 2019 was 76.1% (54/71) and the difference with the previous year was not statistically significant  were due to P. vivax, and 44/788 (5.6%) were due to mixed P. falciparum/P. vivax infections , 656/788 (83.2%) were due to , 88/788 1.2% wereP = 0.7). The proportions of P. falciparum infections confirmed by PCR in 3 different age groups  did not differ significantly (P = 0.07) (Table P < 0.05) of PCR-confirmed malaria-infected patients among 0–9 year olds and 60–79 year olds. On the other hand, patients in the 10–29 year old age group were proportionally overrepresented  age of patients with symptomatic malaria infection confirmed by PCR was 30.5 ± 18.2) years. There was no significant difference with the mean age of the patients with PCR-negative results included in the study  times higher than in females.Concerning the sex of the patients, of the 851 patients with a PCR result and their sex documented, 75.4% (392/520) of males and 64.7% (214/331) of females had malaria. The OR of malaria prevalence among males was 1.67 , compared to those who did not use bed nets (Table Information on the reported use of bed net was obtained from 600 of 1113 (53.9%) febrile patients. Of these respondents, 64.2% (n = 385) reported that they do not possess a bed net or they have at least one bed net in the household but never use it. Only 215 of 600 (35.8%) reported to sleep under a bed net, of whom 34 (5.7%) were occasional users and 181 (30.2%) were regular users. The proportion of malaria infections was 69.9%, 61.8%, and 59.1% among non-users, occasional users, and regular users, respectively  of the samples tested. During the 4-year period of the study, the percentages of RDT-positive P. falciparum were 84.6% in 2018, 17.3% in 2019, 23.0% in 2020, and 53.5% in 2021   in 2020 with CareStart® HRP2-based RDT. However, 17.1% of false negatives (64/374) were observed in 2021 with Biosynex RDT (pf-HRP2/pan/LDH). In contrast, the proportion of false-positive CareStart® RDTs was 14% (11/78), 3% (6/187) and 2% (6/288) in 2018, 2019, and 2020, respectively. In 2021, 6.7% (25/374) of Biosynex® RDTs were false positives , but the specificity was low . However, in 2019, the sensitivity of CareStart® RDT decreased to 28.1% . The sensitivity and specificity of Biosynex® RDT in 2021 was 73.1%  and 81.6% , respectively. The difference in the sensitivity (P < 0.0001) and the specificity (P < 0.01) in the detection of P. falciparum with two RDTs was statistically significant.The sensitivity of CareStart® RDT in 2018–2020, compared to PCR, for the detection of P. vivax infections, the proportion of false-negative RDTs compared to PCR as the reference method was low throughout the study period with the maximum of 4.8% (9/187) in 2019. By contrast, a high proportion of false positives  was observed in 2021. Of these false positives, 45 (67.2%) mixed infections detected by Biosynex® RDT showed only P. falciparum by PCR. The sensitivity of RDTs to detect P. vivax ranged from 60 to 75% depending on the year , but a significant difference was observed in the specificity of the two RDTs (P < 0.0001). PCR and RDT results showed poor agreement for both P. falciparum and P. vivax .As for P. falciparum, the proportion of false-negative RDT cases (i.e. PCR-positive but RDT-negative) varied from one district to another , followed by Arhiba district , “Quartier 7” district , and “Quartier 6” district . Patients from Balbala district had consulted the referral hospital, health centre 1, or health centre 2. Each of these three health facilities had reported false negative RDTs from this district . This difference seems to be associated with nocturnal outdoor activities. Indeed, males are more likely to be infected, especially in this age group. This is consistent with the traditional activities of females who tend to stay home more frequently, leading to a lower level of exposure to Anopheles mosquitoes [In the present study, it was observed that malaria affected all age groups in Djibouti city. The age distribution of the patients included broadly corresponded to the Djiboutian population structure observed in the latest census . The proportion of elderly patients was higher among those who were not infected with malaria . It is unlikely that these differences can be attributed to the acquisition of immunity in older people [The third statistically significant difference observed between r people  because r people . Moreover people . Althougr people , the recr people .Anopheles bites and limit malaria transmission in regular users. Given the current re-emergence of malaria in Djibouti, it will be important for the country to accelerate their vector control policies with massive distribution of LLINs and maintain a tight follow-up on its utilization, as recommended by the WHO. There is also a need to raise community awareness with health education campaigns focusing on the correct use and maintenance of bed nets to optimize their protective effects [An appropriate use of LLINs has been shown to result in the reduction of malaria-associated morbidity and mortality, especially among children, pregnant women, and other vulnerable people . However effects .P. falciparum (83.3%) in Djibouti city, followed by P. vivax (11.2%). No other Plasmodium spp. was detected by PCR in the present study in concordance with the results of previous studies [In line with earlier studies conducted in Djibouti , 10, 11,P. vivax in Djibouti over a period of 4 years using PCR. The results showed that P. vivax accounts for 16.8% of malaria-confirmed cases in Djibouti city, of which 5.6% are mixed P. falciparum-P. vivax infections. Previous monitoring based on RDT or microscopy reported higher percentages of P. vivax. The latest available data from the NMCP reported 13,373 P. vivax infections in 2019, which represents 27% of malaria infections [This is the first study that assessed the prevalence of P. vivax malaria in the present study.i.P. vivax became the predominant species, presumably because of relapses, even though its transmission was also greatly reduced [The present study was mainly conducted during the period of high malaria transmission in Djibouti. In the last two years (i.e. 2020–2021), very few patients were included during the dry season when malaria transmission decreases sharply. During this period,  reduced , 42.ii.P. vivax in the management of patients may also have been accompanied by improved effectiveness of malaria control programmes. The use of primaquine allows the elimination of hypnozoites in the liver. It is prescribed for the treatment of P. vivax-infected patients implemented in Djibouti since 2019 [The recent attention of the specificities of nce 2019 . Althougiii.P. vivax on the continent [P. vivax in the general population of Ethiopia is over 8%, with wide variations between regions [P. vivax has been observed in certain regions of Ethiopia, sometimes even leading to a reversal of the trends, with P. falciparum becoming predominant in an area where P. vivax was previously predominant [The regional context may have had an effect on the situation in Djibouti. The Horn of Africa is the region with the highest prevalence of ontinent . Ethiopiontinent . The pre regions . Given t regions , the evo regions . A trenddominant –50.iv.P. vivax reported in the previous studies [P. falciparum due to the propagation of strains with deleted P. falciparum hrp2 (Pfhrp2) gene, which will not be detected by HRP2-based RDTs [P. falciparum. Strains without the Pfhrp2 gene will not be detected or will be detected late . This situation offers an intra-species selective advantage to Pfhrp2-deleted P. falciparum strains, as evidenced by their spread throughout malarious areas [Plasmodium falciparum strains with deleted Pfhrp2 gene, which is less detectable by many currently available RDTs, would then have an advantage over the P. vivax strains in the face of control strategies based increasingly on RDTs and post-diagnostic treatment only policy [Finally, the failure of the RDTs used in Djibouti may have produced two effects which are consistent with a higher proportion of  studies . The firsed RDTs . This dius areas , 51, buty policy .Four factors could explain the lower proportion of P. vivax. This problem occurred after the introduction of the new test in 2021. Mixed P. falciparum-P. vivax infections had been reported in patients infected with P. falciparum only, as confirmed by PCR. In such cases, surveillance based mainly on RDTs will overestimate the proportions of P. vivax compared to P. falciparum. In contrast, the present study based on PCR avoids this bias.The current study also showed that errors in RDT readings could lead to false positives for P. vivax in the present study, with a decreasing prevalence observed in the last two years of the study (2020–2021). On the other hand, it is difficult to predict how the importation and establishment of An. stephensi in Djibouti could modify the relative transmission of these two plasmodial species. Preliminary data suggested that the arrival of this Asian vector may be accompanied by ecological changes favourable to the spread of P. vivax [These different factors could explain the lower prevalence of P. vivax . In thisP. falciparum were observed during the last three years of the present study. The highest proportion of false-negative P. falciparum (50%) was found when diagnosis was established using HRP2-based RDT. This could be explained by several factors, including low parasite density, low target antigen concentration, poor quality of RDTs due to poor stockage conditions or incorrect warehousing, incorrect use of RDTs by technicians or Pfhrp2 gene deletion [Pfhrp2 gene deletion is of potentially great concern as a recent study in Djibouti detected false-negative P. falciparum by HRP2-based RDT caused by a deletion of the Pfhrp2 gene [P. falciparum strains by HRP2-based RDT because of Pfhrp2 gene deletion [Pfhrp2 gene deletion has reached high levels (9.4–83.5%) in the Horn of Africa [Using PCR as the reference method, the present study highlights the shortcomings of malaria diagnosis that relies on HRP2-based RDT. High proportions of false-negative deletion –55. Due rp2 gene . Moreovedeletion , 57. Indf Africa , 56–58.P. falciparum strains undetected by HRP2-based RDT. This is all the more interesting as patients from the same district consulted in different health centres of this study, thus limiting an introduction of a possible bias due to recruitment and practice in different health centres . There may be an oligoclonal expansion of Pfhrp2-deleted P. falciparum strains, leading to microstructures in Djibouti city as observed in the previous epidemic in 1999 [P. falciparum populations in Djibouti is required to establish the numbers of different circulating strains and characterize their genetic profiles, origins, and evolution in time and place. In addition, a small proportion (2.3%) of false-negative P. vivax by RDT was also confirmed by PCR. This result could probably be explained by low parasitaemia [The distribution of the proportions of false-negative RDTs across Djibouti city suggested a structuring of these strains in certain districts of the city. Balbala, Quartier 6, Quartier 7, and Arhiba were most affected by sitaemia .P. falciparum was observed by HRP2-based RDT. The highest proportion of false positives was 14% in 2018 and 7% in 2021. This could be due mainly to the persistence of HRP2 in recently recovered P. falciparum-infected patients or those presenting the rheumatoid factor, which cross-reacts with HRP2 [P. vivax (7%) were mainly due to misinterpretation of the new RDT introduced in one of the health centres. These results may also suggest an effect of sample storage prior to molecular analysis. However, the overall sensitivity and specificity showed poor performance of RDTs used, with sensitivity for detection of P. falciparum and P. vivax of 58% and 81%, respectively, and specificity of 80% and 97%, respectively. A high rate of misdiagnosis at country level and the accompanying inappropriate anti-malarial drug treatment are potential sources and reasons for ineffective malaria control as well as the spread of drug-resistant P. falciparum (and P. vivax) strains. Indeed, recent studies have documented ACT treatment failure in Djibouti [P. falciparum kelch 13 (Pfk13) gene associated with reduced susceptibility to artemisinins in Eritrea [False-positive ith HRP2 , 61. In Djibouti  and the  Eritrea . To regaP. falciparum was high in two other districts of Djibouti [P. falciparum with pLDH pan. The issue of sensitivity and specificity of RDTs needs to be further investigated on a wider scale in the country. Given the rapidly changing malaria situation and the latest WHO recommendations, a new RDT RapiGEN Biocredit Pf/Pv (pLDH/pLDH), subsidized by the Global Fund, is currently being deployed in 26 health centres in Djibouti and requires further evaluation to assess and adjust malaria control interventions in the country.There are several limitations in this study. First, due to scarcity of qualified microscopists in the country and lack of compliance to the national diagnostic guidelines, parasitaemia was not available. Parasitaemia plays an important role in RDT detection. Parasite density below the RDT detection threshold or very high parasite density can influence the performance of RDT . SecondlDjibouti . Third, P. falciparum is confirmed, and P. vivax, to a lesser extent, remains a public health concern. The performance of HRP2-based RDT to detect P. falciparum was poor and led to its successive changes in February 2021 to pLDH-based RDTs. It is imperative to evaluate regularly the performance of RDTs in Djibouti as there are few well-trained microscopists in the country and false-negative RDTs lead to inadequate or inappropriate treatment and increased malaria mortality and morbidity. Further molecular studies are required to characterize P. falciparum populations that yield false-negative HRP2-based RDT results in order to understand their distribution, origin, and dynamics at both country and regional level.PCR verification of suspected malaria cases in Djibouti city suggested that 29% of non-malarial cases were misdiagnosed by microscopy and/or RDT and mistakenly treated for malaria. The predominance of Additional file 1: Data S1. Number and percentage of positive samples for Plasmodium falciparum and Plasmodium vivax by RDT and PCR in Djibouti city, 2018–2021.Additional file 2: Figure S1. Age of patients by malaria status from four health facilities in Djibouti city between 2019 and 2021.


