The use of rapid diagnostic tests (RDTs) to diagnose malaria is common in sub-Saharan African laboratories, remote primary health facilities and in the community. Currently, there is a lack of reliable methods to ascertain health worker competency to accurately use RDTs in the testing and diagnosis of malaria. Dried tube specimens (DTS) have been shown to be a consistent and useful method for quality control of malaria RDTs; however, its application in National Quality Management programmes has been limited.Plasmodium falciparum strain was grown in culture and harvested to create DTS of varying parasite density . Using the dried tube specimens as quality control material, a proficiency testing (PT) programme was carried out in 80 representative health centres in Togo. Health worker competency for performing malaria RDTs was assessed using five blinded DTS samples, and the DTS were tested in the same manner as a patient sample would be tested by multiple testers per health centre.A All the DTS with 100 parasites/µl and 50% of DTS with 200 parasites/µl were classified as non-reactive during the pre-PT quality control step. Therefore, data from these parasite densities were not analysed as part of the PT dataset. PT scores across all 80 facilities and 235 testers was 100% for 0 parasites/µl, 63% for 500 parasites/µl and 93% for 1000 parasites/µl. Overall, 59% of the 80 healthcare centres that participated in the PT programme received a score of 80% or higher on a set of 0, 500 and 1000 parasites/ µl DTS samples. Sixty percent of health workers at these centres recorded correct test results for all three samples.The use of DTS for a malaria PT programme was the first of its kind ever conducted in Togo. The ease of use and stability of the DTS illustrates that this type of samples can be considered for the assessment of staff competency. The implementation of quality management systems, refresher training and expanded PT at remote testing facilities are essential elements to improve the quality of malaria diagnosis. Plasmodium falciparum parasite [The use of rapid diagnostic tests (RDT) contributes to achieving the World Health Organization (WHO) recommendations that all patients with suspected malaria have a confirmed diagnosis prior to anti-malarial treatment initiation. Globally, 93% of malaria cases are found in sub-Saharan Africa (SSA), with 213 million cases reported in 2018 [Plasmodium species, in those who have been infected with malaria. RDTs are now the most widely used test to diagnose malaria in suspected cases, comprising 75% of all malaria tests performed [Unlike blood smear, which is considered the gold standard method and which has been used to diagnose malaria for decades, RDTs do not require specific equipment or highly skilled personnel and can be used in field settings by health workers without extensive laboratory training. Malaria RDTs, which are lateral flow immunochromatography assays, detect the presence of circulating antigens, such as HRP2 and pLDH from different erformed . EspeciaWhile testing at or near the point-of-care (POC) can accelerate potentially life-saving medical decisions, procedural challenges, due in part to varied competency of health workers who conduct testing, can result in misinterpretation and misuse of RDTs, poor accuracy and misdiagnosis. Data from studies in the Democratic Republic of Congo have shown that only 18% of health workers correctly reported RDT results obtained as part of an external quality assurance (EQA) programme . UnaccepWhen RDTs are used and stored under optimal conditions , they can accurately detect malaria antigens. Recognizing that optimal conditions do not exist in many POC settings, it is important to monitor RDT usage and identify opportunities to maintain high standards of patient care.In Togo, where malaria remains a major public health problem, children and pregnant women are disproportionately affected. The introduction of RDTs has been gradual in Togo, they were first deployed into health facilities in 2006 with expansion into community settings in 2007. As this rollout has scaled up and the associated training of personnel has been expanded, the rate of malaria cases confirmed by RDT has rapidly risen in Togo, from 0.39 million in 2010 to 0.98 million in 2018, with 2.1 million estimated cases of malaria in 2018 nationwide [Currently, there is a lack of universal, field-ready malaria QC material to test the quality and performance of RDTs, or ascertain staff competency . While sPlasmodium falciparum infected blood, have been reported to be suitable for quality control and EQA in malaria testing facilities [Dried tube specimens (DTS), comprised of dried cilities , 12. ThiThis was a nationwide proficiency testing survey for health workers at 80 health centres across 40 districts and six regions, selected as described below, that use RDTs to diagnose malaria cases representing a subset of all testers within Togo. These 80 health centres represent 9.6% of the total 835 health centres in Togo which use RDTs and have been accredited by the National Malaria Control Programme (NMCP). A cut off of 80 sites for this activity was necessary based on the total number of DTS vials available. The programme was performed in Togo in October 2018.The PT activities were planned and implemented by the National Quality Assessment Programme Management (NQAPM) team of the Division of Laboratories of Togo Ministry of Health. Three field teams from the central level conducted the activity in the following health regions: Team 1: Lomé and Maritime health regions; Team 2: Plateaux health region and Team 3: Centrale, Kara and Savanes health regions.Based on the information from the NMCP and the number of PT samples available for use, a subset, selected as described below, of the total number of health centres using RDT to diagnose malaria were selected to be included in the malaria PT activity. As sites are unevenly distributed within Togo, facilities in locations with high capacities of microscopy diagnosis (e.g. Lomé) were eliminated to give priority to locations with lower level facilities or without a dedicated laboratory. A second review of the list of sites was undertaken to identify those sites with higher throughput of patients and group them geographically to ensure representation of all six health regions of Togo while maintaining ease of access by the field teams. Finally, after applying these criteria, 80 of these sites were randomly selected as participant sites with all workers present on the day of the field team visit being evaluated. The number of facilities meeting these criteria and selected to participate in the PT programme varied across 6 regions, with Centrale Region represented by 10 facilities, Kara Region by 13 facilities, Lomé Region by 7 facilities, Maritime Region by 17 facilities, Plateaux Region by 24 facilities, and Savanes Region by 9 facilities . Parasite preparation was then diluted with washed  parasite negative blood at a similar haematocrit to generate preparations with 0, 100, 200, 500 or 1000 parasites/µL. Parasite negative blood was procured from a US-based blood bank and were collected from US resident donors with no reported overseas travel and confirmed by PCR to have no malaria infections. Blood was determined to be HIV and HBV negative. Blood was also confirmed in the CDC malaria laboratory to not produce a positive test on RDTs before being used to produce DTS. The diluted parasite preparations were then tested on a histidine-rich protein 2 (HRP2)-based RDT brand with performance characteristics that met World Health Organization (WHO) procurement criteria [DTS were prepared in the USA as previously described . Brieflycriteria . After cTo check that baseline reactivity was not affected by drying, 1 vial of DTS at each of the parasite densities was re-hydrated with a solution of PBS-Tween20  and tested on the same RDTs brands as prior to drying. All DTS vials were from the same batch and stored at + 4°C before shipping via commercial air service at ambient temperatures to Togo and, similarly, were stored centrally in Lomé at + 4°C prior to field activities. As previously described, short term storage (up to 4 weeks) at ambient temperatures does not impact the reactivity of the DTS .A batch of SD Bioline® Malaria Ag Pf RDT  used in Togo was obtained from the NMCP for use in preliminary QC of DTS and in the PT. All procured kits were stored centrally until use. This allowed for the same lot number of kits to be used throughout the evaluation and provided an opportunity for baseline assessment prior to using the kits for the PT as well as eliminating the potential impact of variable storage conditions at testing sites. RDTs and DTS were provided to each health centre by the NQAPM team at the time of PT.Before distributing DTS vials to sites, the DTS panels were evaluated by the NQAPM in collaboration with the NMCP in the malaria reference laboratory in Lomé. Six aliquots of each parasitaemia level , 30 vials in total, were reconstituted in 50 µL of PBS-Tween rehydration buffer according to standard procedures provided by the malaria laboratory, CDC. Tubes were allowed to rehydrate for 1.5 h, gently shaken and 5 µL were then tested using SD Bioline® Malaria Ag Pf RDT  in the same manner as patient blood samples would be tested.To standardize reconstitution and remove a potential source of error, all DTS were reconstituted by the NQAPM team in the field according to the procedure provided by the CDC malaria laboratory and described in previous paragraph. Each reconstituted DTS contained 50 µl of sample and thus provided sufficient sample to evaluate multiple health workers at a given site.To evaluate the performance of the health workers using RDTs, PT panels comprised of five reconstituted samples with different parasite densities  were distributed to health centres and selected laboratories. One facility received only three samples containing 0, 100, and 1000 parasites/µl due to an insufficient number of samples with parasite density of 200 and 500 parasites/µl. DTS vials were rehydrated onsite as described above. A total of 235 health workers participated in the PT activity, with three, two and one health workers testing samples in 76, three and one health facility, respectively. The number of participants at each site was a function of the number of staff on duty at the time of the supervisory visit. All personnel with a role in RDT work and present on the day of the visit participated in the PT. The NQAPM team explained the PT procedure to the health workers prior to providing reconstituted DTS. To record the observations, each evaluated health worker received a results form designed by the NQAPM. Completed forms were collected by the field teams and returned to the NQAPM for analysis.t-test was used to compare the means of two paired samples. P-value of < 0.05 was considered significant. For the assessment of individual tester performance, PT scores were calculated as percentage of samples identified by a tester correctly . For the assessment of health centre, district, or regional performance, correct results were aggregated as percentage of tests performed correctly at corresponding level from a total number of tests performed at corresponding level. For this analysis, a level of 80% correct PT result was selected as a value to mark acceptable proficiency. While 80% correct is used as a threshold for malaria microscopy testing and Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) 5-star certification, ideally testers would achieve 100% correct results on PT to offer appropriate patient care when conducting malaria testing.XLSTAT Basic , a statistical application for Microsoft Excel®, was used for statistical analysis of data and Tableau Desktop  was used for data visualization. The difference between proportions was assessed using Chi-square test, followed by Marascuilo procedure to identify proportions responsible for statistically significant differences. Correlation between proportions of qualified testing personnel and regional performance was