Early Diagnosis Of Malaria Research Articles

Novel Plasmodium falciparum histidine-rich protein 2/3 repeat type in Ethiopian malaria infection: does this affect performance of HRP2-based malaria RDT?

BackgroundRapid diagnostic tests (RDTs) provide quick, easy, and convenient early diagnosis of malaria ensuring better case management particularly in resource-constrained settings. Nevertheless, the efficiency of HRP2-based RDT can be compromised by Plasmodium falciparum histidine-rich protein 2/3 gene deletion and genetic diversity. This study explored the genetic diversity of PfHRP2/3 in uncomplicated malaria cases from Ethiopia.MethodsA cross-sectional study was conducted from June 2022 to March 2023 at Metehara, Zenzelema and Kolla Shele health centres, Ethiopia. Finger-prick blood samples were collected for RDT testing and microscopic examination. For molecular analysis, parasite genomic DNA was extracted from venous blood. Plasmodium falciparum was confirmed using VarATS real time PCR. Additionally, PfHRP2/3 was amplified, and DNA amplicons were sequenced using Oxford Nanopore technology.ResultsPfHRP2/3 sequences revealed small variations in the frequency and number of amino acid repeat types per isolate across the three health centres. Twelve and eight types of amino acid repeats were identified for PfHRP2 and PfHRP3, respectively, which had been previously characterized. Repeat type 1, 4 and 7 were present in both PfHRP2 and PfHRP3 amino acid sequences. Type 2 and 7 repeats were commonly dispersed in PfHRP2, while repeat types 16 and 17 were found only in PfHRP3. A novel 17 V repeat type variant, which has never been reported in Ethiopia, was identified in six PfHRP3 amino acid sequences. The majority of the isolates, as determined by the Baker’s logistic regression model, belonged to group C, of which 86% of them were sensitive to PfHRP2-based RDT. Likewise, PfHRP2-based RDT detected 100% of the isolates in group A (product of type 2 × type 7 repeats ≥ 100) and 85.7% in group B (product of types 2 × type 7 repeats 50–99) at a parasitaemia level > 250 parasite/μl.ConclusionThis study highlights the significant diversity observed in PfHRP2 and PfHRP3 among clinical isolates of Plasmodium falciparum in Ethiopia. This emphasizes the necessity for monitoring of PfHRP2- based RDT efficacy and their repeat type distribution using a large sample size and isolates from various ecological settings.

Characterization of urinary metabolites associated with malaria infection using infra-red spectroscopy and liquid chromatography–mass spectrometry in South Western Uganda

Early malaria diagnosis improves outcomes during malaria treatment; routine diagnostic techniques rely on blood samples obtained invasively. Therefore, this study used infra-red (IR) spectroscopy coupled with Principle Component Analysis (PCA) to study the urinary profile of malaria patients and that of controls aimed at understanding metabolite perturbation during malaria infection so as to contribute towards development of non-invasive malaria diagnosis methods. Freeze dried human urine samples form malaria infected individuals (cases) and controls were screened in the IR region of 4000 cm−1 to 600 cm−1 and overall spectral differences were observed at wave numbers 1618 cm−1, 1679 cm−1 (amino acids). Peaks at 3030 cm−1 (NH4+) and 940 cm−1 (O–H of carboxylic acids) showed high absorbance in patients compared to controls. Liquid-chromatography–mass spectrometry (LC–MS/MS) was used to quantify amino acids in the urine samples and the results indicated a significant increase of amino acid cystine (P = 0.012). Lysine and tyrosine also increased in patients compared to controls. The use of IR-PCA differentiated clusters of urine samples from patients with malaria from control and the demonstrated amino acid perturbation is consistent with malaria infection. This data provides baseline information for application in development of a non-invasive diagnostic tests for malaria.

Comparison of SD Bioline Malaria Ag Pf/Pan and Acro Malaria P.f./P.v./Pan with Microscopy and Real Time PCR for the Diagnosis of Human Plasmodium Species.

