Molecular Diagnostics and Treatment Failures in Pediatric Malaria: a prospective observational study from Omdurman, Sudan

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Despite the dominance of P. falciparum , increasing cases of P. vivax and mixed infections complicate the diagnosis and management. This study aimed to analyze the demographic, clinical, diagnostic and treatment characteristics of recurrent malaria in children, highlighting diagnostic challenges and treatment outcomes. Methods We conducted a prospective observational study from October 2021 to April 2023, at the Tropical Diseases Teaching Hospital in Omdurman, Sudan among all consecutive pediatric patients with recurrent malaria (n=81). Data on demographics, clinical history, diagnostic and treatment were collected. Species identification was performed using Blood Film for Malaria (BFFM), and PCR. Treatment adherence to Sudan’s national malaria/WHO protocol was assessed, and outcomes were tracked over 28-day follow-up periods. Results Patients had a mean age of 6.78±4.7 years, with 53.1% male. Most cases originated from Khartoum (60.5%), with a malaria recurrence duration of 9.7±8.8 months. PCR detected P. falciparum (55.6%), P. vivax (6.2%), and mixed infections (38.2%), whereas BFFM primarily identified P. falciparum (93.8%). Recrudescence was seen in 55.6% of cases and relapses in 44.4%. When the BFFM is compared to PCR, it has a sensitivity of 11.1% for diagnosing relapsing recurrent malaria. Conclusion This study highlights the diagnostic limitations of traditional methods compared to PCR and underscores the need for protocol adherence and tailored therapies. Enhanced diagnostic tools and management strategies are crucial for addressing recurrent and resistant malaria in pediatric populations in Sudan. Recurrent malaria Pediatric malaria Molecular diagnostics Malaria treatment failure P. falciparum and P. vivax Figures Figure 1 Introduction Malaria is a life-threatening infectious disease transmitted through the bite of a female Anopheles mosquito, with sporozoites entering the bloodstream and traveling to the liver. After infection, individual symptoms may appear as early as 7 days, depending on the species of the parasite. The symptoms begin during the erythrocytic phase of the parasite's life cycle, as infected red blood cells rupture and release merozoites, leading to fever and other clinical manifestations. Most infections caused by Plasmodium falciparum ( P. falciparum ) become symptomatic within a month of exposure with high parasitemia ( 1 – 3 ). Non-falciparum or relapsing species including Plasmodium vivax ( P. vivax ) and Plasmodium ovale ( P.ovale ), these infections can present weeks to months after the initial exposure due to the activation of dormant hypnozoites in the liver ( 1 – 3 ). Relapses often occur within two to three years or even longer ( 4 ). Recurrences of malaria can be the result of treatment failure (recrudescence) or relapse, and distinguishing between the two can be challenging in endemic areas like Sudan. Recrudescent infections, usually caused by P. falciparum , occur when the disease re-emerges after an apparent recovery. This is usually due to the survival of parasites in the bloodstream and it can occur when the initial treatment is insufficient to fully clear the infection. Factors contributing to recrudescence include inadequate drug regimens, drug resistance, and incomplete clearance of parasites ( 1 – 4 ). Moreover, P. vivax can relapse weeks to months after the primary blood-stage infection has resolved, due to their hypnozoite forms. The likelihood of relapse is reduced when patients receive presumptive anti-relapse therapy, such as primaquine ( 5 ), which targets the dormant liver stages. Also, there have been instances of P. vivax infection in the bone marrow, even without detectable parasitemia. One case series from India, involving 108 patients with fever and hematological abnormalities like anemia, thrombocytopenia, or pancytopenia, reported that P. vivax infection in the bone marrow was present in about 40% of cases ( 6 – 9 ). The detection threshold for malaria using microscopy is approximately 25-parasites per micro-liter in the hands of experts. However, individuals who have never been exposed to malaria before can develop acute symptoms at parasitemia levels that fall below this limit, making detection difficult in such cases ( 10 ). Microscopy is less sensitive when it comes to detecting very low-density parasitemia, which can result in missed diagnoses of asymptomatic, submicroscopic infections that serve as potential reservoirs for transmission. This challenge is particularly pronounced in cases of mixed infections which may go undetected ( 11 ). Sudan is highly endemic to P. falciparum but not P.vivax ( 12 ), the latter presents a new diagnostic challenge due to its characteristic of having low pyrogenic density, especially with a co-infection with P. falciparum . As a result, standard microscopy may not be effective for accurate diagnosis of P. vivax , especially when high-density P. falciparum infections are present, as these can overshadow the lower levels of P. vivax . Moreover, The Deletion of histidine-rich protein 2 (PfHRP-2) in the P.falciparum genome will impact the accuracy of PfHRP-2 based rapid diagnostic tests in Sudan ( 13 ). In such instances, molecular techniques like polymerase chain reaction (PCR), which amplify DNA, offer a more sensitive alternative to microscopy and it is more capable of detecting low parasitemia levels ( 14 ). This study investigated patient with recurrent malaria and focusing on diagnosing relapses and recrudescence and investigating the characteristics of treatment failure. The research assessed the mixed Plasmodium infections on the same host, such as P. vivax and P. falciparum on the diagnostic accuracy of microscopy vs PCR. Materials and methods Study Design: This prospective cohort study was conducted over 80 weeks, from October 2021 to April 15, 2023 at the Tropical Diseases Teaching Hospital in Omdurman, Sudan. Established in 1974 by the Ministry of Health in collaboration with the National Council of Research, the hospital serves as a reference center for tropical diseases, receiving patients from Sudan, South Sudan, Ethiopia, Chad, and Somalia. It has three wards, each accommodating 100–140 patients. In February 2019, the Pediatrics Tropical Department was established in collaboration with the Ministry of Health and the hospital administration. This department includes an outpatient tropical diseases clinics, a tuberculosis (TB) management unit and pediatric multidrug-resistant TB unit, a malaria diagnostic and treatment referral center, two pediatric wards with 12 beds each, and a quarantine area for TB patients. This setup makes it the first specialized center for pediatric tropical and infectious diseases in Sudan. Patients: This study included all consecutive pediatric patients referred to the hospital with a history of recurrent malaria. Each patient underwent a detailed history, physical examination, and complete blood count to assess symptoms of recurrent malaria. Patients with recurrent malaria episodes occurring more than 28 days apart were not included. All patients were diagnosed using thin and thick blood films for malaria (BFFM) to determine the baseline parasite species and parasite count. Molecular detection via PCR was also performed to confirm parasite species. Patients were classified based on the cause of malaria treatment failure: either recrudescence ( P.falciparum ) or relapsing species ( P.vivax ). Patients were treated according to the National Malaria Treatment/WHO Protocol for uncomplicated and severe malaria ( https://www.who.int/publications/i/item/guidelines-for-malaria ). Also, we controlled factors that could contribute to antimalarial treatment failure by ensuring weight-appropriate dosing, administering medication with a fatty meal, promoting compliance with the full treatment course, and providing guidance on when to repeat a dose if vomiting occurs. Additionally, we ensure proper preparation and infusion of IV antimalarials by trained nurses within our hospital setting and exclusively use antimalarial drugs supplied under the Sudan National Malaria Protocol. Follow-up assessments included BFFM and parasite counts on days 0, 3, 7, 14 and 28 until parasite clearance was achieved. Based on these results, patients were classified as having early or late clinical, or parasitological failure, according to the WHO and national malaria protocol definitions. Patients who experienced treatment failure during follow-up were transitioned to the next treatment step as per WHO and national malaria protocols. These patients were monitored with repeat parasite counts until parasite clearance was achieved, confirmed by negative BFFM results. Molecular and microscopic detection for malaria: Blood sample DNA was extracted following the guanidine chloride extraction method described by Ciulla et al ( 15 ), with minor modifications. Briefly, 1 ml of blood samples were washed with a red blood cell (RBC) lysis buffer, and then 1 ml of 6 M guanidine chloride was added to lyse the cell walls and release DNA. Proteinase K solution was then added, and the lysate was incubated at 37°C overnight. The following day, 3 ml of chloroform was added to the samples to separate DNA from proteins using centrifugation at 8,000 rpm. The DNA-containing supernatant was then added to 3 ml of cold absolute ethanol to precipitate the DNA. After precipitation, the DNA pellet was dissolved in 100 microliters of deionized distilled water. The quality and purity of the extracted DNA were assessed using a spectrophotometric method with an Implen Nanodrop (Implen, Germany). DNA was then stored at -20°C until molecular detection of Plasmodium species. Polymerase chain