assessed using Pearson’s correlation coefficient. Paired Student’s The results of the QC conducted at the malaria reference laboratory showed that all aliquots of DTS containing 0, 500 and 1000 parasites/µl produced expected results with the SD Bioline® Malaria Ag Pf RDT. All DTS with 100 parasites/µl and 50% of DTS with 200 parasites/µl were classified by NQAPM team as non-reactive  = 8.2; p = 0.004; effect size phi = 0.19). Forty-seven (59%) of the 80 testing facilities that participated in the PT programme received a score of 80% or higher on a set of 0, 500 and 1000 parasites/ µl DTS samples.PT scores across all 80 facilities and 235 testers was 100% for 0 parasites/µl, 63% for 500 parasites/µl and 93% for 1000 parasites/µl  while senior health technicians or medical assistants represented only 5%   = 5.6; p = 0.590; effect size Cramer’s V = 0.03) nor when comparing only the results of tests on samples containing 500 parasites/µl  = 3.9; p = 0.787; effect size Cramer’s V = 0.05) or those with 1000 parasites/µl  = 13.5; p = 0.061; effect size Cramer’s V = 0.09).All qualification groups correctly identified negative DTS and performed better in the detection of malaria in samples with parasite densities of 1000 parasites/µl than those with 500 parasites/µl (t-test for two paired samples st) Fig. . There wχ2 = 9.9; p = 0.078; Cramer’s V = 0.05)  despite differences, although with small effect size, in the percentage of health workers drawn from these three groups between the regions  = 26.4; p < 0.0001; Cramer’s V = 0.15).The 80 health centres were distributed amongst multiple regions in the country Fig. . The difχ2df = ; N = 702χ2  = 6.3; p = 0.278; Cramer’s V = 0.07). While a difference was detected between regions in testing 1000 parasites/µl specimens, the effect size was negligible and the data were not sufficient to identify significantly different regions  = 15.0; p = 0.010; Cramer’s V = 0.11)  at or near 200 parasite/µl, RDT sensitivity is reduced at low parasite densities and cases of low sensitivity of RDTs in samples of parasite densities less than 200 parasites/µl have been reported , 13.The parasite densities of 0, 500 and 1000 parasites/µl are consistent with those used by other groups for PT using malaria DTS . BecauseWith the increased use of RDTs, shifting the role and location of diagnostic testing from laboratory technicians in higher-level facilities to community health workers in peripheral or point-of-care settings has become common practice. Other studies showed that community healthcare workers can successfully follow the RDTs procedural steps to diagnose malaria using RDTs when they are provided with adequate training and job materials , 15. WhiIncorrect results and incorrect interpretation of results have negative clinical implications. While no false positive results were reported by study participants, 34 (43%) of 500 parasites/µl and 13 (16%) of 1000 parasites/µl samples were incorrectly deemed negative by at least one tester at a site. At a site level, 13 (16%) of 500 parasites/µl but none of 1000 parasites/µl samples were designated as negative by all testers at a site. This creates a case for testing of samples by multiple staff members or for results to be confirmed via microscopy where personnel and reagent resources allow.Variation in results was also seen amongst testers of different qualifications  and amongst different parasite density PT samples: as expected, 1000 parasites/µL DTS were more commonly identified as reactive than the 500 parasites/µL DTS. Although most clinical malaria cases are often associated with densities of 1000 parasites/µL or higher, clinical cases with lower densities are not uncommon –18. Low Many factors can impact the performance of RDTs such as lack of adherence to the manufacturer’s instruction including misreading or misinterpretation of results by health workers. Errors in procedures can result in mismanaged care for patients with malaria. Such human errors include using the wrong amount or type of sample or buffer or incubating the test strip for an incorrect amount of time, especially shorter times can result in provision of incorrect test results . In manyUsing malaria DTS, the Division of Laboratories of Togo Ministry of Health was able to conduct a nationwide proficiency testing activity across multiple health worker categories. This PT programme validates the feasibility of the use of malaria DTS for PT in a field setting. The ease of preparation, dissemination, shipping and reconstitution of the DTS, along with reportable results from testers, demonstrates that DTS can serve as well-characterized, stable blinded samples for PT at parasite densities above the LOD of the rapid tests.The PT data from Togo demonstrates that there is room for adherence to QMS and use of QC to assess health worker competency to improve malaria rapid testing across all testing settings. This programme showed that regardless of education level, the challenge for RDT users lies closer to lower parasitemia levels where minor errors in technique during testing can become much more impactful.High-quality and routine (at minimum yearly) health worker assessments, training, site supervision and monitoring visits to follow adherence to the testing protocol can contribute to higher levels of health worker performance. These practices coupled with other QMS principles can standardize testing, thus improving patient outcomes , 26.


Arriving in the pediatrics department, I realized my expectations of medicine in Uganda were worlds away from reality. I had agreed to go on call with the pediatric intern that evening. “Just for emergencies” he had said, which had given me the image of sitting in the ward waiting for any acutely unwell children who may or may not appear. On reaching the ward, I was instead met by more than 50 mothers with children waiting at the emergency bench to see the one doctor.Our first patient had blood results with them, showing a blood smear positive for malaria and a hemoglobin of 3.1 g/dL, the lowest hemoglobin I had ever seen and one which requires swift transfusion. A femoral tap was carried out immediately for blood grouping and crossmatch, without hesitation or any attempts at cannulation for fear of wasting time. The intern took me to the laboratory, showed me how to sign in our samples, who to give them to, and then left me there, so he could return to the queue on the ward. I waited for the blood to be analyzed, feeling considerably out of my depth but trying to appear confident. “He’s O+” said the laboratory technician “and I’m sorry, we don’t have any.” I was stunned that one of the biggest hospitals in Uganda could just run out of blood. I thought this was going to be some sort of crisis, and people on the ward would be appalled that running out of O+ could possibly be allowed to happen. “Do you have any O−?” I asked. “We have just one bag, but if you really need it then you can take it.” As medical students in Europe, we have absolutely no responsibility for decision-making and are heavily supervised even through the early years of our careers; and now, someone was asking me to decide whether to take the last unit of the universal donor from a bank supplying a whole hospital. Deciding I was definitely not qualified to make this decision, I ran to the ward and asked the intern what to do. Looking at me like I had just fallen from the sky, he said “of course, the hemoglobin is 3.1, he will die without it.” And that was that, I emptied the blood bank on my first day in the hospital.I quickly learned that the situation on that first night was not uncommon; it was the norm. Every night, at least five children younger than 5 years came in with malaria and a hemoglobin below 5 g/dL, and most nights, the blood bank ran dry. This meant that relatives had to donate to keep their children alive, but the blood groups are not always compatible or the relative is HIV+. Almost every night, some children do not receive the transfusions they need and some of them die for this reason.“How much malaria had you seen before coming here?” one of the interns asked during my second week. I told him I’d never seen it before and that we don’t really have it where I’m from.“There’s whole hospitals without any malaria?” he questioned me further, sounding skeptical.“Yes, there’s whole countries, virtually a whole continent,” I replied.Unconvinced, he continued, “So what’s your malaria? What kills the most children where you’re from?” This was another wake-up call to me; a world without malaria, a world without children dying every night, was unfathomable to him.I kept pushing myself on through the work on the ward, trying to keep up with the interns. They work 7 days a week plus two nights, resulting in levels of tiredness that are incompatible with providing the best care for patients or themselves. By my third week, I had seen more, knew more, and was less shocked by the things on the ward; then I had an evening call shift, which will forever remain in my mind.pm, I was met by the medical officer. He said, “Put on gloves, this one bites.” We restrained the struggling child to take blood, and I was sent to the laboratory to get a group and crossmatch, and bring back the transfusion. She was so clinically anemic; we did not need the hemoglobin result to know that transfusion was essential to her survival. It took 20 minutes for the blood to be confirmed as compatible; when I returned to the ward, the 3-year-old girl was already dead. The full blood count and blood film confirmed malaria and a hemoglobin of 2.6 g/dL. I returned the unused blood to the laboratory, utterly dejected, but there was no time to stop to process this. Work does not, and cannot, pause here when you lose a patient.Arriving on the ward at 8 Ten minutes later, the intern asked me to check a patient’s vitals. The boy was 4 years old, with a diagnosis of cerebral malaria. He was not breathing, sats were not registering; he did not respond to a sternal rub, and his pupils were dilated and not reactive to light. I knew what this meant, but I could not tell the mother who is anxiously looking up at me with her eyes full of fear. I told the intern. He came and spoke a few soft words in the local language. The mother began to cry, and a mix of rage and sadness rose in me at the injustice of it. How is it possible that in the same world where babies born at 24-weeks gestation can survive and technology such as extracorporeal membrane oxygenation exists that a child still dies every 2 minutes from this preventable disease?My final malaria lesson was a personal one, hailed by a combination of fever, freezing chills, the worst headache I have ever experienced, a variety of miserable GI symptoms, and the very real concern I may not physically be able to fly home. On my last night in Uganda, I got a first-hand reminder that atovaquone/proguanil is only 90% effective as prophylaxis. Being infected with this monstrosity of a parasite is not an experience I would ever like to relive, but I am thankful that I am a healthy nonpregnant 23-year-old and I came out the other side of it with no major complications, a happy ending for me.The story of malaria in Uganda, however, is far from its happy ending. In 2017, according to the WHO, the country had an estimated 8.6 million cases and 14,400 deaths, accounting for 4% of the global malaria burden. The impact of malaria is clear when you look at these statistics, but what I failed to understand before experiencing it is the real human cost. I saw the humanity of it in the frightened eyes of children struggling to breathe, the desperation of parents helplessly watching their child seize, the misery of those who lost their children, and the exhausted faces of the doctors who cannot do any more than attempt to put out each fire as it comes through the door.Humanity’s fight against malaria has spanned decades, and objectively, progress has been made. However, the first time I met malaria, it felt like the disease was winning. We must not forget how much work is still needed and what is at stake.We cannot relent.


