Plasmodium vivax and Plasmodium falciparum mixed infections in human and mosquito hosts: the impact of multi-species infection on parasite densities and transmission to mosquitoes

Plasmodium vivax and Plasmodium falciparum mixed infections in human and mosquito hosts: the impact of multi-species infection on parasite densities and transmission to mosquitoes | medRxiv Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Plasmodium vivax and Plasmodium falciparum mixed infections in human and mosquito hosts: the impact of multi-species infection on parasite densities and transmission to mosquitoes Wakweya Chali , Legesse Alamirie Ejigu , Tigist Atele , Solomon Sisay , Gutema Jebessa , Getnet Habtamu , Melat Abdo , Amanuel Shimelash , Mulugeta Demisse , Migbaru Keffale Bezabih , Desalegn Nibret , Addisu Gizat , Kebede Getachew , Kassahun Abi , Ayalew Jejaw Zeleke , Banchayehu Getnet , Zewdu Solomon , Teressa Bayena , Tirhas Endale , Meskele Dechasa , Fekadu Massebo , Fikregabrail Aberra Kassa , Sagni Challi , Hassen Mamo , Chris Drakeley , View ORCID Profile Teun Bousema , View ORCID Profile Fitsum G. Tadesse doi: https://doi.org/10.64898/2026.03.16.26348472 Wakweya Chali 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia 2 Radboud University Medical Centre , Nijmegen, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Legesse Alamirie Ejigu 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia 3 London School of Hygiene and Tropical Medicine , London, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tigist Atele 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Solomon Sisay 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gutema Jebessa 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Getnet Habtamu 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Melat Abdo 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amanuel Shimelash 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mulugeta Demisse 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Migbaru Keffale Bezabih 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia 2 Radboud University Medical Centre , Nijmegen, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Desalegn Nibret 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Addisu Gizat 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kebede Getachew 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kassahun Abi 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ayalew Jejaw Zeleke 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia 6 Department of Medical Parasitology, School of Biomedical and Laboratory Sciences, University of Gondar , Gondar, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Banchayehu Getnet 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia 6 Department of Medical Parasitology, School of Biomedical and Laboratory Sciences, University of Gondar , Gondar, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zewdu Solomon 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Teressa Bayena 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tirhas Endale 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Meskele Dechasa 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Fekadu Massebo 4 Department of Biology, Arba Minch University , Arba Minch, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Fikregabrail Aberra Kassa 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sagni Challi 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hassen Mamo 5 Department of Microbial Sciences and Genetics, College of Natural and Computational Sciences, Addis Ababa University , Ethiopia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chris Drakeley 3 London School of Hygiene and Tropical Medicine , London, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Teun Bousema 2 Radboud University Medical Centre , Nijmegen, The Netherlands 3 London School of Hygiene and Tropical Medicine , London, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Teun Bousema Fitsum G. Tadesse 1 Armauer Hansen Research Institute , Addis Ababa, Ethiopia 2 Radboud University Medical Centre , Nijmegen, The Netherlands 3 London School of Hygiene and Tropical Medicine , London, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fitsum G. Tadesse For correspondence: fitsum.tadesse{at}ahri.gov.et Abstract Full Text Info/History Metrics Data/Code Preview PDF Abstract In co-endemic regions, mixed Plasmodium vivax (Pv) and Plasmodium falciparum (Pf) infections are commonly reported. How mixed species infections compare to single species infections in terms of parasite densities and transmission to mosquitoes is incompletely understood. Parasitemia, gametocytemia, and mosquito infectivity were evaluated among Pv mono-infections (n=284), Pf mono-infections (n=150), and mixed- Pv-Pf infections (n=77) recruited at four Ethiopian health facilities. Parasitemia and gametocytemia were quantified in patient blood samples by qPCR. Mosquito infectivity was assessed using direct membrane feeding assays (DMFA), with Plasmodium species confirmation by PCR. Pf gametocyte prevalence was lower in mixed-infections (64.5%, 49/76) compared with mono-infections (91.8%, 135/147); among gametocyte-positive parasite carriers gametocyte density was also lower in mixed-infections ( p <0.001). Pv gametocyte prevalence was similar in mono- and mixed-infections despite lower asexual parasite density in mixed infections ( p <0.001). Statistically significant positive correlations between asexual parasitemia and gametocytemia were observed in mono-infections ( p <0.001), but not in mixed-species infections ( p =0.120 for Pv ; and p =0.570 for Pf ). Transmission to mosquitoes was high across infections and infection combinations. Among infectious Pv-Pf parasite carriers, 56.3% (27/48) transmitted both species, often with both species being transmitted to individual mosquitoes. The associations between (species specific) gametocyte density and mosquito infection rates was unaffected by the concurrent presence of the