Plasmodium falciparum and Plasmodium vivax among indigenous Orang Asli communities in Kelantan, Peninsular Malaysia. A community-based cross-sectional survey was conducted in three Orang Asli communities  in Kelantan from June to July 2019. Antibody responses to malaria were assessed by enzyme-linked immunosorbent assay (ELISA) using two P. falciparum (PfAMA-1 and PfMSP-119) and two P. vivax (PvAMA-1 and PvMSP-119) antigens. Age-adjusted antibody responses were analysed using a reversible catalytic model to calculate seroconversion rates (SCRs). Multiple logistic regression was used to investigate factors associated with malaria exposure. The overall malaria seroprevalence was 38.8% for PfAMA-1, 36.4% for PfMSP-119, 2.2% for PvAMA-1, and 9.3% for PvMSP-119. Between study areas, the proportion of seropositivity for any P. falciparum and P. vivax antigens was significantly highest in Pos Kuala Betis with 34.7% (p < 0.001) and 13.6% (p < 0.001), respectively. For all parasite antigens except for PvAMA-1, the proportion of seropositive individuals significantly increased with age . Based on the SCR, there was a higher level of P. falciparum transmission than P. vivax in the study area. Multivariate regression analyses showed that living in Pos Kuala Betis was associated with both P. falciparum  and P. vivax  seropositivities. Significant associations were also found between age and seropositivity to P. falciparum and P. vivax antigens. Analysis of community-based serological data helps describe the level of transmission, heterogeneity, and factors associated with malaria exposure among indigenous communities in Peninsular Malaysia. This approach could be an important adjunct tool for malaria monitoring and surveillance in low malaria transmission settings in the country.Malaria remains a public health problem in many parts of the world. In Malaysia, the significant progress towards the national elimination programme and effective disease notification on malaria has resulted in zero indigenous human malaria cases since 2018. However, the country still needs to determine the extent of malaria exposure and transmission patterns, particularly in high-risk populations. In this study, a serological method was used to measure transmission levels of  Plasmodium knowlesi continue to be reported in the country , of which the EIR is considered the gold standard for assessing malaria transmission intensity, are no longer applicable in areas with low-intensity malaria transmission due to their low sensitivity . Unlike P. falciparum and P. vivax among the most vulnerable population to malaria infection in Malaysia, namely, the indigenous Orang Asli. Results from this study could be an important additional tool for malaria monitoring and surveillance in high-risk and low-transmission settings in Malaysia and other countries.As countries go through the epidemiological transition from high through moderate to low transmission, it becomes challenging to assess intervention programme performance and to know when it is appropriate to conduct an impact evaluation. Therefore, the seroepidemiological analysis would benefit malaria control programmes if implemented and evaluated in regions on the path to elimination as Malaysia. This study was designed to determine serological transmission levels of The study was conducted in accordance with the Declaration of Helsinki and was approved by the Medical Ethics Committee of the National University of Malaysia (reference number UKM PPI/111/8/JEP-2019-148). Permission for fieldwork was obtained from the Regional Health Office of Kelantan (reference number JKNK/BPA/KV 01/2/1). Respondents were sensitised to the study objectives and procedures by the local health district personnel for the study participation.The study was conducted in three indigenous Orang Asli settlements, namely, Pos Kuala Betis , Pos Bihai , and Pos Gob  located in Gua Musang district, Kelantan state, Peninsular Malaysia Figure 1A cross-sectional survey was carried out between June and July 2019. All villagers were invited to take part in the study. The study protocol and consent were explained to the participants, and their voluntary consent was documented. For the illiterate participant, written informed consent was obtained in the presence of an independent literate witness. Written informed consent was obtained from parents or legal guardians for children  and adolescents (between 10 and 18 years old). Finger-prick blood samples were obtained from each participant for microscopy smears and filter-paper blood spots, and the latter were stored at −20°C until processing. District healthcare providers and community leaders  were purposely involved in the study to facilitate participation and cooperation among the community.Giemsa-stained thick and thin malaria films reading was performed by trained laboratory technicians to identify active infections. All blood films were stained with 3% Giemsa solution  for 30 min and examined under oil emersion (10 × 100 magnification). Blood smears were negative if no parasites were found after examining 100 high-power microscopy fields.P. falciparum  and P. vivax (PvAMA-1 and PvMSP-119) were determined by ELISA as previously described  of a hyper-immune plasma pool (n = 15) were added per plate. Optical density (OD) values were measured at 450 nm with a Multiskan Go ELISA microplate reader .Blood spots were reconstituted as previously described, and the solution was equivalent to a 1:200 dilution of serum . Antibodescribed . Brieflypost-hoc tests. Duplicate ODs per individual were averaged, adjusted for background reactivity, and normalised against the positive control curve as previously described to adjust for plate variation . The median and interquartile range (IQR) were used to provide data for continuous variables, while frequencies and percentages were used to present data for categorical variables. Differences in proportions were tested using the chi-squared test. Antibody levels between categories were compared using the Mann–Whitney U test or the Kruskal–Wallis test with Dunn’s multiple comparison ariation . Threshoariation . Individkelihood . Infantskelihood . FactorsSerum was successfully eluted and serologically tested for 645 participants in this study Table 1.p < 0.001). Except for PfMSP-119 (p = 0.043) and PvAMA-1 (p = 0.008), no significant differences between genders were observed in PfAMA-1 and PvMSP-119 median antibody levels. As expected, median antibody levels significantly differed between age groups and increased significantly with age (p < 0.001) for all antigens. Furthermore, median antibody levels to all antigens also differed significantly (p < 0.05) between settlements. For P. falciparum, the anti-AMA-1 and anti-MSP-119 levels were significantly (p < 0.05) higher among the population in Pos Bihai and Pos Gob than in Pos Kuala Betis. For P. vivax, the anti-AMA-1 antibody level was significantly (p < 0.001) higher among the population in Pos Bihai than in the other two settlements. In comparison, only the anti-MSP-119 antibody level was significantly (p = 0.042) higher in Pos Bihai than in Pos Kuala Betis.Antibody levels as measured in optical densities are shown in P. falciparum and P. vivax antigens among indigenous Orang Asli communities in Kelantan. Overall, malaria seroprevalence was 38.8% for PfAMA-1, 36.4% for PfMSP-119, 2.2% for PvAMA-1, and 9.3% for PvMSP-119. Seropositivity to all parasite antigens was generally higher in women than in men, although no significant difference was observed (p = 0.378). For all parasite antigens, the proportion of seropositive individuals significantly increased with age (p = 0.004), highest in the >30 age group and lowest in the ≤5 age group. Between study areas, the proportion of seropositive individuals was significantly different (p < 0.001) for all parasite antigens. The highest proportion of seropositive individuals was observed in Pos Kuala Betis for PfAMA-1, PvMSP-119, and PvAMA-1 antigens, with 26.5%, 8.8%, and 1.2%, respectively. In Pos Bihai, 18.7% and none were seropositive to PfMSP-119 and PvMSP-119 antigens, respectively.P. falciparum or P. vivax, seropositivity to species-specific AMA-1 and MSP-119 antigens was combined. In total, 58.3% of participants were seropositive for P. falciparum (375/645), 10.1% for P. vivax (65/645), and 7.9% for both species (51/645). Relatedly, the annual probability of malaria seroconversion curves based on the combination of seropositivity to species-specific AMA-1 and MSP-119 antigens are shown in P. falciparum transmission than P. vivax using all tested antigens, ranging from 0.032 year−1 (0.026–0.04) to 0.122 year−1 (0.093–0.16) for any P. falciparum and 0.000 year−1 (0.000–0.002) to 0.019 year−1 (0.011–0.035) for any P. vivax. The SCRs were statistically significant between species-specific antigens, as evidenced by the non-overlapping confidence intervals.To define any serological evidence of exposure to P. falciparum- and P. vivax-specific antigens are shown in P. falciparum and P. vivax antigens . Significant associations were also found between settlement and seropositivity in the adjusted model for P. falciparum and P. vivax antigens. For P. falciparum, a significantly higher risk of seropositive was observed in Pos Bihai  but a lower risk of seropositive in Pos Gob , when compared to Pos Kuala Betis. In contrast, a significantly lower risk of P. vivax seropositivity remained apparent in the adjusted model for Pos Bihai  and Pos Gob , when compared to Pos Kuala Betis. No association was found between gender to seropositivity for P. falciparum and P. vivax antigens.Univariate and multivariate logistic regression analyses to identify factors associated with seropositivity to any P. falciparum and P. vivax among the Orang Asli communities in four states in Peninsular Malaysia, namely, Kelantan . This finding is consistent with previously described levels in other countries in the Southeast Asia region with P. falciparum–P. vivax co-endemic, such as Thailand (79% and 40%)  in patients from Malaysia  , Vietnamand 40%) , Myanmarand 40%) , and Indand 40%) . The hig species . FurtherP. vivax , and thiP. vivax . Moreovever time . Given rMalaysia , furtherP. vivax in children up to the age of 5 years  for nfection . Furthernfection , SCR hasnfection .P. falciparum remained twice as high for Pos Bihai compared with Pos Kuala Betis in the adjusted model. In contrast, the likelihood of seropositivity to P. vivax remained low for both Pos Bihai and Pos Gob compared with Pos Kuala Betis in the adjusted model . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.Conceived and designed the study: ZI, KT, PD, IL, SC and MK. Collected and prepared samples: MAR, MM, ND, MSR, NG, NH, SM, MK, AM, EO, WW and ZI. Performed the laboratory assays: MAR, MM, ND, SM and ZI. Analysed the data: MAR, MM, ND and ZI. Contributed reagents, materials, and analysis tools: AK, KT and ZI. Drafted the manuscript: MAR. Provided critical revisions: ZI, KT, AK, PD, IL, SC, EO and WW. All authors contributed to the article and approved the submitted version.