reaction Molecular detection was performed using a nested PCR protocol previously developed by Snounou et al. for the detection of plasmodium species infections ( 16 ). Initially, an outer PCR was performed using the primers rPLU6: 5'- TTA AAA TTG TTG CAG TTA AAA CG-3' and rPLU5: 5'- CCT GTT GTT GCC TTA AAC TTC − 3'. For the nested PCR to detect P.falciparum and P.vivax infections, the primers used were rPF1: 5'-TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT − 3', rPF2: 5'-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC − 3', rPV1: 5'-CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC − 3', and rPV2: 5'-ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA − 3'. The PCR reaction mixture was prepared using a ready-to-use PCR mastermix (Intron Biotechnology, South Korea) following the manufacturer's instructions. In the outer PCR reaction, 1 microliter of outer primers was added to 1 microliter of the DNA sample. For the nested PCR reaction, 1 microliter of each species-specific primer was added to 1 microliter of the first PCR amplicons, and the reaction volume was adjusted to 20 microliters using deionized distilled water (DDW) as recommended. Thermal cycling conditions were adjusted in the AB 2700 Thermal Cycler Gene Amp PCR machine (Applied Biosystems, Singapore) according to Snounou et al ( 16 ). Positive control samples (previously known as P. falciparum and P. vivax DNA samples) were included in each run. Negative controls were prepared by adding primers to tubes without DNA. Amplified PCR products were then loaded onto a 2.5% agarose gel (Intron Biotechnology, South Korea), and electrophoresis was conducted at 100 mA and 30 volts for 1 hour using a gel electrophoresis device (Major Sciences, Taiwan). The results were visualized using a gel documentation system (Major Sciences, Taiwan). A 100 bp molecular ladder (Intron Biotechnology, South Korea) was loaded with the samples to determine PCR amplicon lengths. PCR results were interpreted based on the size of the amplified PCR product. Positive samples for P. falciparum showed a band of 205 bp, while positive samples for P. vivax showed a band of 120 bp. Moreover, for the microscopic diagnosis of malaria, we utilized similar techniques in our study as described in a study in White Nile state in Sudan. Specifically, 2.5 mL of venous blood was collected from each participant. Malaria diagnosis was performed using blood film microscopy from two laboratories and confirmed via PCR. Both thick and thin blood films were prepared, with fields examined at least twice ( 17 ), following the WHO's quality control guidelines to maintain diagnostic accuracy. Data collection and analysis: The data was collected for this study by using data collection tools that included the following variables: gender, age, and state of residence. Also, the duration of recurrent malaria in months, and the frequency of recurrent malaria episodes. The severity of malaria was assessed for all patients, along with a history of recurrent malaria in the family or among neighbors. Data on previous and current species identification was recorded, as well as details of previous diagnostic tests and previous failed malaria drugs. We also recorded the malaria species identified by our lab throughout combined PCR and BFFM. Follow-up data included BFFM, along with basic clinical assessments covering symptoms, and treatment information. The data were analyzed by SPSS 26 and Jamovi 2.5 using descriptive statistics and presented as frequency tables with percentages or in graphical form. To assess associations between variables, the Chi-square test (χ² test) and binary logistic regression were employed. A p-value of ≤ 0.05 was considered statistically significant. Result Table 1 Demographic and clinical/Diagnostic/treatment history of recurrent malaria in 81 pediatric patients. Count % Mean ± Std Gender (N = 81) Male 43 53.1% Female 38 46.9% Age by years 6.78 ± 4.7 States (N = 81) Khartoum 49 60.5% Al-Gazira 8 9.9% White Nile 10 12.3% River Nile 3 3.7% Kassala 3 3.7% Kordofan 4 4.9% Sennar 3 3.7% Darfur 1 1.2% Duration of recurrent malaria (months) 9.7 ± 8.8 Frequency of recurrent Malaria (N = 81) Every week 4 4.9% Every two weeks 34 41.9% Every three weeks 7 8.6% Every four weeks 15 18.5% Variable duration 21 25.9% Malaria severity (N = 81) Severe 14 17.3% Uncomplicated 67 82.7% History of recurrent malaria in the family/neighbors (N = 81) Yes 11 13.6% No 70 86.4% History of Previous Species identification confirmed (N = 81) Yes 45 55.5% No 36 44.4% Previous diagnostic tests for Species identification and their results (N = 45) By BFFM P.falciparum 20 100.0% By both BFFM and immunochromatographic assay P.falciparum 25 100.0% Previous Follow-up by BFFM (N = 81) Yes 7 8.6% No 74 91.4% Table 2 Assessment, investigations, and treatments of the current malaria infection. Count % Basic clinical assessment BMI (N = 81) Normal 61 75.3% Underweight 20 24.7% HB (N = 81) Normal 27 33.3% Anemic 47 58.0% Not Done 7 8.6% Hepatomegaly (N = 81) Yes 3 3.7% No 78 96.3% Splenomegaly (N = 81) Yes 6 7.4% No 75 92.6% Diagnosis Species identification by BFFM (N = 81) P.falciparum 76 93.8% P.vivax 4 4.9% Negative 1 1.2% Species identification by PCR (N = 81) P.falciparum 45 55.6% P.Vivax 5 6.2% Mixed 31 38.2% Causes of recurrent malaria (N = 81) Relapsing species/P.vivax + mixed 36 44.4% Recrudescence/ P.falciparum 45 55.6% Classification of Recrudescence treatment failure (N = 36), Nine patients responded from the first time Early parasitological failure 7 19.4% Late parasitological failure 4 11.1% Early Clinical and parasitological failure 21 58.3% Late Clinical and parasitological failure 4 11.1% Treatment Clearance drug (N = 81) Dihydroartemisinin–Piperaquine/Primaquine 32 39.5% Artesunate/Primaquine 3 3.7% Artesunate/Chroquine prophylaxis 1 1.2% Dihydroartemisinin–Piperaquine 25 30.9% Quinine/Doxycycline 2 2.5% Quinine/Clindamycin 3 3.7% Artesunate 1 1.2% Artesunate/Dihydroartemisinin–Piperaquine 6 7.4% Fansidar 4 4.9% Artemether 1 1.2% Quinine/Artemether 1 1.2% Atovaquine 2 2.5% In this study, the demographic and clinical history data of 81 pediatric patients with recurrent malaria showed a nearly equal gender distribution (53.1% male, 46.9% female), with an average age of 6.78 ± 4.7 years. Most cases originated from Khartoum (60.5%), with varying representation from other states. The average duration of recurrent malaria was 9.7 ± 8.8 months, and 41.9% experienced episodes every two weeks. The majority of cases were uncomplicated (82.7%), and 55.5% had previously confirmed species identification using traditional methods like BFFM and rapid immunochromatographic assay (ICT). Among the 45 patients who underwent these diagnostic tests, 100% confirmed P. falciparum via BFFM, and those tested with both BFFM and ICT also showed only P. falciparum , with no detection of P. vivax or mixed infections (Table 1 ). Notably, prior to being referred to our unit, only 8.6% had follow-up diagnostic tests, indicating a gap in continuous and appropriate follow-up care in primary care centers for malaria. There, all patients received the same treatment for each malaria episode without moving to other lines, which is against the Sudan national malaria/WHO protocol: the first line (Artemether-lumefantrine) for uncomplicated malaria, and (artesunate/quinine) for severe malaria. The clinical assessments revealed that 75.3% of patients had a normal BMI (according to the WHO definition of BMI), while 24.7% were underweight. Hemoglobin levels indicated anemia in 58% of the children (defined by HB, hematocrit, or red blood cell count levels lower than the normal age and sex-adjusted ranges). Hepatomegaly was observed in 3.7% and splenomegaly in 7.4%. Fever was the most common symptom, present in 79, followed by persistent vomiting in 30, headache in 18, and diarrhea in 14 patients (Fig. 1 ). Species identification using BFFM by expert microbiologists showed that 93.8% of cases involved P. falciparum , 4.9% P. vivax , and 1.2% were negative. In comparison, advanced PCR testing provided more detailed results, identifying 55.6% P. falciparum , 6.2% P. vivax , and 38.2% mixed infections, highlighting a broader detection range. The causes of recurrent malaria were attributed to P. falciparum recrudescence in 55.6% of patients and P. vivax relapses in 44.4% (both p.vivax and mixed infection). Among those with relapsing infections identified by PCR, 86.1% had mixed infections and 13.9% had P. vivax alone (Table 2 ). Interestingly, BFFM results for relapsing species showed only P. falciparum (86.1%) and P. vivax (11.1%) without detecting mixed infections, which were only revealed by advanced PCR. In terms of P. falciparum treatment failure, 58.3% experienced early clinical and parasitological failure, 19.4% had an early parasitological failure, and 11.1% had late parasitological failure. All patients who were found to have malaria in the last 2 weeks and received Artemether lumfantrin were treated with Dihydroartemisinin–Piperaquine (DHAP) according to the Malaria treatment protocol (9 patients had achieved clearance from the first time with negative BFFM day 3,7, 14 and 28 day). All patients who were found to have P.vivax malaria were treated according to the protocol (uncomplicated or severe) then followed by radical treatment by primaquine except for one patient who was younger than 6 months and received weekly chloroquine prophylaxis after clearance of malaria infection. Eight patients developed failure of treatment many times during their follow up and received all treatment options in malaria protocol then treated with antimalarial medications outside treatment protocol to achieve parasite clearance (Table 2 ). Discussion In tropical regions with limited specialized diagnostic and treatment facilities recurrent malaria emerged as a significant