other Plasmodium species. Although mixed-species infections may have different parasite and gametocyte densities compared to mono-infections, we observed no evidence for competition between species in mosquitoes. Because mixed infections are often undetected, they represent a hidden risk for sustaining malaria transmission. Author Summary Malaria remains a major public health challenge in Ethiopia, where Plasmodium vivax and Plasmodium falciparum often occur together in the same patient. While mixed-species infections are common, little is known about how they compare to single-species infections in terms of parasite levels and transmission to mosquitoes. In this study, we examined patients with P. vivax , P. falciparum , or mixed infections across four health facilities. We measured parasite and gametocyte densities in blood samples and tested whether patients could infect mosquitoes. We found that P. falciparum gametocyte levels were lower in mixed infections compared to single-species infections, while P. vivax gametocyte levels were similar regardless of infection type. Importantly, patients with mixed infections were still highly infectious to mosquitoes, and many transmitted both parasite species simultaneously. These findings show that mixed infections, which are often missed by routine diagnosis, can contribute substantially to malaria transmission. Recognizing and addressing mixed-species infections is therefore critical for malaria control and elimination efforts. Competing Interest Statement The authors have declared no competing interest. Clinical Trial NA Clinical Protocols NA Funding Statement Yes Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Not Applicable The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval was obtained from the AHRI/ALERT Ethics Review Committee (Ref. No. P042/18, and Ref. No. PO-058-24), the National Research Ethics Review Committee (Ref. No. MoSHE/RD/141/1097/19), and the London School of Hygiene and Tropical Medicine (LSHTM) (Ref. No. 22518). Before sample collection, all participants and/or the parents/guardians of the children provided written informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Not Applicable I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Not Applicable I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Not Applicable Data and code availability All the data used in the paper will be made available on Dryad (linked with the ORCID: https://orcid.org/0000-0003-1931-1442 ). Raw data from the study will be available in the future upon written request to the corresponding author following the signing of the data-sharing agreement, abiding by institutional and international data-sharing guidelines. The R codes used to run the analyses reported in this study will be made available at (GitHub link). Copyright The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . Back to top Previous Next Posted March 18, 2026. 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Share Plasmodium vivax and Plasmodium falciparum mixed infections in human and mosquito hosts: the impact of multi-species infection on parasite densities and transmission to mosquitoes Wakweya Chali , Legesse Alamirie Ejigu , Tigist Atele , Solomon Sisay , Gutema Jebessa , Getnet Habtamu , Melat Abdo , Amanuel Shimelash , Mulugeta Demisse , Migbaru Keffale Bezabih , Desalegn Nibret , Addisu Gizat , Kebede Getachew , Kassahun Abi , Ayalew Jejaw Zeleke , Banchayehu Getnet , Zewdu Solomon , Teressa Bayena , Tirhas Endale , Meskele Dechasa , Fekadu Massebo , Fikregabrail Aberra Kassa , Sagni Challi , Hassen Mamo , Chris Drakeley , Teun Bousema , Fitsum G. Tadesse medRxiv 2026.03.16.26348472; doi: https://doi.org/10.64898/2026.03.16.26348472 Share This Article: Copy Citation Tools Plasmodium vivax and Plasmodium falciparum mixed infections in human and mosquito hosts: the impact of multi-species infection on parasite densities and transmission to mosquitoes Wakweya Chali , Legesse Alamirie Ejigu , Tigist Atele , Solomon Sisay , Gutema Jebessa , Getnet Habtamu , Melat Abdo , Amanuel Shimelash , Mulugeta Demisse , Migbaru Keffale Bezabih , Desalegn Nibret , Addisu Gizat , Kebede Getachew , Kassahun Abi , Ayalew Jejaw Zeleke , Banchayehu Getnet , Zewdu Solomon , Teressa Bayena , Tirhas Endale , Meskele Dechasa , Fekadu Massebo , Fikregabrail Aberra Kassa , Sagni Challi , Hassen Mamo , Chris Drakeley , Teun Bousema , Fitsum G. Tadesse medRxiv 2026.03.16.26348472; doi: https://doi.org/10.64898/2026.03.16.26348472 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Infectious Diseases (except HIV/AIDS) Subject Areas All Articles Addiction Medicine (637) Allergy and Immunology (917) Anesthesia (348) Cardiovascular Medicine (4974) Dentistry and Oral Medicine (493) Dermatology (426) Emergency Medicine (674) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1682) Epidemiology (16207) Forensic Medicine (36) Gastroenterology (1238) Genetic and Genomic Medicine (7250) Geriatric Medicine (751) Health Economics (1092) Health Informatics (5262) Health Policy (1463) Health Systems and Quality Improvement (1825) Hematology (599) HIV/AIDS (1375) Infectious Diseases (except HIV/AIDS) (16466) Intensive Care and Critical Care Medicine (1199) Medical Education (696) Medical Ethics (156) Nephrology (747) Neurology (7552) Nursing (377) Nutrition (1120) Obstetrics and Gynecology (1293) Occupational and Environmental Health (1038) Oncology (3730) Ophthalmology (1087) Orthopedics (406) Otolaryngology (463) Pain Medicine (499) Palliative Medicine (143) Pathology (720) Pediatrics (1852) Pharmacology and Therapeutics (752) Primary Care Research (784) Psychiatry and Clinical Psychology (6099) Public and Global Health (9957) Radiology and Imaging (2515) Rehabilitation Medicine and Physical Therapy (1506) Respiratory Medicine (1264) Rheumatology (657) Sexual and Reproductive Health (799) Sports Medicine (595) Surgery (799) Toxicology (109) Transplantation (318) Urology (307)

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