Plasmodium falciparum population diversity and transmission dynamics provides information on the intensity of malaria transmission, which is needed for assessing malaria control interventions. This study aimed to determine P. falciparum allelic diversity and multiplicity of infection (MOI) among asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo (DRC).Understanding pfmsp1 block 2 and pfmsp2 block 3.A total of 438 DNA samples (248 asymptomatic and 190 symptomatic) were characterized by nested PCR and genotyping the polymorphic regions of pfmsp1 block2. The K1-type allele was predominant with 78% (229/293) prevalence, followed by the MAD20-type allele  and RO33-type allele . Twelve alleles were detected in pfmsp2, and the 3D7-type allele was the most frequent with 84% (256/304) prevalence, followed by the FC27-type allele . Polyclonal infections were detected in 63%  of the samples, and the MOI (SD) was 1.99 (0.97) in P. falciparum single-species infections. MOIs significantly increased in P. falciparum isolates from symptomatic parasite carriers compared with asymptomatic carriers , p = 0.046) and parasitaemia > 10,000 parasites/µL compared to parasitaemia < 5000 parasites/µL , p < 0.001).Nine allele types were observed in P. falciparum, which reflects a moderate intensity of malaria transmission in the study areas. MOIs were more likely to be common in symptomatic infections and increased with the parasitaemia level. Further studies in different transmission zones are needed to understand the epidemiology and parasite complexity in the DRC.This survey showed low allelic diversity and MOI of The online version contains supplementary material available at 10.1186/s12936-023-04528-z. Plasmodium falciparum remains the most prevalent malaria parasite species in the WHO African region, and accounts for almost all malaria cases [P. falciparum [Despite efforts to control and eliminate malaria, the disease continues to threaten the lives of people in 85 countries. Globally, 241 million cases and 627,000 deaths were estimated to have occurred in 2020, of which 96% and 95%, respectively, occurred in the World Health Organization (WHO) African region [lciparum .P. falciparum merozoite surface proteins 1 and 2 (PfMSP1 and PfMSP2) and the glutamate-rich protein have been used to study allelic diversity and estimate the minimum number of different parasite clones present in malaria infections [msp1 gene located on chromosome 9. It comprises 17 blocks with different degrees of conservation among isolates [msp1 gene and can be grouped into three major allelic families (K1 (K), MAD20 (M), and RO33 (R)-types) [P. falciparum malaria infections [Pfmsp1 block 2 combined with a conserved pfmsp1 block 1 sequence has been shown to enhance the repertoire of MSP-1 block 2 antibody responses [P. falciparum, thus protecting against malaria [An efficient malaria vaccine should be part of the arsenal of public health tools. However, the extensive genetic diversity of the parasite is a major obstacle for the acquisition of immunity against malaria, and thus impedes vaccine development , 14. Thefections –19. MSP1fections  and are fections . The majisolates , 21. Blo)-types) . Block 2)-types) . In vaccfections , and Pfmesponses . Human a malaria .msp2 gene located on chromosome 2 and contains 5 blocks, of which block 3 is the most polymorphic [msp2 gene has 2 major allelic families, FC27 (F)-type and 3D7 (D)-type [PfMSP2 was found to be a potential vaccine candidate when its two main allelic types were coupled to PfMSP8 (rPfMSP2/8) [PfMSP1 block 2 and PfMSP2 block 3 are mostly targeted by the immune response due to their higher polymorphisms and are associated with protection from clinical malaria [pfmsp1 block 2 and pfmsp2 block 3 may constitute good indicators for measuring malaria transmission intensity and malaria control interventions. The pfmsp2 gene was previously found to be more genetically diverse than pfmsp1 in the Democratic Republic of Congo (DRC) [MSP2 is an ~ 25-kDa GPI-anchored protein abundantly expressed on the merozoite surface. It is encoded by the ymorphic . The msp(D)-type . PfMSP2 fMSP2/8) . PfMSP1  malaria , 30, 31.go (DRC)  and the go (DRC) –35. MSP2go (DRC) .P. falciparum, constitutes a parameter for measuring the impact of malaria control strategies; and can provide information on malaria transmission intensity that is useful for monitoring and evaluating possible gaps in malaria control interventions [The extent of diversity, estimated in part by polyclonal infection frequency, the number of alleles detected, and the multiplicity of infection (MOI) of ventions , 36–39. ventions , 41.The Democratic Republic of Congo follows only Nigeria in malaria prevalence in sub-Saharan Africa (SSA) and accounted for 12% of both the estimated global malaria cases and deaths in 2020 .School-age children are often neglected in malaria control interventions, and thus constitute a reservoir of infections and contribute substantially to malaria transmission –48. In hP. falciparum in various regions and populations has been extensively studied [pfmsp1 and pfmsp2 genetic diversity among school-age children in the DRC. Understanding the extent of parasite diversity will provide information on malaria transmission intensity in this population, which will be useful for formulating public health policy. Here, the allelic diversity of pfmsp1 and pfmsp2, the MOI and the prevalence of polyclonal infections in malaria parasites isolated from asymptomatic and symptomatic school-age children in Kinshasa Province, DRC were determined.The genetic diversity of  studied , 39, 54.P. falciparum-positive genomic DNA samples were analysed in this study. The details of the study site and sampling procedures have been previously described [Samples for this study were collected as part of a cross-sectional study carried out from October to November 2019 in Kinshasa Province, DRC . The oriescribed .Plasmodium species were identified by amplifying the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene using a modified protocol described by Nundu et al. [falciparum-positive samples, 248 asymptomatic infections  and 190 symptomatic infections  were used for PCR analysis [Genomic DNA was extracted from dry blood spots on Whatman 903 filter paper  using a QIAamp DNA Mini Kit  following the manufacturer’s instructions. The u et al. . In thispfmsp1 and pfmsp2 genes using sequence-specific primers as described, with slight modifications [pfmsp1 and block 3 for pfmsp2. The second reaction used primers specific for allelic families of pfmsp1  and pfmsp2 (F- and D-types). Template DNA (2 µL) was used in all first reactions, while 1 µL of amplicon was used for the second reactions. Cycling conditions and primer sets for the respective alleles used in the nested PCRs have been reported elsewhere , HB3 (M-type MSP1 block 2), and 7G8 (R-type MSP1 block 2) laboratory strains were used as positive controls  and nuclpfmsp1 and 2% agarose gels for pfmsp2. DNA amplicons were visualized under a UV light transilluminator after post staining with Gel Red®  solution for 30 min. The fragment sizes were determined using a 100 bp DNA ladder  and alleles were categorized according to their sizes.All PCR products were analysed by electrophoresis at 100 V for 30 min on 2.5% agarose gels for pfmsp1 and pfmsp2 allelic frequencies were calculated as the proportion of the allele determined for each allele family out of the total alleles detected. The number and size of bands per sample indicated the minimum number of clones in a positive sample. Single infections were samples with only one band and multiple infections were samples with two or more bands. The MOI was determined by calculating the number of different alleles at any locus detected in the sample. The mean MOI was calculated by dividing the respective total number of pfmsp1 or pfmsp2 genotypes by the number of positive samples for each marker [pfmsp1 and pfmsp2 in single-species P. falciparum infections were described with proportions and mean (SD), respectively.Categorical variables are presented as proportions and numerical variables by median and interquartile range (IQR) or by means and standard deviation (SD). The h marker , 34, 56.P. falciparum clones in single and mixed infections were determined for comparison among asymptomatic and symptomatic groups. Evaluations were made to determine the number of pfmsp1 and pfmsp2 genotypes in asymptomatic and symptomatic P. falciparum monoinfections and in mixed-species infections. The chi-square test was used to compare proportions, Student’s t test and ANOVA were used to compare means among categorical groups, and simple and multiple logistic regression with odds ratios (ORs) and 95% confident intervals (95% CIs) were used to establish the associations between independent factors and polyclonal infections. Simple and multiple linear regression with 95% CIs were used to establish the associations between independent factors and the mean MOI. Statistical significance was set at P ˂ 0.05. All data were analysed using Stata version 17 .The MOIs of The ethics committees of the School of Public Health, University of Kinshasa, DRC  and the Institute of Tropical Medicine, Nagasaki University  gave approval for this study .P. falciparum parasites represented 60%, versus 40% for those in the Selembao urban HZ  with children aged 6 to 9 years accounting for 60% and children aged 10 to 14 years accounting for 40%. According to the clinical status criteria of school-age children, asymptomatic infections represented 57% of infections, whereas symptomatic infections were 43% and children living in the Mont-Ngafula 2 rural HZ harbour 1 years wpfmsp1 and 304/438 (69%) for pfmsp2 were successfully amplified by PCR. Of 293 pfmsp1 PCR products, 229 (78%) had at least one K-type allele, 152 (52%) had