We report in this manuscript a case of newborn baby with asymptomatic form of congenital malaria; the screening of the peripheral blood smear of the baby after a positive result in the mother allowed the diagnosis. The authors were permitted through this case to discuss the therapeutic possibility in these cases. Plasmodium falciparum, recent data suggest that this is a more frequent situation than expected [In congenital malaria, infants are infected by the passage of parasites from mother to child before or during birth, it is a fatal condition that occurs at low rates in endemic countries . Malariaexpected . In thesPlasmodium falciparum [We report a case of asymptomatic congenital malaria; to our knowledge the articles available in the literature talk about symptomatic cases of the disease. On the other hand, the authors through this case discuss the therapeutic possibilities of asymptomatic congenital malaria. Congenital malaria is the presence of malarial parasites in the peripheral blood smear of the newborn from the first day until the seventh day of life . It is dlciparum . In theslciparum , 6. The lciparum , 8.th week of gestation, prenatal screening tests were not performed. The clinical examination showed normal fundal height, with perception of uterine contractions and normal fetal heart activity, the cervix was effaced and dilated to 4cm. Trans-abdominal ultrasound was performed; the placenta was localized in the posterior side of the fundus; the amniotic fluid and the fetal growth were normal. A healthy male infant weighing 2600g was successfully delivered via spontaneous vaginal delivery, the apgar score was 8/9/10 with no blood loss during delivery, and no congenital abnormality was detected. 24 hours after delivery, the mother presented acute fever associated to chills and excessive sweating; she had anemia with hemoglobin 8.2g/dl, hematocrit 25.8%, MCV 86.1fl, MCH 28.5pg, MCHC 31.5g/dl, total leucocyte count 5.5×103/ul and platelet count was low 56,000/ul. C-reactive protein (CRP) was normal , with normal range of serum electrolytes. Blood and urine cultures were sterile and peripheral blood smear showed malarial parasites of Plasmodium falciparum parasitemia at 4%.A 32 years old woman was admitted during labor, to the Department of Gynecology of the Moroccan Field hospital deployed in South Sudan. The patient was gravida 3, para 2, abortus 1 (2 living children) at the 39th week of pregnancy, managed with unspecific treatment. The newborn baby was healthy and vigorous, exclusively breastfed, the clinical examination was normal; biological analysis showed hemoglobin 16.3g/dl, total leucocyte count 7.5×103/ul and platelet count 72,000/ul. C-reactive protein (CRP) was normal (< 5mg/l), with normal range of serum electrolytes. Peripheral blood smear was negative for malaria parasites; unfortunately, the other methods of diagnosis  were not available for further investigations, the newborn was placed under close observation and a second examination of the peripheral blood, was done within 3 days; a thick and thin blood film revealed P. falciparum trophozoites, with a parasitaemia of 1,5%. The mother was treated according to local guidelines with arthemeter + lumefantrine and the infant was treated by oral chloroquine 10mg/kg for two days and 5mg/kg in the third day. Four days after treatment haemoparasites were cleared, with normal follow-up.The mother reported after evaluation a possible malaria infection in the 32Congenital malaria was described the first time in 1876; it is defined by the presence of asexual forms of malaria parasites in peripheral blood within the first week of life . The occThe genomic amplification and DNA quantification increases the prevalence of diagnosis but they were not possible in our conditions. There are no clearly guidelines established to treat congenital malaria. Primaquine is not indicated because the infection is acquired congenitally (it doesn't include the persistent hepatic phase); chloroquine has been the drug of choice in case of congenital falciparum malaria . HoweverHowever, in various studies, the cases of congenital falciparum malaria, whether symptomatic or not, should be treated , 27, 28,The repetition of thick drop examination has helped us to assert the diagnosis of congenital malaria in asymptomatic and healthy patient. When close clinical monitoring of patients over a 15-week period is feasible, antimalarial treatment should not be initiated unless a clinical signs of infection occurs and after diagnostic confirmation.The authors declare no competing interests.


Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a bead-based immunoassay for Plasmodium antigens; and/or whole-genome sequencing. Among 3627 symptomatic subjects, 427 (11.8%) had RDT-/microscopy + results. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. 56.8% of subjects had PCR-confirmed malaria. Non-falciparum co-infection with P. falciparum was common (13.2%). Agreement between PCR and HRP2-based RDTs was satisfactory (Cohen’s kappa = 0.66) and superior to microscopy (0.33). Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.The majority of  Plasmodium falciparum strains that evade detection by rapid diagnostic tests (RDTs) threatens progress toward malaria control and elimination in Africa3. These parasites have deletions involving the histidine-rich protein 2 and/or 3 (pfhrp2/3) genes, which encode the proteins detected by widely used RDTs throughout Africa4. Increasing reports of these parasites in select locations across Africa raise concern about the future of HRP2-based RDTs in the region7. Recent events in Eritrea, where a high frequency of false-negative RDTs due to these parasites triggered a change in national malaria diagnostic policy, emphasize the need for surveillance and a coordinated response to pfhrp2/3-deleted P. falciparum9.Emergence of pfhrp2 deletions and reported a 6.4% national prevalence of pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC)10. While this cross-sectional, nationally representative household survey enabled spatial analyses and initial population genetic analyses of pfhrp2-negative parasites in the DRC, it did not sample subjects with symptomatic malaria. In order to inform decisions about national malaria diagnostic testing policy, we undertook a cross-sectional survey of children and adults presenting to government health facilities in three provinces selected based on the prevalence of pfhrp2-negative parasites in our initial study. Based on results from our prior study of asymptomatic children and a mathematical model of the spread of pfhrp2/3-deleted P. falciparum10, we hypothesized that pfhrp2/3-deleted parasites were responsible for missed clinical cases of falciparum malaria in the DRC and would be more commonly identified in regions with lower transmission intensity.We previously performed the first national survey of pfhrp2/3-deleted P. falciparum are difficult due to the challenges of confirming the absence of these genes using conventional approaches12. These challenges are compounded by inconsistent laboratory methodologies across studies and inherent limitations of pfhrp2/3 assays that can suffer from variable performance and cross-reactivity14. In addition, false-negative RDT results are common throughout Africa and typically caused by factors other than pfhrp2/3 deletions, including operator error, lot-to-lot RDT variability, low-density infections below the RDT’s limit of detection, and infection by non-falciparum species16. In order to overcome these challenges, we performed a comprehensive molecular, serological, and genomic evaluation of symptomatic infections to define the causes of false-negative RDTs in the DRC and inform national diagnostic testing policy.Studies of 17. These provinces were selected based on results from our prior survey of asymptomatic children in the nationally representative 2013–2014 DRC Demographic and Health Survey (DHS) and included provinces with both high-  and low-prevalence (Bas-Uele) of pfhrp2-negative parasites10. We previously estimated the weighted prevalence of pfhrp2-negative P. falciparum among asymptomatic children in these provinces: 21.9%  in Kinshasa, 18.3%  in Sud-Kivu, and 0.2%  in Bas-Uele10. Two health zones were selected from each province, including one urban and one rural zone per province. Within each health zone, one general reference hospital and two health centers were selected as study sites, yielding six study sites per province and 18 study sites in total. Subjects of all ages presenting with symptoms compatible with malaria were eligible for enrollment.We conducted a cross-sectional study of subjects presenting to hospitals and health centers across three provinces  with suspected malaria. Cases of suspected malaria were identified according to national guidelines and included those with fever, headache, convulsions, muscle or joint pain, vomiting, and other symptoms. The National Malaria