The early diagnosis of malaria is crucial to controlling morbidity and mortality. The World Health Organization (WHO) recommends diagnosing malaria either using light microscopy or a malaria rapid diagnostic test (RDT). Most RDTs use antibodies to detect two P. falciparum histidine-rich proteins named PfHRP2 and PfHRP3. However, false-negative results are known to occur due to the poor performance of RDTs depending on the species and the deletion of the Pfhrp2 and Pfhrp3 genes. This study evaluated new malaria RDTs for the detection of the human Plasmodium species. The Acro Malaria P.f./P.v./Pan Rapid Test Cassette allows the qualitative detection of parasite antigens, such as PfHRP2 specific to Plasmodium falciparum, PvLDH specific to Plasmodium vivax, and/or panLDH Plasmodium genus lactate dehydrogenase, in the blood of infected individuals. This RDT was assessed against 229 samples collected from imported malaria cases, mainly from Africa. The samples were previously diagnosed using light microscopy and RDT (SD Malaria Ag P.f./Pan, SD Bioline Alere Abbott), then confirmed using real time PCR. The two RDTs were evaluated using a comparison with real time PCR as the reference method, and their performances were compared with each other. Compared to SD RDT, the Acro RDT showed a better sensitivity to P. falciparum (96.8% vs. 89.8%), P. vivax (78.6% vs. 64.3%), P. ovale (73.7% vs. 5.3%), and P. malariae (20.0% vs. 0%). The respective specificities of the Acro RDT and SD RDT are 90.7% vs. 95.3% to P. falciparum, 100% to P. vivax, and 100% vs. 100% to Plasmodium genus. Therefore, Acro RDT showed better performance in the identification of P. ovale and low parasitaemia of P. falciparum. In addition, Acro RDT has the advantage of detecting PvLDH-specific antigens. The Acro Malaria RDT presents the benefits of detecting a P. falciparum antigen (PfHRP2) and a P. vivax antigen (PvLDH) with high sensitivity (96.8% and 73.7%, respectively) and specificity (90.7% and 100%, respectively). Acro Malaria P.f./P.v./Pan rapid diagnostic tests could be effectively used in endemic areas, especially when microscopic examination cannot be performed.

Early Diagnosis of Malaria with Rapid Diagnostic Test

Early Diagnosis of Malaria with Rapid Diagnostic Test

Multienzymatic disintegration of DNA-scaffolded magnetic nanoparticle assembly for malarial mitochondrial DNA detection

Early diagnosis of malaria can prevent the spread of disease and save lives, which, however, remains challenging in remote and less developed regions. Here we report a portable and low-cost optomagnetic biosensor for rapid amplification and detection of malarial mitochondrial DNA. Bioresponsive magnetic nanoparticle assemblies are constructed by using nucleic acid scaffolds containing endonucleolytic DNAzymes and their substrates, which can be activated by the presence of target DNA and self-disintegrated to release magnetic nanoparticles for optomagnetic quantification. Specifically, target molecules can induce padlock probe ligation and subsequent one-pot homogeneous cascade reactions consisting of nicking-enhanced rolling circle amplification, DNAzyme-assisted nucleic acid recycling, and strand-displacement-driven disintegration of the magnetic assembly. With an optimized magnetic actuation process for reaction acceleration, a detection limit of 1 fM can be achieved by the proposed biosensor with a total assay time of ca. 90 min and a dynamic detection range spanning 3 orders of magnitude. The robustness of the system was validated by testing target molecules spiked in 5% serum samples. Clinical sample validation was conducted by testing malaria-positive clinical blood specimens, obtaining quantitative results concordant with qPCR measurements.