at least one M-type allele, and 129 (44%) had at least one R-type allele. The proportions of single K-type, M-type, and R-type infections were 30% (87/293), 7% (21/293), and 10% (29/293), respectively. Fifty-three percent of isolates (156/293) harbour polyclonal infections. The frequencies of samples with K/M-type, K/R-type, and M/R-type infections were 19% (56/293), 9% (25/293), and 5% (14/293), respectively. Triple infection of K/M/R-type alleles accounted for 61 samples (21%)  for pfmsp2, 201 (66%) had at least one F-type allele, and 256 (84%) had at least one D-type allele. The proportions of single F-type and D-type alleles were 16% (48/304) and 34% (103/304), respectively. Half of the samples, 153/304 (50%), harboured polyclonal infections , three M-type alleles , and two R-type alleles (approximate fragment sizes 200 bp and 300 bp) were observed. For the pfmsp2 gene, six F-type alleles  and six D-type alleles  were detected , and in rural study areas 40/177 (23%). In symptomatic infections, the K/M/R types accounted for 27/140 (19%), and 21/116 (18%) in the urban areas . Polyclonal P. falciparum infections accounted for 63% (140/223), of which 59% (114/193) were polyallelic for pfmsp1 and 49% (98/199) for pfmsp2. The overall mean (SD) MOI was 1.99 (0.97)) with the mean (SD) MOI for pfmsp1 being 1.93 (0.98) and the mean (SD) MOI for pfmsp2 being 1.67 (0.78)  yielded a positive result for either Plasmodium falciparum isolates from symptomatic children had harboured significantly more polyclonal infections than those from asymptomatic children , p < 0.001) when using bivariate analysis but this was not significant after adjustment by density (p > 0.05). Higher-density infections with more than 10,000 parasites/µL ) had significantly more polyclonal infections than infections with a parasite density below 5,000 parasites/µL when using bivariate analysis but this was not significant after adjustment for clinical form of malaria (p > 0.05). No significant association of age, sex, or location with polyclonal infections was found ) when using bivariate analysis, and this remained significant after adjustment for density ). Infections with densities of 5000–10,000 parasites/µL ) and those with over 10,000 parasites/µL ) had significantly higher mean MOIs than those with parasite densities below 5,000 parasites/µL when using bivariate analysis, and this remained significant after adjustment for the clinical form of malaria  and adjusted b: 0.89 , respectively)). No significant association of sex, age, or location with MOI was found  and frequencies of polyclonal infections (p = 0.016) compared to single-species infection of P. falciparum. This significantly increased the MOI of msp1 (p = 0.002) and polyclonal infections in msp2 (p = 0.017)  Table .Table 8MP. ovale and/or P. malariae in coinfections with P. falciparum significantly increased the MOIs and polyclonal infections among asymptomatic carriers (p < 0.05), while no significant difference was found among symptomatic carriers  or pfmsp2 (F- and D-types) were exclusively restricted to either rural or urban settings, as well as asymptomatic or symptomatic carriers, although the frequency of some alleles was more common at one site compared to another, in addition to differences based on child health status.The purpose of this study was to evaluate the extent of pfmsp2 gene was found to be genetically more diverse than the pfmsp1 gene in the study areas, in support of the findings of the majority of studies conducted in Africa [pfmsp2 allelic families may be circulating at higher frequencies than those for pfmsp1. MSP1 block 2 has been proposed to be most considerably exposed to host immune selective pressure based on the stable K-M-R frequency distribution in endemic regions [The n Africa –35, 59.  regions . Escapin. [pfmsp1 and pfmsp2 with an increase in parasitaemia but not with clinical status, while in southern Benin, Lagnika et al. [Symptomatic children have been shown to have more polyclonal infections than asymptomatic children and those polyclonal infections increased with the level of parasitaemia regardless of clinical status. In Côte d’Ivoire, Gnagne et al.  found sia et al.  found th. [P. falciparum transmission, which differs from one area to another, resulting in heterogeneous malaria transmission rates throughout the area [This study showed a generally lower MOI than has been previously reported in the DRC, Cameroon, and Republic of Congo , 34, 35.. . These othe area , 62, 63,the area , 52, 62.the area  and parathe area , 33, 52 the area . Conversthe area , 64 and pfmsp1 MOI, or pfmsp2 MOI between the two study areas , although a previous report from the same areas and time period showed that the odds of the transmission of Plasmodium infections were fivefold higher in rural versus urban areas [. [P. falciparum populations and the intensity of transmission in malaria-endemic areas, ii) that P. falciparum genetic diversity is higher in hyperendemic areas than in low-endemic settings [pfmsp1 and pfmsp2 allelic diversities are not influenced by the level of transmission between the two zones. However, a lower number of polyclonal infections and a lower MOI in Selembao urban HZ may reflect the moderate malaria transmission level in that area [This study did not find a significant difference in the overall MOI, an areas . Howeverareas [.  showed tsettings , 65–67, settings . These fhat area , 55, whihat area , 48, 72 hat area –76. Selepfmsp1 K- and M-type alleles in P. falciparum mono-species infections and that of the K/M/R triple pfmsp1 alleles in mixed-species infections with P. falciparum regardless of malaria clinical status. For the pfmsp2 gene, the D-type allele was more frequent in P. falciparum mono-species infections, while the F/D-type double infection was more frequent in mixed-species infections in both symptomatic and asymptomatic carriers. The diverse distribution of pfmsp1/2 alleles within P. falciparum mono- and mixed-species infections may be explained based on findings from experimental [Plasmodium species. This situation may be influenced by within-host competition between species independent of immunity. Tang et al. [Plasmodium within hosts.These findings showed the predominance of single rimental , 77 and rimental –80 studig et al.  showed tpfmsp1 and pfmsp2 were significantly lower in P. falciparum/P. malariae-coinfected asymptomatic carriers compared to those with a single-species infection of P. falciparum, and it was suggested that a decrease in genetic diversity and complexity of infection occurred in cases of coinfection [pfmsp1 and pfmsp2 in infections with P. ovale and/or P. malariae coinfected with P. falciparum among asymptomatic subjects, although it was not significantly different among symptomatic subjects. Further studies are needed to provide more information on the consequences of within-host competition in mixed-species infections based on malaria clinical status.In southern Benin, it has been shown that the prevalence of polyclonal infections and MOIs as measured by polymorphisms in nfection . Converspfmsp1, an allele size of approximately 200 bp dominated for all K-, M-, and R-types, regardless of the presence of symptoms and the rural and urban settings. This finding was also shown in Cameroon [pfmsp2, the 400-bp F-type allele and the 500-bp D-type allele dominated, regardless of the presence of symptoms and the rural and urban settings. Among the F-type pfmsp2 alleles, the 400-bp allele has also been shown to be frequent in the Republic of Congo [pfmsp2 alleles, the 300-bp allele was more frequent in the Republic of Congo [This study found that for Cameroon , Gabon [Cameroon , 83, BurCameroon , Benin [Cameroon , and theCameroon . For pfmof Congo  and Burkof Congo  whereas of Congo , the 600of Congo , and theof Congo . Among tof Congo , Côte d’of Congo , and Burof Congo  while thof Congo  and the of Congo . Future of Congo –84.P. falciparum isolates from asymptomatic and symptomatic school-age children were low in the study areas of Kinshasa Province, DRC. MOIs were more likely to be present in symptomatic infections and increased with the parasitaemia level. There is a need to conduct a countrywide study on the genetic diversity of P. falciparum in different transmission zones to provide a clear picture of parasite diversity and to serve as a baseline for determining the impact of malaria interventions on parasite genetic diversity in the country.The allelic diversities and MOIs of Additional file 1: Figure S1. Allelic frequencies in pfmsp1 block 2 in asymptomatic (a) and symptomatic (b) carriers and in both rural and urban areas. Figure S2. Allelic frequencies in pfmsp1 block 2 in rural (a) and urban (b) areas. Figure S3. Allelic frequencies in pfmsp1 block 2 in asymptomatic and symptomatic infections stratified by rural (a) and urban (b) areas. Figure S4. Allelic frequencies in pfmsp2 in asymptomatic (a) and symptomatic (b) carriers. Figure S5. Allelic frequencies in pfmsp2 in rural (a) and urban (b) areas. Figure S6. Allelic frequencies in pfmsp2 in asymptomatic and symptomatic carriers stratified rural (a) and urban (b) areas