challenge, particularly in pediatric populations, where the burden and the prolonged duration of the disease can severely impact growth, development, education, the family, and the overall health status of the patient. Here, this is one of the first updated prospective studies that assess the demographic, clinical, and treatment for recurrent malaria. We investigated recurrent malaria using BFFM and advanced PCR to detect low P.vivax parasitemia, mixed infection, and recrudescence of P.falciparum to aid the identification cause of recurrent malaria, we followed patients with BFFM for parasite clearance and treated them according to Sudan National Malaria treatment/WHO protocol. In this study 58.0% (n = 47) were anemic and 24.7% (n = 20) were underweight for their age and height. Notably, the relationship between recurrent malaria cases and body mass index (BMI) was analyzed. Among patients with P.vivax malaria, only 3 out of 36 (8.3%) were classified as underweight, compared to 17 out of 45 (37.8%) in patients with recurrent P.falciparum (p-value of ≤ 0.05), also, there is a slightly significant relationship between malaria severity and causes of recurrent malaria (p-value = 0.057) with severe malaria present more in P.falciparum compared to p.vivax with 11 and 3 cases, respectively. The vast majority of severe P. vivax malaria cases are from Southeast Asia and India, with few published data on severe P. vivax from Africa ( 18 ). However, one study from Eastern Sudan highlighted severe P. vivax malaria cases among children at New Halfa Hospital, underscoring similar severe manifestations as seen in regions like India and Brazil. In Sudan, the presence of P. vivax is increasingly reported despite P. falciparum being the dominant species ( 19 ). The rise in P. vivax and mixed-infection malaria cases in Sudan could be linked to the increased movement of Ethiopian and Eritrean populations across the border, facilitated by peace agreements and the construction of paved roads between these countries and Sudan. Recent studies have shown that P. vivax is the predominant malaria species in Ethiopia, with reports of treatment failures, which may also contribute to its spread in Sudan, increasing diagnostic difficulties ( 20 ). Another study in Central and Eastern Sudan utilized molecular techniques like PCR to identify mosquito vectors carrying P. vivax . They found a growing number of P. vivax cases, suggesting a shift in malaria transmission patterns in Sudan ( 21 ). Notably, in regions like South Sudan, recurrent malaria has been linked to factors such as incomplete malaria treatment, socio-economic status, and nutrition. They found that patients who ate at least two meals a day had a significantly lower likelihood of developing severe recurrent malaria compared to those who ate only one meal ( 22 ). Moreover, in terms of geographical distribution, a significant relationship was found in this study between malaria severity and location (p-value ≤ 0.05). Only three patients with severe malaria were reported in Khartoum, compared to 11 cases in other states. Conversely, patients with recurrent uncomplicated malaria were more likely to come from Khartoum, with a nearly twofold difference—46 cases in Khartoum versus 21 cases from other states. This suggests that more cases of uncomplicated malaria are present in other states, but these patients cannot reach Khartoum for treatment and investigations. The analysis using binary logistic regression showed no significant relationship between the duration of malaria and the causes of recurrent malaria in pediatric patients. In Sudan, geographical factors play a critical role in malaria distribution. A spatiotemporal study mapping malaria incidence in Sudan highlights how transmission varies by region, with areas like Khartoum showing lower incidence compared to other states ( 23 ). Such studies suggest that geographical disparities in professional healthcare access and ecological factors significantly affect patient outcomes, and more patients may not reach Khartoum for treatment due to economic issues and transportation challenges. The Tropical Disease Teaching Hospital is a national referral center for treating infectious diseases, including malaria. Notably, all malaria patients referred to the hospital during the study had P. falciparum diagnosed based on BFFM and BFFM/ICT done in primary care centers, with only 8.6% (n = 7) patients receiving BFFM follow-up for their disease after treatment. The treatment given to patients from primary care centers was Artemether-lumefantrine for uncomplicated malaria (no second-line therapy was used) or quinine or artesunate for severe malaria. Diagnostic challenges in malaria treatment are also prominent as none of the previous diagnostic tests were able to detect mixed infection. When the BFFM is compared to PCR, it has a sensitivity of 11.1% for diagnosing relapsing recurrent malaria ( 24 ). Studies have shown that BFFM's sensitivity can be significantly lower than that of PCR techniques. The lower sensitivity of BFFM is particularly concerning for relapsing or recurrent mixed infection, where P.vivax parasite levels may be below the detection threshold of microscopy. PCR, with its higher sensitivity, is more adept at identifying these low-density infections, making it a more reliable diagnostic tool in such scenarios, especially with the emergence of P. vivax in Sudan ( 25 – 26 ). Moreover, increasing awareness of malaria WHO/Local protocols is important. A qualitative study on malaria case management in Northern Sudan identified gaps in adherence to diagnostic and treatment protocols, particularly in non-specialized centers due to resource shortages, training gaps, and lack of malaria expert supervision and consultants. This nonadherence could contribute to the mismanagement of recurrent malaria cases, particularly in resource-limited settings ( 27 ). The lack of awareness about the updated management of recurrent malaria and malaria treatment failure protocols could be the reason for the poor management of these patients in peripheral centers. A 2018 study in Equatorial Guinea found that 63.6% of public health practitioners had low awareness of national malaria treatment guidelines, with significant differences between hospital and health center workers. Strengthening practitioners' knowledge and adherence to guidelines is critical for improving malaria diagnosis, treatment, and disease control in the region ( 28 ). Poor adherence to proper management guidelines could also be due to diagnostic difficulty and lack of resources in treating facilities, as reported ( 27 , 28 ). This study concluded that nonadherence to malaria diagnosis and treatment protocols in Northern Sudan was linked to medication misuse and diagnostic distrust, along with contributing factors like resource limitations, inadequate staff training, and patient demands. These factors may lead to improper diagnostic and treatment practices, which translate to poor malaria control. Interestingly, about 10% (N = 8) of the study cohort experienced a failure to clear the parasite and were treated with antimalarial medications outside the established treatment protocol. With the possibility of resistant parasites, we administered Fansidar to these patients. Four achieved parasite clearance with follow-up from day 3 to day 28 for symptoms and BFFM, but the other four also experienced treatment failure with Fansidar, one of whom developed severe malaria. We treated the four patients with artemether injections, while the patient with severe malaria received quinine along with Artemether. At this point, two patients achieved parasite clearance, while two others continued to show treatment failure. We then used atovaquone-proguanil, after which they finally tested negative for BFFM and remained symptom-free for 28 days. We subsequently cleared them and monitored their progress by phone, with no further cases of malaria. The observed multiple treatment failures in these 8 patients, constituting approximately 10% of our study cohort, suggest a concerning prevalence of resistant malaria strains ( 29 – 31 ). This high rate surpasses the expected prevalence in certain endemic regions and highlights the challenges of managing resistant malaria. Reduced susceptibility to artemisinins, characterized by delayed parasite clearance in clinical studies ( 32 ) and prolonged parasite survival in vitro ( 33 ), is a growing concern. This resistance is associated with point mutations in the P. falciparum kelch protein (PfK13) propeller domain ( 34 ), though evidence suggests a more complex genetic basis ( 35 ). Partial artemisinin resistance, pervasive in Southeast Asia ( 36 ), is compounded by resistance to the long-acting partner drugs in Artemisinin-based combination therapies (ACTs), leading to high rates of treatment failure ( 37 , 38 ). Mutations in PfK13 have also been reported in Africa, with clinical evidence of delayed parasite clearance ( 39 , 40 ). Despite this, ACTs remain effective in most sub-Saharan African regions, where enhanced surveillance is critical ( 41 , 42 ). In our study, all patients who came to us with uncomplicated malaria after receiving Artemether-lumefantrine, which was not effective in clearing the parasite. We used other drugs for treatment. Artemisinins are the key component of modern malaria combination therapies. To prevent resistance from developing, the WHO has urged a global ban on the production and sale of artemisinin-based treatments used alone (without combination with other drugs). Resistance to sulfadoxine-pyrimethamine is mediated by mutations in target enzymes and is widespread in malaria-endemic regions. Similarly, resistance to atovaquone is associated with mutations in the cytochrome b gene ( 43 , 44 ). Notably, atovaquone-resistant parasites are less likely to be transmitted