A novel transfer learning-based model for diagnosing malaria from parasitized and uninfected red blood cell images

Malaria represents a potentially fatal communicable illness triggered by the Plasmodium parasite. This disease is transmitted to humans through the bites of Anopheles mosquitoes that carry the infection. This disease has significant and devastating consequences on the health systems of fragile countries, particularly in sub-Saharan Africa. Malaria affects red blood cells by invading and replicating within them, destroying them, and releasing toxic byproducts into the bloodstream. The parasite’s ability to stick and modify the surface of red blood cells can cause them to become sticky, obstructing blood flow in vital organs such as the brain and spleen. Therefore, efficient approaches for the early detection of malaria are critical to saving patients’ lives. The main aim of this study is to develop an efficient model for early malaria diagnosis. We used malaria images based on parasitized and uninfected red blood cells for the study experiments. We applied neural network-based approaches such as Neural Search Architecture Network (NASNet) and compared its performance with machine learning techniques. Moreover, we proposed a novel NNR (NASNet Random forest) method for feature engineering. The proposed NNR approach first extracts spatial features from input malaria images, then class prediction probability features are extracted from these spatial features. The feature set obtained from the data extraction trains machine learning models. Our comprehensive experiments show that the support vector machine outperformed state-of-the-art models, achieving a high-performance score of 99% and having an inference time near 0.025 s. We validated the performance using k-fold cross-validation and optimized the hyperparameters through tuning. Our proposed research has improved the early diagnosis of malaria and can assist medical specialists in reducing the mortality rate.

Seasonal Trends with the Comparison of Venous and Capillary Blood for Diagnosis of Malaria among Children in Karachi, Pakistan.

More than 250 million people are infected by malaria parasites annually while around one million children less than 5 years of age die every year due to malaria. We aimed to assess the seasonal trends and usefulness of capillary and venous blood for rapid diagnosis of malaria. This cross-sectional study of 18 months duration was conducted at the National Institute of Child Health (NICH), Karachi. All patients reporting fever as chief complaint were recruited as study subjects. A semi-structured questionnaire was used to collect demographic information, presenting complaints, awareness of caregivers regarding malaria, preventive measures and history of malaria fever. Three ml Venous (2-3ml) as well as peripheral blood (3-4 drops) samples of all patients were collected for microscopy and rapid diagnostic tests (RDTs). Out of total 477 patients with fever Venous and Capillary Blood RDTs methods detected 33(6.9%) and 30(6.3%) as the malaria positive while Venous and Capillary Blood Microscopy detected 30(6.1%) and 32(6.7%) cases respectively. Plasmodium Vivax infection was the most prevalent (87.9%) and majority (39.39%) of the cases occurred in the quarter, July to September. July to September is the peak season for malaria and P. Vivax (87.9%) is the predominant strain in Karachi. Venous and capillary blood are equally useful for malaria diagnosis however, convenience and less invasiveness may justify the preference of capillary blood over venous blood for early diagnosis of malaria.

Detection of Malaria Parasite Infection Among Children Attending General Hospital Kawo, Kaduna State, Nigeria

Malaria is one of the most common infectious diseases and a great public health problem worldwide. It is one of the world’s deadliest diseases affecting people particularly in tropical and subtropical regions of the world, especially in sub-Saharan Africa and Southeast Asia. This study was conducted to determine the prevalence of malarial parasite among children attending General Hospital Kawo, Kaduna State. A total of 250 blood sample were collected from children at random for malarial parasite infection using Rapid diagnostic test and microscopy. The result showed a prevalence of 77.6% using method of microscopy and 56.0% RDT. It was found that those between ages of 8-10 had the highest prevalence of 44.1%. Based on gender, male had the highest prevalence of 56.6%.The high prevalence of malaria infection recorded in this study may be attributed to the fact that the favourable environmental conditions enhance mosquito breeding and survival. More effort, proper environmental sanitations, legal dumping of waste are needed in the control of malaria in and within the study area, the parents also need enlightenment in the importance of early malaria diagnosis and control. Result from this study also indicates that the blood smear microscopy seems to be better than Rapid Diagnostic Test (RDT) for malaria diagnosis.