Control Program considers malaria prevalence in these provinces to be urban, hyperendemic, and mesoendemic, respectivelyInformed consent/assent was obtained from all study subjects prior to enrollment. Parental permission and informed consent was obtained from parents or legal guardians for all minors younger than 18 years of age, and assent was obtained from all children and adolescents 7–17 years of age. All subjects received malaria RDT testing and treatment according to DRC national guidelines. Subjects underwent a study questionnaire and finger- or heel-prick whole blood collection for diagnostic testing by RDT and microscopy and DBS collection. RDT testing was performed using the World Health Organization- (WHO-) prequalified, HRP2-based SD BIOLINE Malaria Ag P.f.  according to manufacturer instructions. Thick-smear microscopy slides were read in the field, and thin smears fixed and transported to the National AIDS Control (PNLS) reference laboratory for confirmation and determination of parasite density. All thin smears were read by two microscopists, with discrepancies resolved by a third reader. Dried blood spot (DBS) samples  were allowed to air dry at ambient temperature in the field, and stored in individual ziplock bags with desiccant at − 20 °C prior to and after shipment to the University of North Carolina at Chapel Hill for further testing. This study was approved by the Ethical Committee of the Kinshasa School of Public Health . Analysis of de-identified samples and data was determined to constitute non-human subjects research by the UNC Institutional Review Board (study number 17-0155). The study was determined to be non-research by the Centers for Disease Control and Prevention Human Subjects office (0900f3eb81bec92c). Experiments were performed in accordance with relevant guidelines and regulations.18. All microscopy-positive, RDT-negative samples, in addition to an equal number of microscopy-positive, RDT-positive controls from each province were subjected to quantitative PCR (qPCR) testing targeting the single-copy P. falciparum lactate dehydrogenase (pfldh) gene19. Pfhrp2 and pfhrp3 PCR genotyping was performed as previously described13, using conventional single-step pfhrp2/3 PCR assays and a qualitative real-time PCR assay targeting the single-copy P. falciparum beta-tubulin (PfBtubulin) gene (Supplementary File)23. Only samples with ≥ 40 parasites/µL by qPCR (≥ tenfold higher concentration than the pfhrp2 and pfhrp3 assays’ limits of detection) were subjected to pfhrp2 and pfhrp3 PCR to reduce the risk of misclassification of deletions13. Microscopy-positive, RDT-positive controls with ≥ 40 parasites/µL by qPCR were randomly selected from the same facility for pfhrp2/3 genotyping. Samples were called pfhrp2/3-negative if they had ≥ 40 parasites/µL by pfldh qPCR, their pfhrp2 and/or pfhrp3 PCR assays were negative in duplicate, and they had successful amplification of PfBtubulin during a final confirmatory assay.DNA was extracted from DBS samples using Chelex and saponinpfhrp2/3-negative samples identified during initial testing were further assessed using whole-genome sequencing. DNA from these samples was enriched for P. falciparum prior to library prep using selective whole-genome amplification (sWGA) as previously described24. In brief, two sWGA reactions were performed in parallel, one using a custom primer set designed in our lab (JP9) and another using a primer set designed by Oyola et al. (Probe_10)25. sWGA products were pooled in equal volumes and acoustically sheared using a Covaris E220 instrument prior to library preparation using the Kapa HyperPrep kit . Indexed libraries were pooled and sequenced at the UNC High Throughput Sequencing Facility using the HiSeq 4000 platform  with 150 bp, paired-end chemistry. Sequence reads were deposited into the Sequence Read Archive (BioSample accession numbers: SAMN16711875-82).All trimmomatic and aligned to the P. falciparum 3D7 reference genome (PlasmoDB version 13.0) using bwa mem with default parameters27. Duplicates were marked and mate-pair information corrected using Picard Tool’s MarkDuplicates and FixMateInformation functions, respectively28. Candidate indels were identified and realigned using GATK’s RealignerTargetCreator and IndelAligner functions, respectively29. Genome coverage was calculated using bedtool’s genomecov function and visualized using ggplot2 in R 31. Pfhrp2/3 deletions were called by visualization of aligned reads using the Integrative Genomics Viewer  and assessment of sequencing depth  across the pfhrp2/3 genes and their flanking regions32. Variant calling was performed using the Genome Analysis Toolkit’s HaplotypeCaller29. Calls were subset to biallelic SNPs. Variant quality score recalibration was used to filter suspected false positives using the MalariaGEN P. falciparum Genetic Crosses Project (release 1.0) and Pf3k datasets (v5.1). The Fws statistic describes the relationship between within-host and population parasite heterozygosity and has been used as an index of genome-wide SNP diversity, with lower values correlated with higher multiplicity of infection, and vice versa33. Fws values were calculated using the vcfdo package to identify monoclonal infections; samples with Fws > 0.95 were considered monoclonal34.Adapter sequences were trimmed from raw, paired sequence reads using pfhrp2/3 genotyping by PCR were also assayed for the following Plasmodium antigens: Plasmodium genus-specific aldolase (pAldolase) and lactate dehydrogenase (pLDH), as well as P. falciparum HRP2 by a bead-based multiplex assay as previously described35. Samples were assayed at 1:20 whole-blood concentration after elution from filter paper. Thresholds for antigen positivity for the three targets were determined by assaying 92 blood samples from US resident blood donors without history of international travel and determining the lognormal mean and standard deviation of assay signal from this sample set. The lognormal mean plus three standard deviations of this sample set was used as the antigen positivity threshold  in RStudio . Lilliefors test was performed using the nortest package. Maps and figures were generated using the ggplot2, sf, ggspatial, and gridExtra packages and finished in Google Slides .We made comparisons using the Kruskal–Wallis Rank Sum or Fisher’s exact test for categorical variables and one-way analysis of variance (ANOVA) test or the t-test for normally distributed continuous variables. Prior to statistical testing, normality was assessed by visual inspection of the frequency distributions of the raw data (histograms) for comparisons with sample sizes ≥ 30 per subgroup, and using the Lilliefors normality test for comparisons involving sample sizes < 30 per subgroup. Correlation between parasite density calculations was assessed using the Spearman’s rank-order correlation coefficient. Agreement between assays was assessed using Cohen’s kappa coefficient. Statistical analyses were performed and maps rendered using R software  of the 426 RDT-negative/microscopy-positive samples were PCR-negative, a finding consistent with false-positive microscopy calls in this cohort.Among 3627 subjects tested, 1545 (42.6%) were RDT-positive and 1397 (38.7%) were microscopy-positive, with 426 (11.8%) RDT-negative but microscopy-positive . False-negative RDT results were more likely to occur at low microscopy parasite densities, with geometric means of 959 (geometric standard deviation [SD] 4.2) and 4,675 (geom. SD 6.9) parasites/µL for RDT- versus RDT + samples, respectively 13. We further characterized these samples and 74 RDT-positive controls selected from the same facilities  using a series of PCR assays for pfhrp2 and pfhrp3, and a final confirmatory PCR assay for PfBtubulin. Eight parasites were PCR-negative for pfhrp2 or pfhrp3 in duplicate despite having parasite densities well above the PCR assays’ limits of detection and successful amplification of a second single-copy gene, consistent with pfhrp2/3 gene deletions using conventional PCR genotyping criteria41. PCR genotyping suggested five deletions among symptomatic RDT-/microscopy + subjects (four pfhrp2−/3− and one pfhrp2−/3 +) and three among RDT + /microscopy + subjects  .We performed pfhrp2/3-deleted samples had parasites with intact pfhrp2 and pfhrp3 genes  of the 8 samples subjected to whole-genome sequencing  confirmed that all eight putative nes Fig. . All eigpfhrp2/3-PCR-negative samples had detectable HRP2 antigenemia, providing confidence that the intact genes observed during whole-genome sequencing encoded functional HRP2 and/or P. falciparum histidine-rich protein 3 (HRP3) proteins. Background-subtracted mean fluorescence intensity (MFI) thresholds for positivity were 29, 62, and 72 for HRP2, pLDH, and pAldolase, respectively  was similar between groups for all three antigens tested: HRP2, Plasmodium parasite lactate dehydrogenase (pLDH), and Plasmodium parasite aldolase (pAldolase)   of