through mosquitoes ( 45 ), and the drug retains high clinical efficacy in combination therapies despite antifolate resistance ( 46 , 47 ). In this study, patients who received Atovaquone did not develop clinical resistance to the drug. Notably, the mechanism of quinine resistance remains poorly understood and is thought to be complex ( 48 ). These findings underline the multifaceted challenge of drug-resistant malaria. Our observation of treatment failure in 10% of the study cohort underscores the necessity of ongoing surveillance, molecular characterization of resistance, and the development of robust therapeutic strategies to counteract resistance. This study has several limitations. First, the sample size was relatively small and only covered pediatric patients, which may limit the generalizability of the findings. Second, the study did not include detailed molecular investigations to identify genetic markers of malaria drug resistance, such as mutations associated with resistance to artemisinin-based therapies or other antimalarial drugs. This lack of molecular data prevents a deeper understanding of the mechanisms driving treatment failure, also, we have no details about other factors that may contribute to previous treatment failure. Further research is needed to address these limitations and future studies should include larger and more diverse populations, Additionally, examining the role of liver metabolism in malaria drug resistance development may be essential for a more comprehensive analysis. Recommendations We strongly recommend that healthcare providers in Sudan adhere strictly to the WHO/local malaria treatment protocol to ensure standardized and effective management of cases. Additionally, we highly emphasize the need for the establishment of reference laboratories for malaria in Sudanese states. These labs would facilitate molecular investigations, improve resistance surveillance, and guide evidence-based policy decisions for malaria control and treatment. Conclusion This study highlights the increasing burden of undetected P. vivax malaria emphasizing the need for improved diagnostic tools, adherence to treatment protocols, and surveillance strategies. Addressing these challenges is critical to mitigating the impact of resistant malaria and ensuring effective disease control in the region. Abbreviations ACTs: Artemisinin-based combination therapies BFFM: Blood films for malaria BMI: body mass index DDW: Deionized distilled water ICT: Immunochromatographic assay P. falciparum: Plasmodium falciparum P. ovale: Plasmodium ovale P. vivax: Plasmodium vivax PCR: Polymerase chain reaction PfHRP-2: Histidine-rich protein 2 PfK13: P. falciparum kelch protein RBC: Red blood cell TB: Tuberculosis WHO: World Health Organization χ² test: Chi-square test Declarations Parent's patients consent were obtained and all approved by the Ethical committee. Funding No fund was obtained. Authors' contributions All authors have contributed equally to this work. Conflict of interests and declaration The author reports no conflict of interest. Ethics Approval Ethical approval was obtained from the Tropical Disease Teaching Hospital Ethical Committee serial number (TDTH/A/222/3). Data availability Data is available upon contacting the corresponding author. References Schwartz E, Parise M, Kozarsky P, Cetron M. Delayed onset of malaria--implications for chemoprophylaxis in travelers. N Engl J Med. 2003 Oct 16;349(16):1510-6. https://doi.org/10.1056/NEJMoa021592 PMid:14561793 Jameson JL, Fauci AS, Kasper DL, Hauser SL, Longo DL, Loscalzo J. Harrison's principles of internal medicine. No Title. 2018; Mühlberger N, Jelinek T, Gascon J, Probst M, Zoller T, Schunk M, et al. Epidemiology and clinical features of vivax malaria imported to Europe: sentinel surveillance data from TropNetEurop. Malar J. 2004 Mar 8;3:5. Durante Mangoni E, Severini C, Menegon M, Romi R, Ruggiero G, Majori G. Case report: An unusual late relapse of Plasmodium vivax malaria. 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J Antimicrob Chemother. 2007 Nov;60(5):929-36. https://doi.org/10.1093/jac/dkm337 PMid:17848375 Ferdig MT, Cooper RA, Mu J, Deng B, Joy DA, Su X zhuan, et al. Dissecting the loci of low-level quinine resistance in malaria parasites. Mol Microbiol. 2004 May;52(4):985-97.https://doi.org/10.1111/j.1365-2958.2004.04035.x PMid:15130119 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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After infection, individual symptoms may appear as early as 7 days, depending on the species of the parasite. The symptoms begin during the erythrocytic phase of the parasite's life cycle, as infected red blood cells rupture and release merozoites, leading to fever and other clinical manifestations. Most infections caused by \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (\u003cem\u003eP. falciparum\u003c/em\u003e) become symptomatic within a month of exposure with high parasitemia (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNon-falciparum or relapsing species including \u003cem\u003ePlasmodium vivax\u003c/em\u003e (\u003cem\u003eP. vivax\u003c/em\u003e) and \u003cem\u003ePlasmodium ovale\u003c/em\u003e (\u003cem\u003eP.ovale\u003c/em\u003e), these infections can present weeks to months after the initial exposure due to the activation of dormant hypnozoites in the liver (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Relapses often occur within two to three years or even longer (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Recurrences of malaria can be the result of treatment failure (recrudescence) or relapse, and distinguishing between the two can be challenging in endemic areas like Sudan. Recrudescent infections, usually caused by \u003cem\u003eP. falciparum\u003c/em\u003e, occur when the disease re-emerges after an apparent recovery. This is usually due to the survival of parasites in the bloodstream and it can occur when the initial treatment is insufficient to fully clear the infection. Factors contributing to recrudescence include inadequate drug regimens, drug resistance, and incomplete clearance of parasites (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMoreover, \u003cem\u003eP. vivax\u003c/em\u003e can relapse weeks to months after the primary blood-stage infection has resolved, due to their hypnozoite forms. The likelihood of relapse is reduced when patients receive presumptive anti-relapse therapy, such as primaquine (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), which targets the dormant liver stages. Also, there have been instances of \u003cem\u003eP. vivax\u003c/em\u003e infection in the bone marrow, even without detectable parasitemia. One case series from India, involving 108 patients with fever and hematological abnormalities like anemia, thrombocytopenia, or pancytopenia, reported that \u003cem\u003eP. vivax\u003c/em\u003e infection in the bone marrow was present in about 40% of cases (\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The detection threshold for malaria using microscopy is approximately 25-parasites per micro-liter in the hands of experts. However, individuals who have never been exposed to malaria before can develop acute symptoms at parasitemia levels that fall below this limit, making detection difficult in such cases (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Microscopy is less sensitive when it comes to detecting very low-density parasitemia, which can result in missed diagnoses of asymptomatic, submicroscopic infections that serve as potential reservoirs for transmission. This challenge is particularly pronounced in cases of mixed infections which may go undetected (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSudan is highly endemic to \u003cem\u003eP. falciparum\u003c/em\u003e but not \u003cem\u003eP.vivax\u003c/em\u003e (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), the latter presents a new diagnostic challenge due to its characteristic of having low pyrogenic density, especially with a co-infection with \u003cem\u003eP. falciparum\u003c/em\u003e. As a result, standard microscopy may not be effective for accurate diagnosis of \u003cem\u003eP. vivax\u003c/em\u003e, especially when high-density \u003cem\u003eP. falciparum\u003c/em\u003e infections are present, as these can overshadow the lower levels of \u003cem\u003eP. vivax\u003c/em\u003e. Moreover, The Deletion of histidine-rich protein 2 (PfHRP-2) in the \u003cem\u003eP.falciparum\u003c/em\u003e genome will impact the accuracy of PfHRP-2 based rapid diagnostic tests in Sudan (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In such instances, molecular techniques like polymerase chain reaction (PCR), which amplify DNA, offer a more sensitive alternative to microscopy and it is more capable of detecting low parasitemia levels (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This study investigated patient with recurrent malaria and focusing on diagnosing relapses and recrudescence and investigating the characteristics of treatment failure. The research assessed the mixed Plasmodium infections on the same host, such as \u003cem\u003eP. vivax\u003c/em\u003e and \u003cem\u003eP. falciparum\u003c/em\u003e on the diagnostic accuracy of microscopy vs PCR.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design:\u003c/h2\u003e\u003cp\u003eThis prospective cohort study was conducted over 80 weeks, from October 2021 to April 15, 2023 at the Tropical Diseases Teaching Hospital in Omdurman, Sudan. Established in 1974 by the Ministry of Health in collaboration with the National Council of Research, the hospital serves as a reference center for tropical diseases, receiving patients from Sudan, South Sudan, Ethiopia, Chad, and Somalia. It has three wards, each accommodating 100\u0026ndash;140 patients. In February 2019, the Pediatrics Tropical Department was established in collaboration with the Ministry of Health and the hospital administration. This department includes an outpatient tropical diseases clinics, a tuberculosis (TB) management unit and pediatric multidrug-resistant TB unit, a malaria diagnostic and treatment referral center, two pediatric wards with 12 beds each, and a quarantine area for TB patients. This setup makes it the first specialized center for pediatric tropical and infectious diseases in Sudan.