Luminescent diagnostics and quantitative assessment of malaria based on a lateral flow immunoassay with cdte quantum dots

A lateral flow immunoassay (LFI) is a simple, low-cost and rapid diagnostic tool to identify various diseases by detecting analytes such as antibodies, parasites, or other relevant biomarkers. However, most LFIs can only confirm the presence or absence of a target analyte being applied as qualitative diagnostic tools. In addtion, the conventional LFIs, which require visual inspection of the test line, may demonstrate insufficient sensitivity for the mild form or early detection of infections and for this reason, the result can be falsely negative. This study presents the results of employing thioglycolic acid capped CdTe quantum dots as a probe to enhance luminescence and subsequently detection sensitivity in LFIs for diagnosis of malaria. The chemical route synthesis of thioglycolic acid capped CdTe quantum dots was optimized systematically by probing optical properties of the quantum dots. These optimized quantum dots of thioglycolic acid capped CdTe have been conjugated with the anti-malaria antibodies against HRP2 protein (P. falciparum) and were then incorporated into LFIs. Further, an image processing code has been developed to carry out the quantification of malaria parasites in terms of the ratio on intensities of control and test lines on the LFIs. The results have been compared with those obtained using the standard, colloidal gold based LFIs. It has been demonstrated that sensitivity and lower detection limits of malaria under low parasite concentration increase significantly due to enhanced luminescence of control and test lines under UV light, owing to the presence of thioglycolic acid capped CdTe quantum dots. It has also been shown that the image processing based quantification of malaria will likely minimize the chances of false negative results under low parasite concentration and assist in early diagnosis of malaria.

Highly sensitive quasi-D-shaped photonic crystal fiber biosensor designed for the detection of RBC parasitized by Plasmodium falciparum for the early diagnosis of malaria

A simple quasi-D-shaped photonic crystal fiber (PCF)-based surface plasmon resonance biosensor is proposed for the early diagnosis of malaria that arises as a result of Plasmodium falciparum parasite development in erythrocytes in the human body. The flat surface of the D-shaped PCF is covered with a thin layer of TiO2 along with a gold layer. The finite element method (FEM) is used to numerically investigate the characteristics of the sensor. With the well-optimized set of parameters, the proposed sensor exhibits maximum spectral sensitivities of 42857.14 nm/RIU, 22105.26 nm/RIU, and 16206.90 nm/RIU with resolutions of 2.33 × 10−06 RIU, 4.52 × 10−06 RIU, and 6.17 × 10−06 RIU for ring, trophozoite, and schizont phases, respectively. The obtained amplitude sensitivities are 784.55 RIU−1, 491.02 RIU−1, and 407.99 RIU−1 and FOMs are 596.90 RIU−1, 423.98 RIU−1, and 341.63 RIU−1 for the three phases, respectively. Therefore, with the promising results and simplified practical realization, the proposed sensor can be an excellent candidate for the identification of Plasmodium falciparum phases in RBC for malaria diagnosis.

Computational Methods for Automated Analysis of Malaria Parasite Using Blood Smear Images: Recent Advances.

Malaria comes under one of the dangerous diseases in many countries. It is the primary reason for most of the causalities across the world. It is presently rated as a significant cause of the high mortality rate worldwide compared with other diseases that can be reduced significantly by its earlier detection. Therefore, to facilitate the early detection/diagnosis of malaria to reduce the mortality rate, an automated computational method is required with a high accuracy rate. This study is a solid starting point for researchers who want to look into automated blood smear analysis to detect malaria. In this paper, a comprehensive review of different computer-assisted techniques has been outlined as follows: (i) acquisition of image dataset, (ii) preprocessing, (iii) segmentation of RBC, and (iv) feature extraction and selection, and (v) classification for the detection of malaria parasites using blood smear images. This study will be helpful for: (i) researchers can inspect and improve the existing computational methods for early diagnosis of malaria with a high accuracy rate that may further reduce the interobserver and intra-observer variations; (ii) microbiologists to take the second opinion from the automated computational methods for effective diagnosis of malaria; and (iii) finally, several issues remain addressed, and future work has also been discussed in this work.