Plasmodium-PCR-positive symptomatic cases. Among the four (0.8%) symptomatic cases involving P. vivax, half involved P. falciparum and all were low density (< 5 parasites/µL by semi-quantitative 18S rRNA PCR). The majority of symptomatic P. malariae infections  occurred as part of mixed infections with P. falciparum , followed by Sud-Kivu (17%), and finally Kinshasa (5%) . Microscopy was 53% sensitive and 81% specific, with fair agreement with PCR (Cohen’s kappa = 0.33). Parasite densities as determined by microscopy and pfldh qPCR had moderate correlation  of 538 pfhrp2- or pfhrp3-deleted P. falciparum in this large, cross-sectional survey across three geographically disparate DRC provinces. The majority of RDT-negative/microscopy-positive results occurred in the setting of low or absent parasitemia. This finding implicates parasite densities below the RDT’s limit of detection and false-positive microscopy results as the primary causes of RDT-microscopy discordance in the present study. Further assessment of RDT performance using microscopy, genus- and species-specific real-time PCR assays, and Luminex-based antigenemia assessment confirmed that RDT failure and/or user error also caused false-negative RDTs in the present study. However, the overall performance of HRP2-based RDTs was superior to microscopy and in good agreement with PCR.We did not observe symptomatic malaria due to pfhrp2/3-deleted parasites. We and others have proposed the hypothesis that parasites with deletions of the pfhrp2 and/or pfhrp3 genes and their flanking regions may be less fit43, and less likely to cause symptomatic disease. Direct assessment of this hypothesis has not yet been performed in vivo or in vitro, to our knowledge, and is limited by the challenges of confirming deletions in low parasite density infections. However, genetic cross experiments of the 3D7 (wild-type), DD2 (pfhrp2-deleted), and HB3 (pfhrp3-deleted) lab strains did not provide definitive evidence of a fitness cost associated with deletion of either gene45. In addition, reports from Eritrea confirm that pfhrp2/3-deleted parasites can cause symptomatic and sometimes severe disease8.These findings support continued use of HRP2-based RDTs in the DRC. They also contrast with the results of our prior study of asymptomatic children enrolled in the 2013–2014 DHS. There are several possible explanations for these differences. The present study enrolled symptomatic subjects in order to directly inform policy decisions about malaria case management. This study design could have inhibited our ability to identify pfhrp2/3-deleted parasites was needed to discern the status of both genes. The use of rigorous parasite density thresholds well above the downstream pfhrp2/3 PCR assays’ limit of detection13, confirmation of successful amplification of multiple single-copy genes, and adherence to commonly accepted criteria5 reduced the risk of inappropriate pfhrp2/3 deletion calls. Only five of 426 (1.2%) RDT-negative/microscopy-positive samples were identified as putative pfhrp2/3 deletions during initial testing. However, we subsequently confirmed HRP2 antigenemia and intact pfhrp2 and pfhrp3 genes in all eight pfhrp2/3-PCR-negative samples using highly sensitive antigen detection methods and WGS, respectively. Pfhrp2/3 sequence variation and resulting changes in the structure of the HRP2 and HRP3 proteins could yield a negative RDT but positive Luminex result, or vice versa, due to differences in the anti-HRP2 monoclonal antibodies employed by both assays. However, previous evaluation of diverse P. falciparum strains failed to identify an association between pfhrp2/3 sequence variation and RDT sensitivity46. Taken together, Luminex and WGS results confirmed that the putative pfhrp2/3-deleted parasites were misclassified during initial PCR testing.Exhaustive analysis of putative pfhrp2/3 gene deletions and support the argument that a portion of pfhrp2/3 deletion calls in our original study of asymptomatic children in the DRC resulted from experimental artifact11. Even complex laboratory workflows conducted in accordance with commonly used deletion classification criteria are not always sufficient to eliminate the risk of misclassification of pfhrp2/3 deletions. The use of advanced serological and next-generation sequencing methods improved the quality of our pfhrp2/3 deletion assessment, allowed for a more robust evaluation of RDT performance, and enabled visualization of the genetic structure of the pfhrp2 and pfhrp3 genes and their flanking regions. While these methodologies are not widely available in resource-limited settings, they are now accessible through a network of laboratories that collaborate with the World Health Organization to support pfhrp2/3 deletion surveillance47 and in select locales in sub-Saharan Africa with advanced laboratory capacity.These findings emphasize the challenges of confirming P. falciparum. Although non-falciparum species are not detected by widely deployed HRP2-based RDTs, co-infection with P. falciparum is expected to trigger a positive RDT result and prompt treatment with artemisinin-combination therapy according to current DRC guidelines. Therefore, complications due to untreated symptomatic, non-falciparum malaria are likely uncommon, although the risk of relapse by P. vivax or P. ovale without proper diagnosis and terminal prophylaxis remains. Our findings are generally in-line with prior reports of non-falciparum infection among asymptomatic subjects in the DRC50.Symptomatic malaria due to non-falciparum species was common but usually occurred as part of mixed infections with pfhrp2-negative parasites from our initial study of asymptomatic subjects. Our experience in the DRC confirms the importance of basing policy decisions on careful studies of the target population—individuals presenting to health facilities with symptomatic malaria—rather than convenience sampling determined by sample availability or access to study sites.Strengths of this study include its geographically diverse sampling locations, robust pipeline of conventional and advanced laboratory methodologies, and relevance to malaria case management. Indeed, these findings directly informed the DRC national malaria control program’s decision to continue the use of HRP2-based RDTs, despite evidence of pfhrp2/3-deleted from pfhrp2/3-intact strains in individuals infected by multiple P. falciparum strains. Neither the conventional methods nor the advanced Luminex-based HRP2 antigenemia assessment and WGS methods employed here are well-suited to identify gene deletions in mixed infections. Indeed, assessment of whole-genome sequencing data confirmed mono-infection in only three of eight samples tested. We therefore cannot exclude the possibility that the five remaining samples included mixed infection that involved pfhrp2/3-deleted strains. Recently developed multiplexed qPCR methods51 and amplicon-based deep sequencing approaches52 have potential to elucidate pfhrp2/3-deleted minor variants in future large-scale surveys. Second, we restricted our pfhrp2/3 deletion analysis to samples with ≥ 40 parasites/µL. This requirement was necessary to reduce the risk of misclassification due to DNA concentrations below the pfhrp2/3 PCR assays’ limits of detection13, but it prevents us from commenting on the prevalence of deletions in lower density infections. Third, enrollment occurred primarily in the rainy season, with only limited dry season enrollment near the end of the study in Bas-Uele. A modeling study predicts that pfhrp2/3-deleted parasite prevalence may be underestimated during the rainy season, when individuals are more likely to be infected by multiple parasite strains53. Fourth, we could not evaluate associations between specific symptoms or temperature and RDT performance, as this data was only available for a subset of subjects. Finally, this study was restricted to three provinces. These provinces spanned a range of malaria prevalences, but they do not capture the full diversity of the DRC, which is Africa’s second largest country by land mass and neighbors nine other countries.Limitations include our inability to discriminate pfhrp2/3 deletions were not observed among symptomatic subjects. Most false-negative results in the DRC are likely due to low parasite densities, RDT failure, or operator error. Only a minority of non-falciparum malaria cases would be missed by an HRP2-based RDT testing strategy; co-infection with P. falciparum and non-falciparum species was common. Careful laboratory workflows are required during pfhrp2/3 gene deletion analyses. Advanced serological and next-generation sequencing approaches can be used to improve the rigor and reproducibility of pfhrp2/3 deletion surveillance efforts and to inform malaria diagnostic testing policy.In conclusion, ongoing HRP2-based RDT use is appropriate in the DRC. False-negative RDT results due to Supplementary Information 1.Supplementary Information 2.