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePatients:\u003c/h3\u003e\n\u003cp\u003eThis study included all consecutive pediatric patients referred to the hospital with a history of recurrent malaria. Each patient underwent a detailed history, physical examination, and complete blood count to assess symptoms of recurrent malaria. Patients with recurrent malaria episodes occurring more than 28 days apart were not included. All patients were diagnosed using thin and thick blood films for malaria (BFFM) to determine the baseline parasite species and parasite count. Molecular detection via PCR was also performed to confirm parasite species. Patients were classified based on the cause of malaria treatment failure: either recrudescence (\u003cem\u003eP.falciparum\u003c/em\u003e) or relapsing species (\u003cem\u003eP.vivax\u003c/em\u003e). Patients were treated according to the National Malaria Treatment/WHO Protocol for uncomplicated and severe malaria (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/publications/i/item/guidelines-for-malaria\u003c/span\u003e\u003cspan address=\"https://www.who.int/publications/i/item/guidelines-for-malaria\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Also, we controlled factors that could contribute to antimalarial treatment failure by ensuring weight-appropriate dosing, administering medication with a fatty meal, promoting compliance with the full treatment course, and providing guidance on when to repeat a dose if vomiting occurs. Additionally, we ensure proper preparation and infusion of IV antimalarials by trained nurses within our hospital setting and exclusively use antimalarial drugs supplied under the Sudan National Malaria Protocol. Follow-up assessments included BFFM and parasite counts on days 0, 3, 7, 14 and 28 until parasite clearance was achieved. Based on these results, patients were classified as having early or late clinical, or parasitological failure, according to the WHO and national malaria protocol definitions. Patients who experienced treatment failure during follow-up were transitioned to the next treatment step as per WHO and national malaria protocols. These patients were monitored with repeat parasite counts until parasite clearance was achieved, confirmed by negative BFFM results.\u003c/p\u003e\n\u003ch3\u003eMolecular and microscopic detection for malaria:\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eBlood sample\u003c/h2\u003e\u003cp\u003eDNA was extracted following the guanidine chloride extraction method described by Ciulla et al (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), with minor modifications. Briefly, 1 ml of blood samples were washed with a red blood cell (RBC) lysis buffer, and then 1 ml of 6 M guanidine chloride was added to lyse the cell walls and release DNA. Proteinase K solution was then added, and the lysate was incubated at 37\u0026deg;C overnight. The following day, 3 ml of chloroform was added to the samples to separate DNA from proteins using centrifugation at 8,000 rpm. The DNA-containing supernatant was then added to 3 ml of cold absolute ethanol to precipitate the DNA. After precipitation, the DNA pellet was dissolved in 100 microliters of deionized distilled water. The quality and purity of the extracted DNA were assessed using a spectrophotometric method with an Implen Nanodrop (Implen, Germany). DNA was then stored at -20\u0026deg;C until molecular detection of Plasmodium species.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePolymerase chain reaction\u003c/h3\u003e\n\u003cp\u003eMolecular detection was performed using a nested PCR protocol previously developed by Snounou et al. for the detection of plasmodium species infections (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Initially, an outer PCR was performed using the primers rPLU6: 5'- TTA AAA TTG TTG CAG TTA AAA CG-3' and rPLU5: 5'- CCT GTT GTT GCC TTA AAC TTC \u0026minus;\u0026thinsp;3'. For the nested PCR to detect \u003cem\u003eP.falciparum\u003c/em\u003e and \u003cem\u003eP.vivax\u003c/em\u003e infections, the primers used were rPF1: 5'-TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT \u0026minus;\u0026thinsp;3', rPF2: 5'-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC \u0026minus;\u0026thinsp;3', rPV1: 5'-CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC \u0026minus;\u0026thinsp;3', and rPV2: 5'-ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA \u0026minus;\u0026thinsp;3'. The PCR reaction mixture was prepared using a ready-to-use PCR mastermix (Intron Biotechnology, South Korea) following the manufacturer's instructions. In the outer PCR reaction, 1 microliter of outer primers was added to 1 microliter of the DNA sample. For the nested PCR reaction, 1 microliter of each species-specific primer was added to 1 microliter of the first PCR amplicons, and the reaction volume was adjusted to 20 microliters using deionized distilled water (DDW) as recommended. Thermal cycling conditions were adjusted in the AB 2700 Thermal Cycler Gene Amp PCR machine (Applied Biosystems, Singapore) according to Snounou et al (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Positive control samples (previously known as \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e DNA samples) were included in each run. Negative controls were prepared by adding primers to tubes without DNA. Amplified PCR products were then loaded onto a 2.5% agarose gel (Intron Biotechnology, South Korea), and electrophoresis was conducted at 100 mA and 30 volts for 1 hour using a gel electrophoresis device (Major Sciences, Taiwan). The results were visualized using a gel documentation system (Major Sciences, Taiwan). A 100 bp molecular ladder (Intron Biotechnology, South Korea) was loaded with the samples to determine PCR amplicon lengths. PCR results were interpreted based on the size of the amplified PCR product. Positive samples for \u003cem\u003eP. falciparum\u003c/em\u003e showed a band of 205 bp, while positive samples for \u003cem\u003eP. vivax\u003c/em\u003e showed a band of 120 bp. Moreover, for the microscopic diagnosis of malaria, we utilized similar techniques in our study as described in a study in White Nile state in Sudan. Specifically, 2.5 mL of venous blood was collected from each participant. Malaria diagnosis was performed using blood film microscopy from two laboratories and confirmed via PCR. Both thick and thin blood films were prepared, with fields examined at least twice (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), following the WHO's quality control guidelines to maintain diagnostic accuracy.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eData collection and analysis:\u003c/h2\u003e\u003cp\u003eThe data was collected for this study by using data collection tools that included the following variables: gender, age, and state of residence. Also, the duration of recurrent malaria in months, and the frequency of recurrent malaria episodes. The severity of malaria was assessed for all patients, along with a history of recurrent malaria in the family or among neighbors. Data on previous and current species identification was recorded, as well as details of previous diagnostic tests and previous failed malaria drugs. We also recorded the malaria species identified by our lab throughout combined PCR and BFFM. Follow-up data included BFFM, along with basic clinical assessments covering symptoms, and treatment information. The data were analyzed by SPSS 26 and Jamovi 2.5 using descriptive statistics and presented as frequency tables with percentages or in graphical form. To assess associations between variables, the Chi-square test (χ\u0026sup2; test) and binary logistic regression were employed. A p-value of \u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographic and clinical/Diagnostic/treatment history of recurrent malaria in 81 pediatric patients.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCount\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;Std\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGender (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53.1%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e46.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eAge by years\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eStates (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eKhartoum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eAl-Gazira\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eWhite Nile\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eRiver Nile\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.7%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eKassala\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.7%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eKordofan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eSennar\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.7%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eDarfur\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.2%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eDuration of recurrent malaria (months)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eFrequency of recurrent Malaria (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eEvery week\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eEvery two weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e41.