Malaria Diagnosis in Non-Endemic Settings: The European Experience in the Last 22 Years.

Accurate, prompt, and reliable tools for the diagnosis of malaria are crucial for tracking the successes or drawbacks of control and elimination efforts, and for future programs aimed at global malaria eradication. Although microscopy remains the gold standard method, the number of imported malaria cases and the risk of reappearance of autochthonous cases stimulated several laboratories located in European countries to evaluate methods and algorithms suited to non-endemic settings, where skilled microscopists are not always available. In this review, an overview of the field evaluation and a comparison of the methods used for the diagnosis of malaria by European laboratories is reported, showing that the development of numerous innovations is continuous. In particular, the combination of rapid diagnostic tests and molecular assays with microscopy represents a reliable system for the early diagnosis of malaria in non-endemic settings.

Correlation between CD 34 and CD 68 expression in placental malaria with maternal anemia.

Background:Malaria is the second most life-threatening infectious disease in Indonesia, causing approximately 1–3 million deaths annually. Histopathologic studies assessing CD 68 and CD 34 protein expression in placental malaria and its association with maternal anemia are essential to determine the prognosis of malaria in pregnancy.Materials and Methods:This cross-sectional study was carried out in 2017. Thirty biopsy samples of human placental tissue were obtained from Timika and Sumba, and ten normal biopsy samples were taken from the Pathological Anatomy Department of Cipto Mangunkusumo General Hospital as comparisons. CD 34 and CD 68 protein expressions were determined using immunohistochemistry, and the resulting data were analyzed using SPSS.Results:Average hemoglobin (Hb) level was 9.5 mg/dL, 11.5 mg/dL, and 9.9 mg/dL in acute infection, chronic infection, and latent infection, respectively. A positive correlation was found between CD 68 protein expression and maternal Hb level. No correlation was found between CD34 expression and maternal anemia.Conclusions:CD 68 expression in placental tissue biopsy from Timika and Sumba residents with placental malaria was shown to be positively correlated with maternal anemia. Immunohistochemical examination of CD 68 may play a role in the early diagnosis of malaria.

Maintaining focus on administering effective malaria treatment during the COVID-19 pandemic.

As September marks the start of the malaria season in South Africa (SA), it is essential that healthcare professionals consider both COVID- 19 and malaria when a patient who lives in or has recently travelled to a malaria area presents with acute febrile illness. Early diagnosis of malaria by either a rapid diagnostic test or microscopy enables prompt treatment with the effective antimalarial, artemether-lumefantrine, preventing progression to severe disease and death. Intravenous artesunate is the preferred treatment for severe malaria in both children and adults. Adding single low-dose primaquine to standard treatment is recommended in endemic areas to block onward transmission. Use of the highly effective artemisinin-based therapies should be limited to the treatment of confirmed malaria infections, as there is no clinical evidence that these antimalarials can prevent or treat COVID-19. Routine malaria case management services must be sustained, in spite of COVID-19, to treat malaria effectively and support SA's malaria elimination efforts.

Malaria Por Plasmodium Falciparum, evolución de un caso

La malaria es una enfermedad parasitaria con una amplia distribución en zonas tropicales y subtropicales, endémica en más de 100 países, producida por cuatro especies de plasmodios. Es una patología presente en zonas con abundantes mosquitos Anopheles, por lo que su prevalencia está directamente relacionada con las condiciones socioeconómicas y ambientales de los pacientes. Al tratarse de una enfermedad capaz de producir la muerte rápidamente, es necesario considerarla como una urgencia médica. En este contexto se presenta paciente masculino que acude al servicio de emergencias con un cuadro clínico cuyos síntomas hacen sospechar de un brote de malaria. El diagnóstico oportuno de malaria por Plasmodium falciparum permitió un abordaje óptimo del paciente al cabo de pocos días de realizada la prueba confirmatoria, el examen de gota gruesa y extensión