The effort, supported by a US$ 2.7–3.2 billion annual investment, to “bend the curve” towards zero malaria by 2030 has stalled over the past five years4. This plateau is reminiscent of the decades-long effort to break through the ~80% ceiling on reaching the goal of fully immunizing every child worldwide. Despite immunization of 116 million children annually, 20 million infants fail to receive a full course of essential vaccines and 13 million infants receive no vaccines whatsoever—these “zero dose” children are highlighted in the Immunization Agenda 20303 (IA2030), recently adopted by the World Health Assembly5. The shared state of stalled progress towards equity and coverage of effective malaria interventions and essential vaccines has a common solution—new tools.Despite a ~30% reduction in clinical cases and ~60% reduction in lives lost from P. falciparum, and the most prevalent, P. vivax, has been assembled6. Novel vaccine platforms , better defined correlates of protection in non-human primates8, and orthogonal learnings from other mosquito-transmitted pathogens 9 portend an even more robust pipeline of needed new tools, if adequate investments are made.The early promise of innovative vector control and single-dose radical cure for malaria have yet to deliver the impact needed to achieve “zero malaria”. That said, steadily over the last two decades, as reviewed by Duffy and Gorres, a pipeline of vaccine candidates against the most lethal human malaria, 6). Despite decades of efforts directed against targets throughout the lifecycle, only one vaccine candidate, the pre-erythrocytic circumsporozoite (CSP)-based RTS,S/AS01E, has advanced through licensure and pilot implementation6. A myriad of biological and technical barriers have been encountered; however, step-by-step, these barriers have been chipped away. Advances include: new insights into the evolution of the immune response to the major pre-erythrocytic target, CSP10, and into the role of complement-fixing antibodies in blood stage clinical immunity11; the ability to express and manufacture full length versions of blood stage targets, e.g., the highly promising, highly conserved reticulocyte-binding protein homolog 5 (PfRH5)12, as well as the 196-kDa merozoite surface protein 1 , the latter of which demonstrated favorable safety and immunogenicity in a first-in human study14; and, the delivery of target malaria parasite antigens by measles vectors to overcome the barrier of waning immune responses, resulting in durable memory and protection, at least in a murine model15.Malaria vaccine targets are typically assigned to one of three sequential stages of the parasite’s lifecycle: blood stage—through which the parasite causes all human pathology; sexual and sporogony stage—through which the parasite is transmitted from human host to female mosquito vector; and, pre-erythrocytic stage—through which the parasite infects humans during a subsequent bloodmeal by an infected mosquito 17. That said, molecular approaches to identify and direct immune responses to specific promising epitopes18 and use of novel particle-forming lipid-based adjuvants19 provide paths forward for poorly immunogenic targets, including those designed to interrupt transmission from human to mosquito.To be clear, significant challenges remain—for example, immune interference when concomitantly administering other vaccines, such as BCG for tuberculosis20. Pregnancy malaria presents an even clearer case for prioritizing vaccine development as both mother and offspring have a well-described higher risk of disease6. More than two decades ago, antibodies to VAR2CSA, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, were associated with protection, identifying VAR2CSA as a promising vaccine target21. Placenta malaria vaccine (PMVs) research and innovation continues to progress6, including through the development of new animal models22 and down-selection of lead PVM candidates23.The longstanding mindset that vaccine evaluation during pregnancy should be delayed to post-licensure studies is obsolete. Even when pregnant women and their offspring do not have a higher risk of disease, there is an ethical rationale to evaluate at least vaccine safety pre-licensureP. vivax vaccine development have been significantly smaller than that in the more lethal, albeit similarly morbid, falciparum cousin. Many P. falciparum vaccine targets have homologs in P. vivax6, so a fast follow-on vivax malaria vaccines based on safe, effective, affordable falciparum malaria vaccines have a reasonable, high likelihood of success. That said, several distinct differences in P. vivax biology, such as liver stage hypnozoites and rapid development of sexual stages directly from liver schizonts, require a P. vivax-specific vaccine research and innovation strategy. A critical differential feature of blood-stage vivax parasites is the use of Duffy antigen receptor for chemokines (DARCs) on human reticulocytes as a major invasion pathway. Recent insights into the structural basis of anti-P. vivax Duffy Binding Domain (PvDBP) immunity25 and early clinical results from a PvDBP vaccine candidate26 are encouraging; however, given the parasite’s gene amplification mechanisms to evade anti-PvDBP immunity27, developing a blood stage vivax malaria vaccine won’t be trivial.Despite being the most prevalent human malaria parasite, investments in P. falciparum and P. vivax vaccine pipeline faces a significant resource shortfall as candidates head into late-stage development—this increasingly more apparent resource gap, or second “Valley of Death”, is also faced by most, if not all, late stage vaccine candidates for pathogens afflicting primarily those living in low resource settings28. Hopefully, the recent global adoption of the Research and Innovation pillar of IA2030 and the growing African continent-led commitment to zero malaria will lead to the investments needed to generate the evidence that support the compelling value proposition required to build bridges for promising malaria vaccine candidates to become affordable, effective, sustainable new tools—part of the solution to regain the prior trajectory towards zero malaria.The promising


Due to the sustained morbidity and mortality that malaria-associated anaemia imposes on patients, malaria is still a global threat, most especially, to residents in sub-Saharan Africa. Merozoite invasion and destruction of erythrocytes, a target for this study, have been necessary due to its unique nature and also since the erythrocytes suffer the most brunt of malarial infection leading to anaemia. The issue of malaria anaemia has to do with why uninfected RBCs get destroyed and even more so than infected ones. Studies have proposed that cytophilic anti-RSP2 (ring surface protein 2—merozoite rhoptry protein 2) antibodies present in sera enhance phagocytosis of RSP2-tagged RBCs by macrophages either directly or via complement, while others have proposed transfer of RSP2 to both infected and uninfected RBCs which may render them susceptible to phagocytosis. What is missing is the agent involved in the transfer of these parasite-induced surface proteins onto the uninfected RBCs, i.e., the mediator molecules. Considering the intracellular location of the parasite in the parasitophorous vacuolar membrane and the absence of a transport mechanism such as the Golgi apparatus within the mature RBC, since the latter has no nucleus, we propose that erythrocyte-derived microparticles (EMPs) may be the possible mediators.  This study aimed at examining the immunological interactions between EMPs released during malarial infections and host erythrocytes that may lead to their lysis possibly through complement mediation. Results and Conclusion. At a fixed volume of 50 μL complement, there were statistically significant (p < 0.01) increases in mean percentage haemolysis as the volume of EMPs increased. Similarly, at a fixed volume of 50 μL EMPs, there were statistically significant (p < 0.01) increases in mean percentage haemolysis with increasing volumes of complement. This was an indication that both complement and EMPs contribute significantly to uninfected erythrocyte haemolysis during malaria infection. This was an experimental study during which malarial EMPs were isolated by differential centrifugation of malaria-positive plasma. This was followed by cell-based in vitro assays where malaria-positive EMPs were added to uninfected blood group “O” negative erythrocytes in the presence of complement and haemolysis checked for.  Haemolysis refers to the abnormal breakdown of red blood cells (RBCs), which could be intravascular or extravascular . There aDuring malaria infection, merozoite invasion and destruction of RBCs contribute significantly to ensuing anaemia where it is believed that the RBCs infected with malaria parasites beside haemolysing also release certain components that are transferred to other nonparasitized RBCs, leading to their phagocytosis and subsequent haemolysis as well. In fact, it is believed that, during malaria infection, the nonparasitized RBCs haemolyse more than the parasitized ones resulting in enhanced haemolysis leading to the ever present anaemia associated with malaria infection . This leMalaria is an acute and sometimes chronic vector-borne protozoan disease with widespread distribution in both tropical and subtropical regions . The totμm carrying molecules (when released) such as phosphatidylserine (PhtdSer) and tissue factors inherent in their parental cells [Erythrocyte-derived microparticles (EMPs) have been found to be very important in malaria pathology, playing various roles in parasite-cellular interactions , 11. Mical cells –14. Theyal cells . Proteomal cells , 17. Agaal cells .Microparticles are derived constitutively from apoptosis and from activated blood cells . They maPlasmodium falciparum malaria, RBCs can be detected that contain ring-infected erythrocyte surface antigen (RESA or Pf155) but no intracellular parasite [Mechanisms leading to haemolysis of infected RBCs include abnormal distribution of membrane phospholipids, such as phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine . Phosphaparasite . This coparasite . This maparasite .In fact, both mathematical modeling and clinical observations suggest that 10 uninfected RBCs are removed from the circulation for each infected RBC that is removed . The incSo, the question is how are these parasite products and other molecules deposit on the uninfected RBCs? Could it be that EMPs that are released from the infected RBCs transfer these complexes through signal transduction to the uninfected RBCs and hence render them susceptible to phagocytosis, haemolysis, and subsequently their removal? It has become prudent, therefore, for in-depth work to be carried out to examine candidate component EMPs to ascertain and understand their role in the pathogenesis of the disease. This work therefore looked at the immunological interactions between EMPs released during malarial infections and host RBCs that may lead to their lysis and thereby aggravating anaemia in malaria infection.The study was an experimental study conducted from January to October, 2018.Blood samples were collected from the Central Laboratory of Korle-Bu Teaching hospital in the Greater Accra Region of Ghana, West Africa.4 ml of human venous blood stored in EDTA tubes and tested to be malaria-positive at the Korle-Bu Central Laboratory was obtained from 10 outpatients who gave their informed consent.Whole blood from each subject was first spun at a low speed of 160 ×g for 5 mins to separate RBCs from platelet-rich plasma. The platelet-rich plasma was in turn centrifuged at a higher speed  to obtain platelet-free plasma (PFP) and to remove any cell debris. The resultant supernatant was sonicated