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eEvery three weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eEvery four weeks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eVariable duration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.9%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMalaria severity (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eSevere\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.3%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eUncomplicated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e82.7%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHistory of recurrent malaria in the family/neighbors (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e86.4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHistory of Previous Species identification confirmed (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e55.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e44.4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePrevious diagnostic tests for Species identification and their results (N\u0026thinsp;=\u0026thinsp;45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBy BFFM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP.falciparum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100.0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBy both BFFM and immunochromatographic assay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP.falciparum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100.0%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePrevious Follow-up by BFFM (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.6%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91.4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssessment, investigations, and treatments of the current malaria infection.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCount\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBasic clinical assessment\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBMI (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75.3%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnderweight\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24.7%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eHB (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNormal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33.3%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAnemic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNot Done\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHepatomegaly (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.7%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96.3%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSplenomegaly (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.4%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e92.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDiagnosis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSpecies identification by BFFM (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP.falciparum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e93.8%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP.vivax\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.9%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNegative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eSpecies identification by PCR (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP.falciparum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eP.Vivax\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMixed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCauses of recurrent malaria (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRelapsing species/P.vivax\u0026thinsp;+\u0026thinsp;mixed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.4%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRecrudescence/ P.falciparum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.6%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eClassification of Recrudescence treatment failure (N\u0026thinsp;=\u0026thinsp;36), Nine patients responded from the first time\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEarly parasitological failure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19.4%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLate parasitological failure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEarly Clinical and parasitological failure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.3%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLate Clinical and parasitological failure\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.1%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTreatment\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e\u003cp\u003eClearance drug (N\u0026thinsp;=\u0026thinsp;81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDihydroartemisinin\u0026ndash;Piperaquine/Primaquine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39.5%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArtesunate/Primaquine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.7%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArtesunate/Chroquine prophylaxis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDihydroartemisinin\u0026ndash;Piperaquine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30.9%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuinine/Doxycycline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.5%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuinine/Clindamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.7%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArtesunate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArtesunate/Dihydroartemisinin\u0026ndash;Piperaquine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.4%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFansidar\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.9%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArtemether\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuinine/Artemether\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.2%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAtovaquine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.5%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn this study, the demographic and clinical history data of 81 pediatric patients with recurrent malaria showed a nearly equal gender distribution (53.1% male, 46.9% female), with an average age of 6.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 years. Most cases originated from Khartoum (60.5%), with varying representation from other states. The average duration of recurrent malaria was 9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8 months, and 41.9% experienced episodes every two weeks. The majority of cases were uncomplicated (82.7%), and 55.5% had previously confirmed species identification using traditional methods like BFFM and rapid immunochromatographic assay (ICT). Among the 45 patients who underwent these diagnostic tests, 100% confirmed \u003cem\u003eP. falciparum\u003c/em\u003e via BFFM, and those tested with both BFFM and ICT also showed only \u003cem\u003eP. falciparum\u003c/em\u003e, with no detection of \u003cem\u003eP. vivax\u003c/em\u003e or mixed infections (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, prior to being referred to our unit, only 8.6% had follow-up diagnostic tests, indicating a gap in continuous and appropriate follow-up care in primary care centers for malaria. There, all patients received the same treatment for each malaria episode without moving to other lines, which is against the Sudan national malaria/WHO protocol: the first line (Artemether-lumefantrine) for uncomplicated malaria, and (artesunate/quinine) for severe malaria. The clinical assessments revealed that 75.3% of patients had a normal BMI (according to the WHO definition of BMI), while 24.7% were underweight. Hemoglobin levels indicated anemia in 58% of the children (defined by HB, hematocrit, or red blood cell count levels lower than the normal age and sex-adjusted ranges). Hepatomegaly was observed in 3.7% and splenomegaly in 7.4%. Fever was the most common symptom, present in 79, followed by persistent vomiting in 30, headache in 18, and diarrhea in 14 patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Species identification using BFFM by expert microbiologists showed that 93.8% of cases involved \u003cem\u003eP. falciparum\u003c/em\u003e, 4.9% \u003cem\u003eP. vivax\u003c/em\u003e, and 1.2% were negative. In comparison, advanced PCR testing provided more detailed results, identifying 55.6% \u003cem\u003eP. falciparum\u003c/em\u003e, 6.2% \u003cem\u003eP. vivax\u003c/em\u003e, and 38.2% mixed infections, highlighting a broader detection range. The causes of recurrent malaria were attributed to \u003cem\u003eP. falciparum\u003c/em\u003e recrudescence in 55.6% of patients and \u003cem\u003eP. vivax\u003c/em\u003e relapses in 44.4% (both p.vivax and mixed infection). Among those with relapsing infections identified by PCR, 86.1% had mixed infections and 13.9% had \u003cem\u003eP. vivax\u003c/em\u003e alone (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, BFFM results for relapsing species showed only \u003cem\u003eP. falciparum\u003c/em\u003e (86.1%) and \u003cem\u003eP. vivax\u003c/em\u003e (11.1%) without detecting mixed infections, which were only revealed by advanced PCR. In terms of \u003cem\u003eP. falciparum\u003c/em\u003e treatment failure, 58.3% experienced early clinical and parasitological failure, 19.4% had an early parasitological failure, and 11.1% had late parasitological failure. All patients who were found to have malaria in the last 2 weeks and received Artemether lumfantrin were treated with Dihydroartemisinin\u0026ndash;Piperaquine (DHAP) according