Impaired utero-placental blood flow due to malaria infection may cause hypoxia-induced altered skeletal muscle fibre type distribution in the offspring, which may contribute to insulin resistance and impaired glucose metabolism. This study assessed muscle fibre distribution 20 years after placental and/or peripheral m. vastus lateralis in the right leg. As previously reported, fasting and 30 min post-oral glucose challenge plasma glucose values were higher, and insulin secretion disposition index was lower, in the PPM+ group. Aerobic capacity (fitness) was estimated by an indirect VO2max test on a stationary bicycle. Muscle fibre sub-type  distribution was analysed, as were muscle enzyme activities (citrate synthase (CS), 3-hydroxyacyl-CoA dehydrogenase, myophosphorylase, phosphofructokinase, lactate dehydrogenase, and creatine kinase activities. Between-group analyses were adjusted for MHC-I %.We traced 101 men and women offspring of mothers who participated in a malaria chemosuppression study in Muheza, Tanzania. Of 76 eligible participants, 50 individuals (29 men and 21 women) had skeletal muscle biopsy taken from No differences in aerobic capacity were found between groups. Despite subtle elevations of plasma glucose levels in the PPM+ group, there was no difference in MHC sub-types or muscle enzymatic activities between the malaria-exposed and non-exposed groups.The current study did not show differences in MHC towards glycolytic sub-types or enzymatic activity across the sub-groups. The results support the notion of the mild elevations of plasma glucose levels in people exposed to placental malaria in pregnancy being due to compromised pancreatic insulin secretion rather than insulin resistance. Several studies over the past seven decades have shown foetal growth restriction due to malaria exposure . GloballSkeletal muscle is an essential tissue for movement and plays a major role in whole body metabolism. The substrates supplying the energy required for contraction are high-energy phosphates, carbohydrate, lipid or protein , 9. SkelThe contractile and metabolic properties in combination provide muscle fibre phenotypes. The major fibre types, namely fast and slow twitch fibres have characteristic contractile speed, as a result of specific myosin heavy chain (MHC) isoforms . The fibSkeletal muscle is adaptable to environmental stimuli such as altitude . PopulatWe, among others , have prWe aimed to determine whether exposure to combined peripheral and placental malaria compared to peripheral malaria exposure only, or no malaria, in the foetal state had an influence on skeletal muscle fibre distribution, and/or on oxidative or glycolytic skeletal muscle enzymatic activities in young adults.We retrieved information from study documents on offspring and their mothers from a study on malaria chemosupression conducted in 1989–92 in Muheza, Tanga region, north-eastern Tanzania . During This follow-up study was conducted in 2010–11, and clinical results have been described . ConsentWe stratified study participants by foetal malaria exposure as follows: (1) both peripheral and placental malaria (PPM+), (2) peripheral malaria (PM+) at least once during pregnancy, and (3) peripheral and placental malaria negative (M-). LBW was defined as <2.5 kg.2) was calculated. Body fat % and fat-free mass (FFM) were estimated using bio-impedance analysis . Furthermore, abdominal fat distribution was measured using ultrasound scanning technique  , weight (kg), and waist circumference (cm) were measured, and body mass index  . Blood puipment) .2/min/kg according to the formula by  was assessed using a Monark 828E stationary bicycle , and calculated as VOrmula by . In briermula by .m. vastus lateralis using the Bergström needle technique with suction , 3-hydroxyacyl-CoA dehydrogenase (3-HAD), myophosphorylase (PHOS), phosphofructokinase (PFK), lactate dehydrogenase (LDH), and creatine kinase (CK) activities were determined using microplate fluorometric  adapted methods as described by . The enz2max/min/kg fat-free mass. Estimated fitness was expressed as VO2/min/FFM to further account for the skewed sex distribution in the three study groups. Differences in LBW proportion between groups were determined by Fisher’s exact test. Continuous data are presented as mean  and as median  for skewed number distribution. All analyses were performed using Stata 17.0  with p-values <0.05 considered as statistically significant.Linear regression analyses were used to test for differences in outcomes of interest and malaria exposure. Furthermore, all regression analyses for the clinical data were adjusted for MHC-I % due to skewed sex distribution, and as we sought to elucidate if this may be related to intrinsic skeletal muscle phenotype. Regression analyses for muscle enzyme activity were adjusted for fitness expressed as estimated VOWe traced 101 out of 337 mothers who participated in the original study, from whom 76 offspring were eligible after pre-study screening procedure. Biopsy samples were taken from 50 individuals .p = 0.074), and based on the OGTT, mean plasma glucose was highest in the PPM+ group at 30 min (p = 0.049) sustaining already published results (p = 0.02).Sex distribution was 21 women and 29 men with a mean age of 19.6 (0.9) years. There was no difference in proportion of LBW between the groups . Background and clinical characteristics of the mothers (while pregnant) are presented in  results . DisposiTwo participants  were overweight by both BMI and central obesity criteria. Estimated fitness did not differ between groups in mean, or spread around the mean, when adjusted for sex. Two participants had hyperglycaemia, 1 DM, and 1 impaired fasting glucose, and both were from the PPM+ group. Two participants had C-peptide levels <300 pmol/L, both from the PPM+ group, and including the participant who had DM.2/min/kg) levels was significant for CS activity at .Skeletal muscle fibre type and enzymatic activities are presented in p = 0.014).MHC-I distribution was significantly different between women and men, with women having 47.6 (10.7)% and men 55.3 (10.2)%, respectively  was 1–3 weeks in the PPM+ group. Second, malaria prophylaxis blunted the impact of malaria parasitic infection, which could be another reason for the lack of impact on skeletal muscle and enzymatic activity. Furthermore, the negative impact on birth weight due to combined peripheral and placental malaria may have been counteracted due to the use of malaria prophylaxis.Interestingly, there was a significant difference in the distribution of MHC-I between women and men. The literature is not consistent with regard to the possibility that women have less type I fibre proportion, as we have found. The current data is in agreement with Komi and Karlsson , but conn = 76) , is most likely less sensitive than when muscles high in a particular fibre type are assessed either in rats or humans. This suggested explanation for our results and those of Koh et al. (In line with our recent study from a slightly larger group of study participants (n = 76) , we founn = 76) . Cross-sn = 76) ; thus aln = 76) . The youn = 76) , hence tn = 76)  specificn = 76)  study haolerance . Although et al.  may be oa priori exclude the possibility of reduced insulin sensitivity either at hepatic or skeletal muscle level. Hepatic insulin resistance is partly included in the homoeostasis model assessment of insulin resistance, but sensitivity is not reduced in the PPM+ group. However, disposition index does not adjust for peripheral insulin resistance in the skeletal muscle. In brief, the data we present do not indicate insulin resistance in the skeletal muscle, which supports that impaired insulin secretion rather than insulin resistance, explains the mild elevations of plasma glucose levels in the PPM+ group.Nevertheless, our OGTT-stimulated plasma glucose results show that the PPM+ group had an abnormal glucose metabolism. The reduced disposition index indicates that compromised insulin secretion is the main factor behind the increased glucose levels. However, as disposition index is calculated via adjustment for insulin resistance, the reduced disposition index cannot Apart from an effect on blood flow from the placenta causing compromised oxygen supply to the foetus, malaria infection has also been shown to affect the microvasculature of several organs of the host , 51, herWe acknowledge several limitations to the current study. First, malaria prophylaxis/suppression may have blunted adverse physiological effects of placental and peripheral malaria infection. As the biopsies were taken in a fasting and not in a stimulated state, we may have missed the detection of higher response related to enzyme activity and thereby differences between groups. Importantly, the study may have been underpowered by small sample sizes, which may have resulted in a statistical type II error. Finally, placental malaria infection changes persist endlessly. Whereas placental swab (thick blood smear) detects active infection, past infection is detected through histopathological changes . UnfortuIn conclusion, skeletal muscle fibre types or enzymatic activities were not different across groups. Mean fasting and OGTT-derived glucose levels were higher in the group exposed to foetal peripheral and placental malaria and detection of hyperglycaemia as well as low C-peptide levels at the individual level were all from the peripheral and placental malaria exposed group. Compromised insulin secretion rather than insulin resistance seems to be the primary defect, but this needs to be determined in larger and/or methodologically more sophisticated studies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by National Institute of Medical Research (NIMR/HQ/R.8a/vol.IX/916). The patients/participants provided their written informed consent to participate in this study.DC, IB, TM, KR, AV, and KM: contributors conceptualization. DC, AV, JN, and TM: data collection. DC and KM: formal analysis. DC, AV, and KM: writing . DC, TM, IB, AV, LG, FL-T, JN, CS, KR, and KM: writing (review and editing). DC and KM take full responsibility for the work. All authors contributed to the article and approved the submitted version.The study was funded by Thorvald Madsen’s Foundation for the Advancement of Medical Sciences and Brdr Hartmann’s Foundation.DC has received payment from Novo Nordisk Mexico for consultancy work. AV is a shareholder in Astra Zeneca.The remaining 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.