in a sonicating water bath  5 times per 1 minute prior to further centrifugation in order to disperse aggregated exosomes. The supernatant was then centrifuged at 25,000 ×g for 90 minutes to pellet the EMPs. The EMPs were quantified and frozen.10 EMPs/mL) using flow cytometry technique and stored at −80°C in 0.2 mL aliquots in Eppendorf tubes until they were used. Quantification allowed us to estimate the amount of EMPs in a volume of 50 μL.The EMPs, now described as malaria-positive EMPs, were characterized and quantified  negative whole blood (in order to avoid any serologic reaction) was obtained from the Korle-Bu Blood Centre after screening. Before use, the blood group was confirmed using the standard tube method of blood grouping. An aliquot was taken and washed four times in 0.85% phosphate-buffered saline and diluted to 3.0%.oC in the presence of complement and checked for haemolysis after 45 minutes. The experimental set up was done in duplicate, and the mean percentage haemolysis was determined for each set. Controls were set up using anti-C9 .Using 3.0% blood group “O” negative washed RBCs , an amount of malarial EMP suspension was added to uninfected  blood group “O” negative RBCs at 37μL) of complement, there was an increase in mean percentage haemolysis when the volume of EMPs was increased. Using one-way ANOVA, the difference among them was statistically significant (p < 0.05). The post hoc analyses however showed no statistically significant differences in percentage haemolysis during the successive increases of EMP volumes from 10 μL (p=1.00), 20 μL (p=1.00), 30 μL (p=1.00), and 40 μL (p=1.00), though, at an EMP volume of 50 μL, there was a statistically significant increase in mean percentage haemolysis (p=0.01). There was, however, a sharp drop in mean percentage haemolysis by about 50% when anti-C9 was added which was also statistically significant (p=0.01) (At a fixed volume (50 (p=0.01) .μL), there was increasing mean percentage haemolysis as complement volume increases. In multiple comparisons of the variation by one-way ANOVA, the difference among them was statistically significant (p < 0.05). The post hoc analyses also showed statistically significant differences in mean percentage haemolysis among the successive increases of complement volumes from 10 μL (p=0.01), 20 μL (p=0.03), 30 μL (p < 0.01), 40 μL (p < 0.01), and 50 μL (p < 0.01) (At a fixed volume of EMPs (50  < 0.01) .Anaemia is a reduction in haemoglobin concentration or circulating erythrocytes below normal reference ranges taking into consideration the age, sex, altitude of the individual, and also the alteration of plasma volume, which can lead to false changes in haemoglobin concentration. Anaemia may result from either increased destruction or decreased production of erythrocytes. Malaria-associated anaemia, in particular, is a global health problem, causing enormous morbidity and mortality. Nonetheless, the extent of erythrocyte destruction, especially nonparasitized ones, is much greater than observed in other parasite-induced haemolysis. The basis of this is not completely known, though associated immune-mediated haemolysis has been postulated. Host-parasite interactions however offer opportunities for further research to clarify mechanisms that could be explored to fully understand why this happens for a better management of the disease. This study discusses how plasma membrane-derived extracellular vesicles and complement jointly induce erythrocyte haemolysis during malaria infection.μL) of complement while increasing EMP volume, as seen in p=0.01). This is an indication that though EMPs may directly or indirectly contribute to invasion and destruction/haemolysis of red blood cells during malaria infection, the associated mechanisms may involve complement activation as well. This is because during complement activation, the cytolytic process is greatly accelerated by the attachment of C9. That is, C5–8 polymerize C9 to form a tubule referred to as the membrane attack complex (MAC) traversing (destroying) the membrane. C9 is thus the ultimate orchestrator of complement-mediated cell destruction, and since addition of anti-C9 (a C9 inhibitor) subsequently reduced mean percentage haemolysis, the haemolytic event can also be said to be complement-mediated.The increase in mean percentage haemolysis at a fixed volume (50 μL) but increasing volume of complement was used when the erythrocytes were subjected to haemolysis  negative cells which facilitated their recognition by complement and hence resulted in their haemolysis. The drop in haemolysis after the addition of anti-C9 confirms the comediation of complement in haemolysis.


Plasmodium falciparum and P. vivax malaria patients and controls were examined, together with household members and immediate neighbors. Rapid diagnostic test and quantitative PCR (qPCR) were used for the detection of infections that were genetically characterized by a panel of microsatellite loci for P. falciparum (26) and P. vivax (11), respectively. Individuals living in households of clinical P. falciparum patients were more likely to have qPCR detected P. falciparum infections  compared to individuals in control households . Genetically related P. falciparum, but not P. vivax infections showed strong clustering within households. Genotyping revealed a marked temporal cluster of P. falciparum infections, almost exclusively comprised of clinical cases. These findings uncover previously unappreciated transmission dynamics and support a rational approach to reactive case detection strategies for P. falciparum in Ethiopia.The distribution of malaria infections is heterogeneous in space and time, especially in low transmission settings. Understanding this clustering may allow identification and targeting of pockets of transmission. In Adama district, Ethiopia,  Transition from control to elimination requires improved understanding of transmission patterns within communities2 to better target the remaining infections that sustain or seed transmission. In low endemic settings, malaria infections are often characterized by low parasite density, commonly below the detection limit of rapid diagnostic tests (RDT) and microscopy4. These low-density infections are typically asymptomatic and may contribute substantially to onwards transmission to mosquitoes5.Recent success in malaria control in endemic countries has re-ignited elimination efforts8. If this clustering reflects pockets of active transmission, finding and targeting these clustered infections may accelerate targeted elimination efforts9. Based on this assumption, individuals living in close proximity to passively detected cases are screened and treated in reactive case detection (RCD) strategies in low transmission settings8. The optimal and logistically feasible population that needs to be targeted in RCD remains unclear11. Several studies have attempted to determine whether geographical clusters of infections show evidence of increased genetic relatedness and thus truly reflect related transmission events18. Few studies have coupled household-level case clustering studies with information on the genetic relatedness of infections. Such studies can provide a better understanding of fine-scale transmission patterns and estimate the contribution to transmission of infections that are not detected by routine interventions.As malaria transmission intensity declines, the distribution of malaria infections becomes increasingly heterogeneous, and infections may cluster within villages or householdsP. falciparum and P. vivax infections surrounding passively detected RDT-confirmed clinical malaria patients was assessed using RDT and quantitative PCR (qPCR) in Batu Degaga, Ethiopia. Spatial and temporal relatedness and clustering of infections within and between households of passively detected malaria infections and controls were investigated by genotyping 26 and 11 microsatellite loci for P. falciparum and P. vivax, respectively, which had been shown previously to be useful to describe population structure25.In this study, the prevalence and distribution of asymptomatic P. falciparum (n = 10), P. vivax (n = 6) or mixed species infections (n = 2) and 18 controls without malaria infections participated in this study. In total, 499 individuals from 128 households around index cases and 454 individuals from 108 households around controls were successfully sampled  compared with their neighbors  and those from and around control HHs . In the screened population surrounding index cases and controls (excluding the index cases), prevalence of infection was 7.9% (72/913) by RDT and 21.6% by qPCR (189/877). At least one infected individual was found in 28% (66/236) and 48%  of households by RDT and qPCR, respectively. P. falciparum infections detected in households of neighbors of index cases had higher parasite density  than infections detected in households of neighbors of controls ; whilst such difference was not detected in P. vivax infections (P = 0.27). Fewer P. falciparum infections were detected by qPCR in households of controls  compared to index case households  after excluding index cases. There was no evidence for differences in parasite prevalence by PCR between households of control and neighbors of index cases  and population level genetic diversity were compared for infections around index and control cases for each species. In P. falciparum infections, the mean MOI was 1.5 (range: 1–3) and infections detected in and around households of index cases had higher MOI compared to those detected in and around control households , with no difference between index and control cases . Similar to P. falciparum, infections detected around index cases showed moderately higher genetic diversity compared to those detected around controls  and 91 P. vivax samples from 79 households  were compared using the IBS metric. These data were used to determine how genetic relatedness of infections varied in space and time. P. falciparum infections sampled from the same household were more strongly related than infections from different households  and sampled from different households  and group-1 overrepresented in controls . Infections assigned to group-3 were identified in five independent RCD investigations and included one individual in the control group whose infection was genetically related to one of the index cases (IBS = 0.7). Analyses of the elapsed time between samples in each group showed that infections assigned to group-3 were clustered temporally  compared to group-1  and group-2 . These observations support the hypothesis that infections identified through investigation around index cases are linked possibly due to recent, local spread of group-3 haplotypes during the study period. No P. vivax genetic groups were overrepresented either to the control or index cases , with the highest model evidence for both nts Fig. . P. falcons Fig. A, with iPlasmodium infections in a low endemic area in Ethiopia with a focus on asymptomatic infections in the vicinity of passively detected clinical index cases. Asymptomatic P. falciparum infections were clustered in index case households whilst there was no such evidence for clustering of P. vivax infections. P. vivax infections were more genetically complex and diverse than P. falciparum infections, with no detectable spatial or temporal clustering. We identified fine-scale focal transmission and a genetic cluster around index cases that may represent a clonal P. falciparum expansion, highlighting the added value of genotyping to understand the contrasting transmission epidemiology of the two Plasmodium species.In the current study, we examined spatial clustering of 27 and other infectious diseases30. Several countries have adopted RCD approaches that take advantage