to the Malaria treatment protocol (9 patients had achieved clearance from the first time with negative BFFM day 3,7, 14 and 28 day). All patients who were found to have \u003cem\u003eP.vivax\u003c/em\u003e malaria were treated according to the protocol (uncomplicated or severe) then followed by radical treatment by primaquine except for one patient who was younger than 6 months and received weekly chloroquine prophylaxis after clearance of malaria infection. Eight patients developed failure of treatment many times during their follow up and received all treatment options in malaria protocol then treated with antimalarial medications outside treatment protocol to achieve parasite clearance (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn tropical regions with limited specialized diagnostic and treatment facilities recurrent malaria emerged as a significant challenge, particularly in pediatric populations, where the burden and the prolonged duration of the disease can severely impact growth, development, education, the family, and the overall health status of the patient. Here, this is one of the first updated prospective studies that assess the demographic, clinical, and treatment for recurrent malaria. We investigated recurrent malaria using BFFM and advanced PCR to detect low \u003cem\u003eP.vivax\u003c/em\u003e parasitemia, mixed infection, and recrudescence of \u003cem\u003eP.falciparum\u003c/em\u003e to aid the identification cause of recurrent malaria, we followed patients with BFFM for parasite clearance and treated them according to Sudan National Malaria treatment/WHO protocol. In this study 58.0% (n\u0026thinsp;=\u0026thinsp;47) were anemic and 24.7% (n\u0026thinsp;=\u0026thinsp;20) were underweight for their age and height. Notably, the relationship between recurrent malaria cases and body mass index (BMI) was analyzed. Among patients with \u003cem\u003eP.vivax\u003c/em\u003e malaria, only 3 out of 36 (8.3%) were classified as underweight, compared to 17 out of 45 (37.8%) in patients with recurrent \u003cem\u003eP.falciparum\u003c/em\u003e (p-value of \u0026le;\u0026thinsp;0.05), also, there is a slightly significant relationship between malaria severity and causes of recurrent malaria (p-value\u0026thinsp;=\u0026thinsp;0.057) with severe malaria present more in \u003cem\u003eP.falciparum\u003c/em\u003e compared to \u003cem\u003ep.vivax\u003c/em\u003e with 11 and 3 cases, respectively. The vast majority of severe \u003cem\u003eP. vivax\u003c/em\u003e malaria cases are from Southeast Asia and India, with few published data on severe \u003cem\u003eP. vivax\u003c/em\u003e from Africa (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). However, one study from Eastern Sudan highlighted severe \u003cem\u003eP. vivax\u003c/em\u003e malaria cases among children at New Halfa Hospital, underscoring similar severe manifestations as seen in regions like India and Brazil. In Sudan, the presence of \u003cem\u003eP. vivax\u003c/em\u003e is increasingly reported despite \u003cem\u003eP. falciparum\u003c/em\u003e being the dominant species (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The rise in \u003cem\u003eP. vivax\u003c/em\u003e and mixed-infection malaria cases in Sudan could be linked to the increased movement of Ethiopian and Eritrean populations across the border, facilitated by peace agreements and the construction of paved roads between these countries and Sudan. Recent studies have shown that \u003cem\u003eP. vivax\u003c/em\u003e is the predominant malaria species in Ethiopia, with reports of treatment failures, which may also contribute to its spread in Sudan, increasing diagnostic difficulties (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Another study in Central and Eastern Sudan utilized molecular techniques like PCR to identify mosquito vectors carrying \u003cem\u003eP. vivax\u003c/em\u003e. They found a growing number of \u003cem\u003eP. vivax\u003c/em\u003e cases, suggesting a shift in malaria transmission patterns in Sudan (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNotably, in regions like South Sudan, recurrent malaria has been linked to factors such as incomplete malaria treatment, socio-economic status, and nutrition. They found that patients who ate at least two meals a day had a significantly lower likelihood of developing severe recurrent malaria compared to those who ate only one meal (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Moreover, in terms of geographical distribution, a significant relationship was found in this study between malaria severity and location (p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05). Only three patients with severe malaria were reported in Khartoum, compared to 11 cases in other states. Conversely, patients with recurrent uncomplicated malaria were more likely to come from Khartoum, with a nearly twofold difference\u0026mdash;46 cases in Khartoum versus 21 cases from other states. This suggests that more cases of uncomplicated malaria are present in other states, but these patients cannot reach Khartoum for treatment and investigations. The analysis using binary logistic regression showed no significant relationship between the duration of malaria and the causes of recurrent malaria in pediatric patients. In Sudan, geographical factors play a critical role in malaria distribution. A spatiotemporal study mapping malaria incidence in Sudan highlights how transmission varies by region, with areas like Khartoum showing lower incidence compared to other states (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Such studies suggest that geographical disparities in professional healthcare access and ecological factors significantly affect patient outcomes, and more patients may not reach Khartoum for treatment due to economic issues and transportation challenges. The Tropical Disease Teaching Hospital is a national referral center for treating infectious diseases, including malaria. Notably, all malaria patients referred to the hospital during the study had \u003cem\u003eP. falciparum\u003c/em\u003e diagnosed based on BFFM and BFFM/ICT done in primary care centers, with only 8.6% (n\u0026thinsp;=\u0026thinsp;7) patients receiving BFFM follow-up for their disease after treatment. The treatment given to patients from primary care centers was Artemether-lumefantrine for uncomplicated malaria (no second-line therapy was used) or quinine or artesunate for severe malaria. Diagnostic challenges in malaria treatment are also prominent as none of the previous diagnostic tests were able to detect mixed infection. When the BFFM is compared to PCR, it has a sensitivity of 11.1% for diagnosing relapsing recurrent malaria (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Studies have shown that BFFM's sensitivity can be significantly lower than that of PCR techniques. The lower sensitivity of BFFM is particularly concerning for relapsing or recurrent mixed infection, where \u003cem\u003eP.vivax\u003c/em\u003e parasite levels may be below the detection threshold of microscopy. PCR, with its higher sensitivity, is more adept at identifying these low-density infections, making it a more reliable diagnostic tool in such scenarios, especially with the emergence of \u003cem\u003eP. vivax\u003c/em\u003e in Sudan (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMoreover, increasing awareness of malaria WHO/Local protocols is important. A qualitative study on malaria case management in Northern Sudan identified gaps in adherence to diagnostic and treatment protocols, particularly in non-specialized centers due to resource shortages, training gaps, and lack of malaria expert supervision and consultants. This nonadherence could contribute to the mismanagement of recurrent malaria cases, particularly in resource-limited settings (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The lack of awareness about the updated management of recurrent malaria and malaria treatment failure protocols could be the reason for the poor management of these patients in peripheral centers. A 2018 study in Equatorial Guinea found that 63.6% of public health practitioners had low awareness of national malaria treatment guidelines, with significant differences between hospital and health center workers. Strengthening practitioners' knowledge and adherence to guidelines is critical for improving malaria diagnosis, treatment, and disease control in the region (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Poor adherence to proper management guidelines could also be due to diagnostic difficulty and lack of resources in treating facilities, as reported (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). This study concluded that nonadherence to malaria diagnosis and treatment protocols in Northern Sudan was linked to medication misuse and diagnostic distrust, along with contributing factors like resource limitations, inadequate staff training, and patient demands. These factors may lead to improper diagnostic and treatment practices, which translate to poor malaria control.