Study of haematological parameters in malaria: a prospective study

Background: A blood parasites plasmodia is responsible for malaria hence there are haematological alterations in malaria. The haematological changes that have been reported to accompany malaria include anaemia, thrombocytopenia, leucocytosis and leucopenia. Methods: Total 585 smear positive malaria cases were taken and various haematological parameters were studied. Results: Out of 585 smear positive cases, P. vivax was positive in 422 (72%) cases, while P. falciparum was positive in 160 (27%) cases and mixed infection was found in 3 (1%) cases. Anaemia was seen in 420 (71.79%) cases. Normocytic normochromic blood picture was the most common type in anaemic patients 223 (38.11%). Next common finding was Normocytic hypochromic RBCs 185 (31.62%). Microcytic hypochromic RBCs found in 152 (25.98%) cases. Thrombocytopenia was seen in total 490 (83.76%) of the patients. Moderate thrombocytopenia was more common and present in 409 (70%) of patients while Severe thrombocytopenia was seen in 81 (13.84%) of cases. In falciparum malaria thrombocytopenia was present in 90% of the patients while it was present in 81.27% of the patients in vivax malaria. 129 cases (22.05%) shows leucopoenia out of which 81 (13.84%) belong to P. vivax, 45 (7.69%) belong to P. falciparum and 3 (1%) were belong to mixed infection. Total Leucocyte Count was normal in 77.95 % of the patients. Conclusions: Various haematological findings can help in early diagnosis of malaria which is essential for timely and appropriate treatment which can limit the morbidity and prevent further complications.

Antimalarial drug sensitivity test: A new approach toward management of severity of Plasmodium falciparum

Background: Malaria despite everything represents a danger to the strength of occupants and voyagers in tropical nations. The Plasmodium species that are known to infect humans worldwide are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Of these, P. falciparum causes high morbidity and mortality, and it is during the asexual erythrocytic stages that the majority of the indications of malaria are shown. Aim: The aim of this study was to standardize an in vitro assay for antimalarial drug sensitivity of P. falciparum isolates. Materials and Methods: This prospective study was carried out over a period of 1 year from July 2014 to June 2015. We effectively completed in vitro drug susceptibility testing on 34 isolates of P. falciparum, which were collected from the patient admitted in Mahatma Gandhi Mission’s Teaching Hospital, Navi Mumbai, Maharashtra, India. We obtained the P. falciparum 3D7 strain from Haffkine Institute for Training, Research and Testing, and Indian Institute of Technology (IIT) Bombay, Mumbai, Maharashtra, India. World Health Organization (WHO) standard in vitro micro-test (Mark III) method was used for the assessment of the response of P. falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine, and artemisinin. The method was partially modified by replacement of leukocytes with AB serum in Roswell Park Memorial Institute (RPMI)-1640 medium. Results: The development of P. falciparum 3D7 chloroquine-sensitive strain from the ring stage to schizonts (not fewer than six merozoites) in control wells containing chloroquine was 100% recorded. This level of development in the control wells empowered fast minute assurance (5min for isolate per drug) of the Minimum inhibitory concentrations (MICs) of chloroquine. The test well shows the development from the ring stage to mature schizont stages were 67.65% in well containing chloroquine, 17.65% in well containing amodiaquine, and 14.71% in well containing sulfadoxine/pyrimethamine, it means these drugs are resistant to P. falciparum, and however, there is no development of the P. falciparum from the ring stage to mature schizont (with at least six merozoites) stage in the well containing artesunate, mefloquine and quinine were equal to the control well, it means these antimalarial drugs are sensitive to the P. falciparum. Conclusion: Antimalarial drug resistance has developed as a result of prescribing this medication without having malaria. Early diagnosis of malaria and drug sensitivity testing may prevent the emergence of resistance to antimalarial drugs. Monotherapy ought to be kept up a key decent way from; the combined antimalarial drug therapy may reduce the chances of emergence of antimalarial drug resistance.