of infection clustering and assume that transmission chains, or at least transmission sources, can be interrupted by targeting infected household members. In some settings where symptomatic and asymptomatic malaria cases do not overlap spatially, clusters of infections can be missed by RCD31. This highlights the need for detailed studies on Plasmodium transmission patterns in settings aiming for elimination32. In the current study, we moved beyond spatial patterns in infection prevalence and examined small-scale transmission patterns by genotyping microsatellite markers. The overall genetic diversity (i.e. the mean expected heterozygosity) in P. falciparum infections was lower (Mean HE ± SE: 0.47 ± 0.03) than previously observed in other low transmission areas of Eswatini, Zambia, and Namibia (Mean HE: 0.71–0.79)33 suggesting infrequent recombination and smaller parasite population size in the study site.Spatial and temporal heterogeneity in the occurrence of infections is widely acknowledged in malariaP. falciparum infections; the odds of detecting an infection within the household of clinical patients was significantly higher than in neighboring households35. MOI and genetic diversity of infections were higher around passively detected P. falciparum compared to controls, indicative of higher transmission. We observed that the very strong household-level clustering for P. falciparum was matched with strong genetic relatedness of infections within a household. Density of asymptomatic P. falciparum infections surrounding clinical cases was higher than in infections detected in control households. This might indicate that the former infections were acquired more recently compared to older, very low density infections detected in controls. All of these findings corroborate that RCD is useful to detect clusters of recently acquired P. falciparum infections. Results from control households, however, show that a substantial number of infections is not linked to index cases. As found in other studies37, RCD alone is thus unlikely to achieve elimination.We observed a clear overlap of symptomatic and asymptomatic P. falciparum infections, present in multiple households, that was overrepresented in and around index cases. These findings suggest a possible population expansion of a single parasite genotype. We can hypothesize that possible expansion, occurring against the background of wide-spread asymptomatic infections, could indicate introduction of a clone from another site. Its association with clinical cases suggests that some infections disproportionally give rise to symptoms18.We detected a genetic cluster of P. falciparum and P. vivax are co-endemic, P. vivax was more diverse, showed high MOI, and exhibited lower levels of population structure41. P. vivax diversity was similar to previous studies from Madagascar (Mean HE: 0.80), PNG highlands (Mean HE: 0.82) and lowlands (Mean HE: 0.80) and higher than South Korea (Mean HE: 0.56)42. There was no significant difference in the odds to detect a asymptomatic P. vivax infection in the household or neighbors of an index case compared to a control household. There was no difference in the MOI and heterogeneity of infections around passively detected P. vivax infections and controls. No spatial clustering was observed for P. vivax. The loss of relatedness and household level clustering may reflect differences in the biology of these two Plasmodium species where the majority of clinical P. vivax cases are attributable to relapsing episodes and do not necessarily reflect an active circulating infection that involves mosquito bites44. This would contribute to considerable variation in the time between the original inoculation by mosquito bite and parasite detection in the bloodstream, and thus diffuse any genomic signal. In summary, RCD is not more effective than random screening to identify asymptomatic P. vivax infections in the study site.As in other sites where P. falciparum infections. Whilst infections are prevalent outside households of index cases, and thus may be missed by RCD approaches, a disproportionate number of infections may be targeted by RCD45. Our finding of a possible population expansion of a single parasite genotype arising from a clone that was frequently detected in clinical cases further supports RCD approaches that may interrupt clinically relevant transmission chains. The lack of spatial (genetic) clustering in P. vivax may be attributable to the limitations of the methods used in this study, in addition to its unique biology discussed above, calling for more robust approaches. The biology of P. vivax; existence of hypnozoites47, generation of gametocytes at the very early stage48, lower parasite densities50, and higher vectoral capacity53, makes it a harder species to control and one that may require a different strategy to that of P. falciparum55. Future studies may benefit from more sample size and including genotyping of mosquitoes paired with the human sampling in study households56. In summary, for P. vivax RCD may not be more efficient than untargeted screening to identify asymptomatic infections in the study site. Control strategies against P. vivax need to target hypnozoites using radical cure, as the majority of infections detected are attributable to relapsing episodes rather than active infections57. Justified by the very low prevalence of glucose-6-phosphate dehydrogenase deficiency64. Ethiopia is currently implementing a 14-day dose primaquine for radical cure in P. vivax67.Our findings highlight the value of genotyping approaches to infer spatial and temporal clustering of P. falciparum and P. vivax are co-endemic in the district68 with 60% of the infections attributed to P. vivax5. The area is characterized by unstable seasonal transmission that peaks following the two rainy seasons: September to November (major) and April to May (minor). Two health posts, the lowest level governmental heath facilities, serve the study area. Only one of the two health posts . P. falciparum and chloroquine for P. vivax.The study was approved by the ethics review boards of the Department of Medical Biochemistry at Addis Ababa University (DRERC 19/16), Armauer Hansen Research Institute (PO52/14), the National Research Ethics Review Committee (310/109/2016), and the London School of Hygiene and Tropical Medicine (10628). Written informed consent was obtained from all participants and/or parents/guardians. All experiments were performed in accordance with permission obtained from participants on the consent forms, following relevant guidelines and regulations. If participant was found RDT positive while being febrile during household level sampling, he/she was referred to the nearest health post to be treated with current first-line antimalarial drugs according to the Ethiopian national malaria treatment guidelines, artemether–lumefantrine for Patients with (history of) fever that presented to the two health posts and tested positive by RDT  were classified as index cases. Patients that were suspected of malaria but tested RDT negative on the same day were included as controls. Individuals who attended the health post for vaccination, trauma, pregnancy test, antenatal care, and family planning were not considered. Index cases were independent of one another (i.e. they wouldn't be found in the same cluster of households assessed for another index case). A total of 147 patients attended the clinic during the study period of whom 52 presented with main complaint of fever. Fever was confirmed in 44 of them of whom 24 were RDT positive for malaria. Patients who were referred to the nearby health center for better treatment (n = 4), were not residents of the study area (n = 2), or did not have completed information (n = 2) were excluded from the study. Controls were recruited among patients that presented to the same health post where the index case was recruited.8 and had their household geo-located with a handheld GPS receiver . Neighboring households were selected based on proximity to the respective index case or control household upon obtaining permission from the head of the house. Socio-demographic and epidemiological data were collected, together with finger prick blood samples to diagnose malaria using RDT and to prepare dried blood spots (DBS) on 3MM Whatman papers  from all study participants including index cases, controls, neighbors and household members.During the study period, 0–6 patients attended the clinic in a day and the health extension workers were available for 2–3 days a week from 9:00–12:00 a.m. The northern health post was opened occasionally and the same health extension workers were responsible and two index cases and two controls were recruited. Household members and members of the six nearest neighboring households of index cases and controls were tested for malaria by RDT within 2 days after the index or control cases were identified5. Parasites were quantified using species-specific qPCR that targeted the 18S small subunit rRNA gene69. qPCR positive samples were genotyped using 26 microsatellite markers for P. falciparum and 11 markers for P. vivax using previously established laboratory and data analyses protocols23. Mixed species infections were genotyped with both P. falciparum and P. vivax markers. Genotyping was successful in 92/93 P. falciparum and 99/123 P. vivax positive samples. For haplotype reconstruction in multiclonal infections, the predominant peak was included.DNA was extracted from a 6 mm diameter punch, using MagNa Pure LC 2.0 Instrument and Total Nucleic Acid Kit—High Performance (Roche Life Sciences) with prior treatment using Buffer ATL (QIAGEN) and Proteinase K (QIAGEN)http://www.graphpad.com); STATA 13  and R  were used for data analysis. The QGIS software version 3.14.16  was used to map study households. Continuous variables were presented as median and interquartile range (IQR). Tests of association between two categorical variables were performed using the Chi squared test, and comparison of non-normally distributed continuous variables by Mann–Whitney U test. Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using Generalized Estimating Equations to determine the association between being parasite positive and living in households of index cases or surrounding houses. These estimates were adjusted for clustering of species-specific observations from the same household with robust standard errors and used community members not belonging to index cases or neighboring households as a reference.GraphPad Prism version 5.03 for Windows  of each locus. HE was calculated for each locus using the formula ith allele in the population. Mean HE was calculated by taking the average of HE across all loci. To assess the genetic relatedness and clustering of P. falciparum and P. vivax infections, pairwise genetic relatedness was determined for each pair of samples using a modified identity by state (IBS) metric22. Briefly, pairwise IBS was calculated based on the number of shared alleles between isolates, allowing estimation of genetic relatedness from monoclonal as well as polyclonal samples. Samples were considered related if the pairwise genetic relatedness was greater than or equal to 0.5 . This threshold reflects the degree of relatedness of meiotic siblings, which are the result of sexual recombination of different clones in the mosquito. Population structure was assessed using the program MavericK70, under the admixture model with 500 burn-in iterations, 5000 sampling iterations and 20 thermodynamic rungs.Multiplicity of Infection (MOI) was defined as the highest number of alleles detected by at least two loci, reducing the impact of false positive allele calls from a single, outlier locusThis work does not contain any individual person’s data in any form .