\u003c/p\u003e\u003cp\u003eInterestingly, about 10% (N\u0026thinsp;=\u0026thinsp;8) of the study cohort experienced a failure to clear the parasite and were treated with antimalarial medications outside the established treatment protocol. With the possibility of resistant parasites, we administered Fansidar to these patients. Four achieved parasite clearance with follow-up from day 3 to day 28 for symptoms and BFFM, but the other four also experienced treatment failure with Fansidar, one of whom developed severe malaria. We treated the four patients with artemether injections, while the patient with severe malaria received quinine along with Artemether. At this point, two patients achieved parasite clearance, while two others continued to show treatment failure. We then used atovaquone-proguanil, after which they finally tested negative for BFFM and remained symptom-free for 28 days. We subsequently cleared them and monitored their progress by phone, with no further cases of malaria. The observed multiple treatment failures in these 8 patients, constituting approximately 10% of our study cohort, suggest a concerning prevalence of resistant malaria strains (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). This high rate surpasses the expected prevalence in certain endemic regions and highlights the challenges of managing resistant malaria. Reduced susceptibility to artemisinins, characterized by delayed parasite clearance in clinical studies (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) and prolonged parasite survival in vitro (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), is a growing concern. This resistance is associated with point mutations in the \u003cem\u003eP. falciparum\u003c/em\u003e kelch protein (PfK13) propeller domain (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), though evidence suggests a more complex genetic basis (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Partial artemisinin resistance, pervasive in Southeast Asia (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), is compounded by resistance to the long-acting partner drugs in Artemisinin-based combination therapies (ACTs), leading to high rates of treatment failure (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Mutations in PfK13 have also been reported in Africa, with clinical evidence of delayed parasite clearance (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Despite this, ACTs remain effective in most sub-Saharan African regions, where enhanced surveillance is critical (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn our study, all patients who came to us with uncomplicated malaria after receiving Artemether-lumefantrine, which was not effective in clearing the parasite. We used other drugs for treatment. Artemisinins are the key component of modern malaria combination therapies. To prevent resistance from developing, the WHO has urged a global ban on the production and sale of artemisinin-based treatments used alone (without combination with other drugs). Resistance to sulfadoxine-pyrimethamine is mediated by mutations in target enzymes and is widespread in malaria-endemic regions. Similarly, resistance to atovaquone is associated with mutations in the cytochrome b gene (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Notably, atovaquone-resistant parasites are less likely to be transmitted through mosquitoes (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), and the drug retains high clinical efficacy in combination therapies despite antifolate resistance (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In this study, patients who received Atovaquone did not develop clinical resistance to the drug. Notably, the mechanism of quinine resistance remains poorly understood and is thought to be complex (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). These findings underline the multifaceted challenge of drug-resistant malaria. Our observation of treatment failure in 10% of the study cohort underscores the necessity of ongoing surveillance, molecular characterization of resistance, and the development of robust therapeutic strategies to counteract resistance. This study has several limitations. First, the sample size was relatively small and only covered pediatric patients, which may limit the generalizability of the findings. Second, the study did not include detailed molecular investigations to identify genetic markers of malaria drug resistance, such as mutations associated with resistance to artemisinin-based therapies or other antimalarial drugs. This lack of molecular data prevents a deeper understanding of the mechanisms driving treatment failure, also, we have no details about other factors that may contribute to previous treatment failure. Further research is needed to address these limitations and future studies should include larger and more diverse populations, Additionally, examining the role of liver metabolism in malaria drug resistance development may be essential for a more comprehensive analysis.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRecommendations\u003c/h2\u003e\u003cp\u003eWe strongly recommend that healthcare providers in Sudan adhere strictly to the WHO/local malaria treatment protocol to ensure standardized and effective management of cases. Additionally, we highly emphasize the need for the establishment of reference laboratories for malaria in Sudanese states. These labs would facilitate molecular investigations, improve resistance surveillance, and guide evidence-based policy decisions for malaria control and treatment.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the increasing burden of undetected \u003cem\u003eP. vivax\u003c/em\u003e malaria emphasizing the need for improved diagnostic tools, adherence to treatment protocols, and surveillance strategies. Addressing these challenges is critical to mitigating the impact of resistant malaria and ensuring effective disease control in the region.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACTs: Artemisinin-based combination therapies\u003c/p\u003e\n\u003cp\u003eBFFM: Blood films for malaria\u003c/p\u003e\n\u003cp\u003eBMI: body mass index\u003c/p\u003e\n\u003cp\u003eDDW: Deionized distilled water\u003c/p\u003e\n\u003cp\u003eICT: Immunochromatographic assay\u003c/p\u003e\n\u003cp\u003eP. falciparum: Plasmodium falciparum\u003c/p\u003e\n\u003cp\u003eP. ovale: Plasmodium ovale\u003c/p\u003e\n\u003cp\u003eP. vivax: Plasmodium vivax\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003ePfHRP-2: Histidine-rich protein 2\u003c/p\u003e\n\u003cp\u003ePfK13: P. falciparum kelch protein\u003c/p\u003e\n\u003cp\u003eRBC: Red blood cell\u003c/p\u003e\n\u003cp\u003eTB: Tuberculosis\u003c/p\u003e\n\u003cp\u003eWHO: World Health Organization\u003c/p\u003e\n\u003cp\u003e\u0026chi;\u0026sup2; test: Chi-square test\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eParent\u0026apos;s patients consent were obtained and all approved by the Ethical committee.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo fund was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests and declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author reports no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from\u0026nbsp;the\u0026nbsp;Tropical Disease Teaching Hospital Ethical Committee serial number (TDTH/A/222/3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available upon contacting the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSchwartz E, Parise M, Kozarsky P, Cetron M. 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Mol Microbiol. 2004 May;52(4):985-97.https://doi.org/10.1111/j.1365-2958.2004.04035.x PMid:15130119\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Recurrent malaria, Pediatric malaria, Molecular diagnostics, Malaria treatment failure, P. falciparum and P. vivax","lastPublishedDoi":"10.21203/rs.3.rs-6491328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6491328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecurrent malaria is a new significant health challenge in Sudan, particularly among pediatric populations. Despite the dominance of \u003cem\u003eP. falciparum\u003c/em\u003e, increasing cases of \u003cem\u003eP. vivax\u003c/em\u003e and mixed infections complicate the diagnosis and management. This study aimed to analyze the demographic, clinical, diagnostic and treatment characteristics of recurrent malaria in children, highlighting diagnostic challenges and treatment outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted a prospective observational study from October 2021 to April 2023, at the Tropical Diseases Teaching Hospital in Omdurman, Sudan among all consecutive pediatric patients with recurrent malaria (n=81). Data on demographics, clinical history, diagnostic and treatment were collected. Species identification was performed using Blood Film for Malaria (BFFM), and PCR. Treatment adherence to Sudan’s national malaria/WHO protocol was assessed, and outcomes were tracked over 28-day follow-up periods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients had a mean age of 6.78±4.7 years, with 53.1% male. Most cases originated from Khartoum (60.5%), with a malaria recurrence duration of 9.7±8.8 months. PCR detected \u003cem\u003eP. falciparum\u003c/em\u003e (55.6%), \u003cem\u003eP. vivax\u003c/em\u003e (6.2%), and mixed infections (38.2%), whereas BFFM primarily identified \u003cem\u003eP. falciparum\u003c/em\u003e (93.8%). Recrudescence was seen in 55.6% of cases and relapses in 44.4%. When the BFFM is compared to PCR, it has a sensitivity of 11.1% for diagnosing relapsing recurrent malaria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study highlights the diagnostic limitations of traditional methods compared to PCR and underscores the need for protocol adherence and tailored therapies. Enhanced diagnostic tools and management strategies are crucial for addressing recurrent and resistant malaria in pediatric populations in Sudan.\u003c/p\u003e","manuscriptTitle":"Molecular Diagnostics and Treatment Failures in Pediatric Malaria: a prospective observational study from Omdurman, Sudan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 12:42:08","doi":"10.21203/rs.3.rs-6491328/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b2048d16-610a-4fde-81da-6795b6acb853","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-10T12:42:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 12:42:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6491328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6491328","identity":"rs-6491328","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}