Malaria remains a leading public health problem worldwide. Co-infections with other pathogens complicate its diagnosis and may modify the disease’s clinical course and management. Similarities in malaria clinical presentation with other infections and overlapping endemicity result in underdiagnosis of co-infections and increased mortality. Thus, the aim of this study was to determine the seroprevalence of viral and bacterial pathogens among diagnosed malaria patients in malaria-endemic areas in Venezuela.OR) for complications was determined according to CoRE presence with a 95% confidence interval (CI).A cross-sectional study was conducted on malaria patients attending three reference medical centres in Ciudad Bolivar, Venezuela. Clinical evaluation and laboratory tests for dengue virus (DENV), chikungunya virus (CHIKV), viral hepatitis , and leptospirosis (LEP) were performed by enzyme-linked immunosorbent assays. Previous exposure to these pathogens was defined by the presence of specific immunoglobulin (Ig) G, and co-infection or recent exposure (CoRE) was determined by the presence of specific IgM alone or IgM + IgG. Data analysis considered descriptive statistics. Parameter distribution was statistically evaluated using Kolmogorov–Smirnov test and the necessary comparison tests. Odds ratio  and CHIKV (25%) was frequent. CoRE was confirmed in 55 of the 161 malaria patients (34%) and were more frequent in P. falciparum (49%) than in P. vivax (29%) and mixed malaria patients (25%) . The most frequent CoRE was DENV (15%), followed by HAV (12%), HBV (6.2%), CHIKV (5.5%), and LEP (3.7%); HCV CoRE was absent. Complicated malaria was significantly more frequent in patients with CoRE (56%) than those without CoRE .A total of 161 malaria patients were studied, 66% infected with We found high CoRE prevalence in malaria patients as determined by serology in the study region; cases were associated with a worse clinical outcome. Further prospective studies with samples from different infection sites and the use of molecular tools are needed to determine the clinical significance of these findings.The online version contains supplementary material available at 10.1186/s40249-023-01089-w. Plasmodium vivax accounted for 82% of cases in Venezuela, followed by P. falciparum (13%) and P. vivax/P. falciparum mixed infection (4%) [Malaria remains a leading public health problem worldwide, with an estimated 247 million cases and 619,000 deaths in 84 malaria-endemic countries in 2021 . In 2021Malaria is often associated with outdoor occupations, including mining and farming, exposing people to other vector-borne diseases. Although it is often assumed that fever is due only to malaria in malaria-endemic countries, there is evidence of widespread over-diagnosis in people presenting with severe febrile illness . HoweverInfections with more than one pathogen complicate the diagnosis and modify the clinical course of the diseases and their management. In addition, similarities in the clinical presentation of malaria and these arboviruses and icteric febrile diseases, combined with overlapping endemicity, may result in the underdiagnosis of co-infections. Despite limited information on the clinical outcome and the precise interactions of these pathogens in co-infections, it is known that multiple infections confound the malaria course. Diagnosis of non-malaria febrile illness in neglected regions remains a challenge, and delay in diagnosis or treatment initiation for any of these infections could have fatal outcomes . In ruraThe recent decline of the Venezuelan health system capacity has led to the deterioration of the epidemiological surveillance and malaria control program . Additio2. Bolivar state is its largest state, covering an extension of ~ 240,500 km2 and located in the south of the country, bordering Brazil and Guyana, with an approximate population of 1,837,485 inhabitants distributed among 11 municipalities and 46 parishes [Venezuela, located on the northern coast of South America, has a surface area of ~ 916,445 kmparishes . Most ofP. vivax is the most prevalent parasite accounting for 70–80% of malaria cases, whereas P. falciparum causes the remaining cases, with a mean ratio of P. vivax/P. falciparum of 3.04 [The total number of accumulated malaria cases in Venezuela between 2007 and 2017 was 1,207,348 , with overall malaria incidence rates (cases/1000 inhabitants per year) increasing from 5.2 (2007) to 28 (2017). Bolívar contributed ~ 47% of the total cases in Venezuela during 2017, but during the previous years this region accounted for 60–80% of malaria reported for the whole country. This study was conducted in Ciudad Bolivar, the Bolivar state capital, with approximately 427,399 inhabitants . Three rRegarding the epidemiological behaviour of other viral and bacterial pathogens in Venezuela, mainly in Bolivar state, the information is limited and outdated. The average incidence of dengue between 2010 and 2016 was 211 cases per 100,000 inhabitants. Within the country, the temporal increase in dengue cases reflects the national dengue incidence, with the highest incidence in the most densely populated regions  and those bordering Colombia and Brazil (border regions) . The actMinisterio del Poder Popular para la Salud of Venezuela criteria [A cross-sectional study was conducted from June to November 2018. Individuals over 12 years of age and of both sexes with malaria-compatible symptoms and confirmed diagnosis by microscopic examination of thin and thick blood smears, regardless of parasite species and origin, were invited to participate in the study. Trained physicians at each study site performed a detailed physical examination of all study participants according to the local standard of care. Clinical manifestations, socio-demographic information, diagnosis, and treatment of patients were recorded on a standard evaluation form. Blood samples were collected by venepuncture from each patient once diagnosis was confirmed, and patients provided informed consent upon a detailed explanation of the study objectives and methods, followed by antimalarial treatment by the local healthcare provider using the national antimalarial protocol (approved in 2017) [Blood samples (8 ml) drawn from each patient by arm venepuncture were used for haematological analysis, blood chemistry tests , and serological tests against DENV, CHIKV, HAV, HBV, HCV, and LEP. All the laboratory tests were performed once at the local private laboratory “Centro Especializado de Investigación Clínica 42” (Ciudad Bolivar). Enzyme-linked immunosorbent assays (ELISA) were used to determine specific immunoglobulin (Ig) M (IgM) and IgG antibodies to DENV , CHIKV , HAV , and LEP , as well as IgM to HCV , HBV surface antigen, and HBV core antibody , according to the manufacturer’s instructions.These serological tests were selected based on sensitivity and specificity, all higher than 92%. Since plasma antibodies against these diseases persist from a few months for IgM to several years for IgG, we defined co-infection or recent exposure (CoRE) as detection of anti-DENV, anti-CHIKV, anti-HAV, anti-HCV, and anti-LEP IgM alone or IgM plus IgG. HBV CoRE was defined as detection of HBV surface antigen. Previous exposure to any of the pathogens was defined as the presence of IgG and absence of IgM. We were not able to confirm co-infection by other techniques.OR) for complications was determined according to the presence of CoRE with a 95% confidence interval (CI). A P-value < 0.05 was considered statistically significant.Statistical analysis was performed using Statistical Package for the Social Sciences version 25 , and figures were generated with Microsoft Power BI version 2.78  and the statistical software R 4.0.3 . Data analysis considered descriptive statistics. The distribution of the parameters was statistically evaluated using Kolmogorov–Smirnov test and the necessary comparison tests were applied. The odds ratio  P. falciparum, and 12 (7.5%) mixed malaria  infection. Most of the enrolled individuals were male (64%) and younger than 40 years (77%), with no statistically significant differences between the three parasite species groups (P = 0.19). Mineworker (37.2%) and housewife (18.6%) were the most frequent occupations. Self-reported previous malaria episodes were common among the study population, ranging from one to eight episodes in lifetime. Previous exposure to DENV (59.6%) and CHIKV (24.8%), as determined by the presence of specific IgG, was frequent in the study samples, with no differences between malaria parasite species. Previous exposure to HBV (6.8%) and LEP (3.7%) was less frequent, and no samples were positive for HAV or HCV-specific IgG. Fifty-five (34.2%) of the patients had CoRE with at least one pathogen. Whereas a single pathogen was identified in most patients , more than one pathogen was identified in the remaining 11 patients. The prevalence of CoRE was 14.9%, 11.8%, 6.2%, 5.5%, and 3.7% for DENV, HAV, HBV, CHIKV, and LEP, respectively; no patients were found with HCV. In patients with CoRE with two or more pathogens, simultaneous DENV/HAV was found in 4/11 (36.4%), while other combinations like DENV/CHIKV, HAV/HBV, HAV/LEP, CHIKV/LEP, and DENV/LEP were present in one patient each. We found two patients with CoRE by three pathogens, one with DENV/CHIKV/HAV  and the other with DENV/CHIKV/LEP .A total of 161 patients diagnosed with malaria were included, of whom 106 (65.8%) had P. falciparum  than with P. vivax  or mixed malaria infection . Out of the 156 patients from Bolivar state, most  had been in Sifontes municipality during the last month, mainly in “Kilómetro 88” and “El Dorado” towns. The second most frequent municipality of origin was Heres , where mainly came from Ciudad Bolivar . Regarding origin by species, 21/43 (48.8%) of patients with P. falciparum, 6/12 (50%) of those with mixed infection, but only 33/106 (31.1%) of patients with P. vivax came from the Sifontes municipality. In contrast, 41/106 (38.6%), 9/43 (20.9%), and 2/12 (16.6%) of patients with P. vivax, P. falciparum, and mixed infection, respectively, came from the Heres municipality. Most of the mineworkers (36.7%) were from the Sifontes municipality.As shown in Table P = 0.025), cough (P = 0.033), splenomegaly (P = 0.011), and somnolence (P = 0.003). CoRE with HBV was associated with lower pallor (P = 0.023), while CoRE with HAV was associated with stupor (P = 0.038) and seizures (P = 0.038) , chills (100%), and headache (98.1%), without significant differences between the different groups. CoRE with DENV was associated with asthenia P = 0.05, cough P = 0.010). Patients with CoRE generally showed elevated aspartate aminotransferase (AST) levels, but CoRE with HAV was associated with higher AST levels (P = 0.007). Interestingly, we found a significant association between CoRE with LEP and low haematocrit (P = 0.047) and platelet counts (P = 0.019), elevated AST (P = 0.006) and alanine aminotransferase (ALT) (P = 0.034) levels, and low potassium levels (P = 0.043)  by P. falciparum, and 3 (4.3%) by mixed malaria infection. As expected, the proportion of complicated cases over total cases per parasite species was higher for P. falciparum than for P. vivax cases and for mixed malaria infection . Eight patients (11.6%) had two or more complications simultaneously. Taken individually, the most frequent complications were jaundice , cerebral malaria (11.6%), severe anaemia (8.7%), pulmonary oedema (4.4%), and renal failure (2.9%)   patients were classified as having complicated malaria. Of those, 42 (60.8%) were caused by .9% patiePlasmodium spp. co-infections or co-circulation with other pathogens, such as DENV [Several studies, mainly from sub-Saharan Africa and Southeast Asia, report  as DENV , 20, CHI as DENV –23, HAV  as DENV , HBV [10 as DENV , LEP [25 as DENV , human i as DENV , intesti as DENV , and oth as DENV –31. Howe as DENV –35. To t as DENV , but low as DENV . Thus, pThe prevalence of CoRE with DENV (14.9%) was higher than that found in a large cross-sectional study in hospitalised patients with acute febrile syndrome in the Brazilian Amazon 2.8%) , and in .8% , andPlasmodium/DENV co-infection was not associated with disease worsening [Plasmodium species, previous exposure to either pathogen or to both, age and gender, and other epidemiological conditions may explain the discrepancies among the studies.Malaria CoRE with other pathogens does not induce a predictable clinical pattern. While we found that CoRE with DENV was significantly associated with somnolence and splenomegaly, a study conducted in French Guiana showed a worse clinical outcome, with a higher risk of severe thrombocytopenia and anaemia , or induorsening , whereasorsening . Among tPlasmodium parasitaemia correlated inversely with plasma HBV DNA levels [The second most frequent CoRE was HAV (11.8%), higher than that found in children from sub-Saharan Africa (1.7%) . This hiA levels . In contA levels , 41. OurA levels . Our finA levels .CoRE with CHIKV was found in a lower proportion (5.5%) than that found in Tanzanian (7.1%)  and KenyInterestingly, simultaneous CoRE with DENV/HAV was found in four malaria patients. There are few case reports of this concurrent mixed infection . Thus, tP. vivax was more prevalent than P. falciparum in the studied area, CoRE with at least one pathogen was more frequent in patients with P. falciparum, which could be due to several factors including the origin of the patients, with P. falciparum predominating in the Sifontes municipality where most of the mineworkers come from, the occupation with the highest prevalence of CoRE. Unfortunately, there is no information available on the epidemiological behaviour of the studied pathogens in the study region, which does not allow conclusions to be drawn. On the other hand, a high frequency (42.9%) of complicated malaria was found and complications were more likely in CoRE patients compared to no CoRE ones, suggesting that CoRE with another pathogen could exacerbate the malaria clinical course. Nevertheless, as expected, there was a higher proportion of complicated cases in patients with P. falciparum than with P. vivax due to its higher pathogenicity, so this could be a confounding factor for the interpretation of these results. Further investigations are needed to confirm this observation due to the small sample size. Similar results have been found in patients with P. vivax/DENV co-infection, who were more likely to have severe disease than those mono-infected with DENV [Although ith DENV . In contith DENV  found thPlasmodium infection [The limitations of this study include the lack of double testing for viral infection due to economic reasons. Although we evaluated specific IgM antibodies, cross-reactivity cannot be ruled out due to polyclonal activation induced by nfection , 58, as nfection . Anothernfection –62. NoneP. falciparum. The most frequent CoRE was DENV, followed by HAV, HBV, CHIKV, and LEP; HCV CoRE was absent. Complicated malaria was significantly more frequent in patients with CoRE than those without CoRE. The high prevalence of CoRE found in the main Venezuelan endemic state should contribute to the understanding of clinical and paraclinical behaviour to develop guidelines and protocols to optimise early diagnosis and guide treatment in patients with the acute febrile disease. Delay in the diagnosis or initiation of treatment of any of these infections could have fatal outcomes. Prospective studies with samples at different points of infection and the use of molecular tools are needed to clarify these findings.CoRE was confirmed in one third of the malaria patients and were more frequent in Additional file 1: Figure S1. Distribution of malaria cases according to the parasite species. (A) Map of Venezuela. (B) Origin of the malaria cases according to Plasmodium species (pie charts). Only main municipalities in Bolivar state are shown. Map also shows other relevant landscape features, including the localisation of the capital (asterisk), Ciudad Bolivar, in Heres municipality.