English

English

Comparative Analysis of the Serological Reactivity of Individuals with Clinical History of Malaria using Two Different ELISA Tests.

Early diagnosis of malaria reduces disease, prevents deaths, and contributes to decreased malaria transmission. The use of specific and sensitive antigens in the execution of serological diagnostics may have an impact on the transmission of the disease. However, many individuals cannot be easily diagnosed by serological tests due to low levels of antibodies in the serum. Using two different Enzyme-Linked Immunosorbent Assay (ELISA) tests (a commercial and an in-house ELISA), a total of 365 serum samples from individuals with a clinical history of malaria were analyzed. From the serum samples analyzed, 192 (53%) samples from the commercial ELISA and 219 (60%) samples from the in-house ELISA presented positive serological reactivity to malaria. The concordance of the samples tested (n = 365) between both ELISAs was of 67% (n = 242), and with the negative control was 100% (n = 17). We demonstrated that the in-house ELISA showed high antigenic reactivity to Plasmodium falciparum antigens when compared with the commercial ELISA. The degree of concordance of both ELISAs suggested the possibility of existence of other P. falciparum antigens present in the crude extract of P. falciparum that are important in the serological response during malaria infection.

Designing malaria surveillance strategies for mobile and migrant populations in Nepal: a mixed-methods study

BackgroundAs malaria cases have declined throughout Nepal, imported cases comprise an increasing share of the remaining malaria caseload, yet how to effectively target mobile and migrant populations (MMPs) at greatest risk is not well understood. This formative research aimed to confirm the link between imported and indigenous cases, characterize high-risk MMPs, and identify opportunities to adapt surveillance and intervention strategies to them.MethodsThe study used a mixed-methods approach in three districts in far and mid-western Nepal, including (i) a retrospective analysis of passive surveillance data, (ii) a quantitative health facility-based survey of imported cases and their MMP social contacts recruited by peer-referral, and (iii) focus group (FG) discussions and key informant interviews (KIIs) with a subset of survey participants. Retrospective case data were summarised and the association between monthly indigenous case counts and importation rates in the previous month was investigated using Bayesian spatio-temporal regression models. Quantitative data from structured interviews were summarised to develop profiles of imported cases and MMP contacts, including travel characteristics and malaria knowledge, attitudes and practice. Descriptive statistics of the size of cases’ MMP social networks are presented as a measure of potential programme reach. To explore opportunities and barriers for targeted malaria surveillance, data from FGs and KIIs were formally analysed using a thematic content analysis approach.ResultsMore than half (54.1%) of malaria cases between 2013 and 2016 were classified as imported and there was a positive association between monthly indigenous cases (incidence rate ratio (IRR) 1.02 95% CI 1.01–1.03) and the previous month’s case importation rate. High-risk MMPs were identified as predominantly adult male labourers, who travel to malaria endemic areas of India, often lack a basic understanding of malaria transmission and prevention, rarely use ITNs while travelling and tend not to seek treatment when ill or prefer informal private providers. Important obstacles were identified to accessing Nepali MMPs at border crossings and at workplaces within India. However, strong social connectivity during travel and while in India, as well as return to Nepal for large seasonal festivals, provide opportunities for peer-referral-based and venue-based surveillance and intervention approaches, respectively.ConclusionsPopulation mobility and imported malaria cases from India may help to drive local transmission in border areas of far and mid-western Nepal. Enhanced surveillance targeting high-risk MMP subgroups would improve early malaria diagnosis and treatment, as well as provide a platform for education and intervention campaigns. A combination of community-based approaches is likely necessary to achieve malaria elimination in Nepal.