Most data describing severe malaria (SM) in sub-Saharan Africa (SSA) are from research settings outside disease endemic areas. Using routinely collected data from Apac District Hospital, this study aimed at determining the burden and clinical spectrum of severe malaria.This was a retrospective study that reviewed all paediatric admission records for malaria in the 24 months period from Jan 2019 to Dec 2020 at Apac District Hospital. Data on children aged 60 days to 12 years who at admission tested positive for malaria and fulfilled the World Health Organization clinical criteria for surveillance of severe malaria were abstracted using a customized proforma designed to capture variables on social demographic, clinical presentation, treatment, and outcomes. In addition, the tool included laboratory variables for complete blood counts, haemoglobin, and glucose levels. Data were analysed using STATA V15.0. The study had ethical approval from Mbale Regional Referral Hospital REC, Approval No. MRRH-REC 053/2019.P = 0.042) and prostration aOR 8.47 . A majority of admitted children, 735 (98.7%) survived, while 10 (1.3%) died of SM.A total of 5631 admission records were retrieved for this study period. Of these, 3649 (64.8%) were malaria admissions and 3422/3649 were children below 12years, with only 1864 (54.5%) of children having complete data. Of the 1864 children, 745 (40.0%) fulfilled the severe malaria inclusion criteria. Of the 745 children, 51.4% (n = 381) were males. The median age at admission was 31 months (IQR = 17–60). The most common clinical presentations among children with severe malaria were fever 722 (97.3%), cough 478 (64.2%), and difficulty in breathing 122 (17.9%). The median length of hospital stay was 2  days and 133 (17.9%) had prolonged hospital stay (> 4 days). Factors independently associated with prolonged hospital stay were, presenting with difficulty in breathing, aOR 1.83  of malaria admissions were due to SM. Prolonged Hospital stay was associated with prostration and difficulty in breathing. Overall mortality was low, 1.3% compared to mortality in the previously reported series. This study was able to use routinely collected data to describe the burden and clinical spectrum of SM. Improvement in the quality of data from such settings would improve disease descriptions for policy, monitoring of epidemics, response to interventions and to inform research. Plasmodium falciparum [Globally morbidity and mortality due to falciparum malaria remains high. In the pre-COVID-19 era, there were an estimated 229 million malaria cases in 2019 in 87 malaria endemic countries, declining from 238 million in 2000 [lciparum . The onslciparum . In endelciparum . In-patilciparum . In UganCurrent descriptions of SM are predominantly, if not, exclusively from few settings located in low transmission sites and mainly driven by research funding , 9. NoneThis study was a retrospective study for admission records on children aged 2 months to 12 years admitted at Apac district hospital for the period Jan 2019 to Dec 2020. The hospital is located North of Lake Kyoga, 268 km North of Uganda’s Capital city, Kampala and 60 km west of Lira City. About 40% of the district is a marsh land covered with wetlands and swamps, which are conducive for mosquito breeding. The district has a total population of 368,626 people, of which 47.9%  are children less than 15 years of age [P. falciparum species. Children with no clear diagnosis and children admitted with other diagnosis other than malaria were excluded from the study.All records for children aged 2 months to 12 years admitted with a confirmed diagnosis of malaria were included if they met any one or more of the World Health Organization (WHO) 2015 clinical criteria for surveillance of SM . These cUsing a customized proforma, data were abstracted on biological and social demographic characteristics such as age and gender. The study collected clinical features of severe malaria according to the WHO 2015 clinical surveillance criteria for SM described above and as previously applied in Eastern Uganda , 14. In Data were coded and entered in excel where it was cleaned and exported to STATA statistical package for further management and analysis. The data were analysed at three different levels of univariate, bivariate and multivariate. At univariate level, descriptive statistics were summarized as frequency, proportions, mean and median. Binary logistic regression was done to determine the association between independent and outcome  variables, while Fishers’ exact test was used to determine risks associated with death at bivariate level. Multiple logistic regression was used to determine the factors associated with prolonged hospital stay at multivariate level.A total of 5631 admission records were retrieved for this study period. Of these 3649 (64.8%) were malaria admissions and 3422/3649 were children below 12 years. There was high level of data incompleteness as 1558/3422 (45.5%) children admitted had incomplete data as date of discharge and status at discharge and were excluded from further assessment and analysis. Of the 1864 children who had complete data and were assessed for eligibility of diagnosis of severe malaria, 745 (40.0%) fulfilled the severe malaria inclusion criteria  fever 722 (97.3%), (b) cough 478 (64.2%), (c) vomiting 265 (38.7%), (d) diarrhea 192 (28.1%) and (e) difficulty in breathing122 (17.9%), anemia 22 (30.1% among those tested) and hemoglobinuria 3 (0.9% among those tested) , and haemoglobinuria (P = 0.027)  were males. The median age at admission was 31 months (IQR = 17–60). Survival with SM in Apac District Hospital is high, as death rate is low at 1.3%. Despite the low numbers, the factors that had statistical association (Fisher’s exact) with death were fever (P = 0.042) compared to those with normal breathing, and prostration aOR 8.47  compared to those without prostration  days, while the median length was 2  days and 133 (17.9%) of the study participants stayed in the hospital for more than 4 days (Prolonged stay). At multivariate level, factors that were significantly associated with prolonged hospital stay were difficulty in breathing, aOR 1.83  observed in this study is a younger median age compared to the current trend of data reporting increasing age with malaria epidemiological transition . This al–60 obserP = 0.042) and prostration aOR 8.47  to be associated with prolonged hospital stay. Elsewhere, both of these factors have historically been reported to be associated with mortality outcomes [Some data have identified factors affecting length of hospital stay after admission with malaria as sex , age (younger patients), delay seeking treatment, acute renal failure and third line treatment . In thisoutcomes . Mortalioutcomes , 21, 22 outcomes . This cooutcomes . Secondloutcomes , enlistioutcomes . Anotheroutcomes . Other soutcomes . Furtheroutcomes . This stoutcomes , 28. Theoutcomes , 26, 27,outcomes , 27, ceroutcomes .There were limitations that may have affected the study finding. The SM assessments were mainly clinical since laboratory tests were limited. For instance, no tests were reported for serum electrolytes, blood sugar levels and renal function tests. In addition, the clinical data were incomplete in most of the variables except for fever which seems to have been voluntarily reported by caregivers. Very small samples were observed among death, and this made it difficult to reliably determine the predictors of death. This study, however, has been able to report that a high proportion (20.4%) of malaria admissions were due to SM. In addition, a description of SM within the limits of these data has been done. In Apac and most settings in Uganda, just as it is in many parts of SSA, active surveillance of SM is not only costly but also labour-intensive and, therefore, not sustainable. In these settings, routinely collected data represent untapped resource as primary surveillance data that also may complement research data when available. Priority to use routinely collected data needs to be directed to harnessing their potential to contribute to the burden, descriptions, and clinical spectrum of SM. For a complete picture on the burden and disease spectrum, emphasis on high quality clinical and laboratory data are critical.This study was able to use routinely collected data to describe the burden and clinical spectrum of SM. Prolonged hospital stay was associated with prostration and difficulty in breathing. This study has reported a new record low mortality of 1.3% in malaria endemic and high transmission settings. Improvement in the quality of data from such settings would improve disease descriptions for policy, monitoring of response to interventions and to inform research.


Plasmodium ovale malaria, which was previously endemic to tropical Africa and the Southwest Pacific islands is now being reported from parts of Asia. In Sri Lanka, the indigenous transmission of malaria has not been documented since October 2012. Since then, there have been several imported cases of malaria, including P.ovale, which have been detected sporadically. The reporting case of P. ovale was imported and detected incidentally in 2021, with several atypical presentations.Plasmodium ovale with a density of 6535 parasites/microliter on the same day. He never had malaria in the past, but he had worked in South Sudan 1 year ago and visited India six months ago. On the 6th postoperative day, he was treated with chloroquine, and hyperparasitemia reduced rapidly by the next day. As the fever recurred with clinical deterioration, he was treated with different antibiotics. During the course of the illness, he did not develop pallor, or icterus except for a palpable soft spleen. The parasite count was zero on the 9th postoperative day and his fever subsided on the next day. Further, he was treated with primaquine to prevent future relapse and transmission.A 40-year-old Sri Lankan medical doctor developed continuous fever with chills, rigors, and dysuria a day following removal of a large lipoma at the root of the neck under general anaesthesia. When the fever has been responding to antibiotics, on the 4th postoperative day a mild thrombocytopenia on complete blood count was detected. A blood smear which was done on the 5th postoperative day incidentally found a malaria parasite and confirmed as P ovale in a blood smear, and hyperparasitaemia are the atypical presentations of this case. Postoperative bacterial infection and stress may have reactivated the dormant malaria (hyponozoites) in this patient with an unusual picture. Coinfection of malaria with bacterial sepsis is a challenge in the management of the patient. As the Anopheles mosquito vector exists in Sri Lanka, the risk of indigenous transmission is high from such imported cases of P. ovale.A long incubation period, incidental detection of  P. falciparum, P.vivax, P.malariae, P.ovale, and P.knowlesi. Of them, falciparum and vivax are the dominant species of global malaria causing high mortality and morbidity rates. Plasmodium ovale, which was traditionally endemic to tropical Africa, New Guinea, eastern parts of Indonesia, and the Philippines [P.ovale is usually associated with low morbidity and mortality, previous studies have reported several complications that can be fatal [Globally and nationally, malaria is an important tropical disease caused by Plasmodium species. Five Plasmodium parasite species are known to cause malaria in humans. They are lippines , 2 is nolippines –7. Althobe fatal .Plasmodium ovale was detected in Sri Lanka in 2003, and it is considered to be indigenous [Plasmodium ovale infection in Sri Lanka was considered to be imported [P. ovale have been detected in Sri Lanka from 2013 to 19 [P. ovale in Sri Lanka highlighting the way of detection and its atypical presentation.In Sri Lanka, the indigenous transmission of malaria has not been documented since October 2012, and thereafter the World Health Organization (WHO) declared malaria-free status in September 2016 . The firdigenous . In 2017imported . The Ant9/L, hemoglobin of 13 g/dl, and platelet count of 140 × 109/L. The C-reactive protein (CRP) was 40 mg/L. Detection of mild thrombocytopenia on complete blood count had prompted to do a blood smear on the 5th postoperative day which found the malaria parasite incidentally. At this juncture, the anti-malaria campaign in Sri Lanka, was informed about the detection of malaria as a compulsory regulation. On the same day, the regional malaria officer of the anti-malarial campaign visited the patient and obtained blood for thick and thin blood smears and rapid detection tests for malaria. A trained microscopist of AMC identified diagnostic characteristics supporting Plasmodium ovale. Further, higher density of Plasmodium ovale (≥ 1000 parasites/µl) [P. falciparum was performed later, and it was ruled out. On the next day, the patient was started on treatment for malaria according to the National Guideline on Malaria Management 2014 [9/L Escalation of bacterial sepsis was considered, and he was treated with intravenous meropenem on the same day after taking blood for culture. However, due to clinical deterioration and financial constraints, he was transferred to a government hospital where meropenem was not available on that day. Then the antibiotic was changed to Piperacillin-Tazobactam on the 8th postoperative day. His urine and blood cultures gave negative growth probably due to prior empirical antibiotic treatment. His temperature declined and he felt better after 2 days of antibiotics which was continued for 5 more days until full recovery. Afterward, he was given 0.25 mg/Kg body weight per day of primaquine for 14 days with the intention of preventing transmission by gametocytes and preventing relapse by hypnozoites after excluding Glucose-6-phosphatase dehydrogenase (G6PD) deficiency by performing a G6PD assay as G6PD deficiency is prevalent in Sri Lanka [A 40-year-old Sri Lankan medical doctor from Kandy, Sri Lanka developed continuous fever with chills, rigors, and dysuria without loin pain, a day after removal of a large benign subcutaneous lipoma at the root of the neck under general anesthesia in November 2021. He had to remain fasting without drinking water as a prerequisite for general anaesthesia for six hours. His fever was attributed to a postsurgical infection, possibly a urinary tract infection despite a lack of predisposing factors such as urinary bladder catheterization and co-morbidities such as diabetes mellitus. He had received Intravenous Co-amoxiclav for 5 days empirically with temporarily declining fever spikes. The complete blood count done on the 4th day after surgery showed a total white cell count of 5.3 × 10ovale infection but known to have P.vivax and P. falciparum transmission [ovale infection in India [On further inquiry, he never had malaria in the past, but he had worked in South Sudan, until December 2020 for 1 year. During that time, he had taken mefloquine prophylaxis properly according to the National guideline on malaria prophylaxis for travelers 2019 .Being a smission . After rin India .Since thth post-operative day showed resolving features of pyelonephritis. He made a full recovery by the 10th postoperative day, remained asymptomatic, and later migrated to another country. The important days and presentations of the case are depicted as a timeline in the Fig. The investigations including hematological, and biochemical parameters, and parasitic counts during the illness are shown in Tables ovale malaria in a postsurgical patient who developed bacterial sepsis probably due to a urinary tract infection. His travel history suggests that his exposure to malaria would have happened 6 months to 1 year back either in India or South Sudan. Since then, he remained healthy until his recent surgical history. However, along with the postsurgical infection, he has developed hyperparasitemia as detected on the 5th and 6th days after surgery and its contribution to the ongoing fever is not known. His ovale malaria parasitemia cleared quickly with chloroquine but overall sepsis required a strong antibiotic course. He did not have susceptible factors for pyelonephritis, which probably led to sepsis, such as urinary bladder catheterization and immunocompromised states, except for not drinking water for six hours as a prerequisite for general anaesthesia. There are some questions that need inquiry based on this case. They are the asymptomatic long latent phase of the infection, then hyperparasitemia probably augmented by postsurgical stress and infection, and the doubtful contribution of malaria infection to the worsening of sepsis. Additionally, the absence of significant hemolysis was observed despite hyperparasitemia and rapid response to chloroquine suggests the effectiveness of the drug in treating ovale malaria in the background of chloroquine resistance in other species of malaria.We report incidental detection of Plasmodium ovale was first described by a publication in 1922 by J.W.W Stephens by observing the blood of an East African patient [P.ovale consists of two essential subspecies namely P.ovale curtisi and P.ovale wallikeri. The finding was based on analyses of nucleotide deletions and substitutions in the 18S rRNA gene. These two types of P.ovale were shown to have distinct sequences for ookinete surface proteins [ patient . Recent Plasmodium ovale accounts for 0.5% to 10.5% of all malaria cases globally. But, similarities with P.vivax such as similar morphology, ability to relapse, and tertian periodicity give the likelihood of misidentification, with the possibility of underestimating the prevalence of P.ovale [P.ovale infection. The mortality rate of severe P.ovale infection in that study was 0.22% [P.ovale . Howeveras 0.22% .Plasmodium ovale is transmitted by the bites of infected female Anopheles mosquitoes. The only natural host of P.ovale is humans. Therefore, the developmental cycles of P.ovale can be identified in humans and vector mosquitoes. In humans, sporozoites from mosquito bites, rapidly invade the liver, and eventually, two forms are produced. They are merozoites, which invade reticulocytes and initiate the erythrocytic cycle, and hypnozoites which are dormant parasites that remain suspended in the liver. As P.ovale development is restricted to younger erythrocytes, maximum parasitic counts are usually low compared to P. falciparum and P.vivax infection [nfection . In our nfection . In thatnfection .The symptomatic infection shows a tertian pattern of fever (every 48 h) with a feeling of wellness in between fever episodes. Hyperpyrexia accompanied by rigor, profuse sweating with defervescence of fever and gradual recovery over 6–12 h can be seen in typical infection. In the course of illness anemia and splenomegaly may develop. Usually in the natural course of the infection, the illness gradually resolves within a few weeks. On the contrary, our patient had a long latent period after exposure, and the clinical illness was superseded by postsurgical sepsis  BacteriaP.ovale, despite its low morbidity and mortality such as Acute Respiratory Distress Syndrome (ARDS), renal impairment, hyperbilirubineamia, and hypotension [Previous studies have reported several complications associated with otension .P.ovale malaria is approximately 2 weeks. The longest incubation period observed in a recent study was 265.71 weeks [The mean incubation period of primary infection by 71 weeks . A relap71 weeks .P. vivax and P. ovale is closely similar, still differentiation is possible due to characteristic features in parasitic stages found in the blood. Molecular diagnostic methods like PCR and RDT need to be carried out for further confirmation of differentiation [The gold standard of Malaria diagnosis is microscopy of thick and thin blood smears preferably stained by Giemsa stain. While thick blood smears are used to detect the presence of the malaria parasite, thin blood smear is used to identify the species. Though the microscopical appearance of ntiation .P.ovale infection consists of treating the erythrocytic forms. Erythrocytic forms of P.ovale infection are highly susceptible to chloroquine. Hypnozoites that contribute to a relapse of the infection are eradicated by primaquine other than prevention of transmission by gametocytes. Primaquine should be started after the subsiding of fever after confirmation of normal glucose-6-phosphate dehydrogenase status [P.ovale has developed resistance to any antimalarial drugs to date [Treatment of e status . It is u to date . AccordiP.ovale infection in South Sudan where the index patient had worked, the literature reports that P.ovale is extremely rare [P.ovale has presented as mixed infections (0.3%) along with other plasmodium species, such as vivax and falciparum [Considering the prevalence of ely rare . In Delhlciparum , 30.P.ovale infection was detected in a 37-year-old male from Northwestern Province in 2003. He had neither a travel history overseas nor blood transfusions and it was considered as an indigenous case because during that time indigenous transmission of other malaria species was occurring in Sri Lanka. This isolated case raises the possibility of zoonotic P.simiovale infection because the DNA primers have not been evaluated against the DNA of the closely related Simian Parasite [P. simiovale is prevalent in Macaca sinica monkeys in Sri Lanka [In Sri Lanka, the first case of Parasite . Infectiri Lanka . The queP.ovale as it might initiate indigenous transmission due to the presence of the Anopheles mosquito in the island. Findings such as hyperparasitaemia of P. ovale infection and prolonged incubation time with incidental detection need further studies to understand unknown dimensions of the pathogen. Postoperative infection and stress may have surfaced malaria in this patient with an unusual picture.Interestingly, the index case of ovale in Sri Lanka was detected incidentally and managed meticulously with the guidance of the Antimalaria Campaign of Sri Lanka. Coinfection of malaria with bacterial sepsis is a challenge in the management of the patient. Even though the 1st case claimed to be indigenous in origin, the index case has a strong travel history to both Sudan and India. It is important that Sri Lanka should be vigilant about imported cases of