Skip to main content

Plasmodium malariae infections as a cause of febrile disease in an area of high Plasmodium falciparum transmission intensity in Eastern Uganda



Plasmodium falciparum is responsible for the vast majority of (severe) clinical malaria cases in most African settings. Other Plasmodium species often go undiagnosed but may still have clinical consequences.

Case presentation

Here, five cases of Plasmodium malariae infections from Eastern Uganda (aged 2–39 years) are presented. These infections were all initially mistaken for P. falciparum, but Plasmodium schizonts (up to 2080/µL) were identified by microscopy. Clinical signs included history of fever and mild anaemia.


These findings highlight the importance of considering non-falciparum species as the cause of clinical malaria. In areas of intense P. falciparum transmission, where rapid diagnostic tests that detect only P. falciparum antigens are commonly used, non-falciparum malaria cases may be missed.


In 2019, an estimated 229 million cases and 409,000 deaths due to malaria were reported globally [1]. Most of these occurred in sub-Saharan Africa, and were caused by Plasmodium falciparum. Thus, it is not surprising that P. falciparum remains the focus of malaria control programmes and most malaria research in Africa. Other Plasmodium species are endemic in sub-Saharan Africa, including Plasmodium ovale, Plasmodium vivax and Plasmodium malariae [2]. These non-falciparum species also cause clinical symptoms, and lead sometimes to severe consequences. Chronic infections with P. malariae may last for years, and can cause severe complications in approximately 3% of cases, including nephrotic syndrome, splenomegaly and anaemia [3, 4]. Moreover, P. malariae infections have been observed following treatment of P. falciparum infections with artemisinin-based combination therapy (ACT) [5, 6].

Different Plasmodium species can be differentiated by microscopy but this requires laboratory infrastructure and technical expertise. Infections with P. malariae are typically characterized by low parasitaemia [7] and the presence of rosette schizonts (daisy heads) and other parasite life-stages in the peripheral blood [8]. Plasmodium malariae is often missed or misdiagnosed by microscopy [9, 10]. However, polymerase chain reaction (PCR) suggests a non-negligible prevalence of P. malariae infection, either alone or mixed with other species, in population surveys in African countries, where prevalence of up to 12% has been reported [11, 12].

Rapid diagnostic tests (RDTs), which are simple to use and require minimal training, are increasingly being used to diagnose malaria. In Africa, most RDTs detect histidine-rich protein 2 (HRP2), an antigen that is specific for P. falciparum; use of these HRP2 RDTs is likely to contribute further to under-diagnosis of P. malariae infections.

Here, five cases are presented of symptomatic, uncomplicated P. malariae. These cases were coming from an area of intense P. falciparum transmission in Eastern Uganda and were initially misdiagnosed as P. falciparum by microscopy.

Case presentation

Patients described in this case report presented at either Masafu General Hospital or Nagongera Health Centre IV. Masafu General Hospital is a district referral facility in Busia District, an area in south-eastern Uganda that borders Kenya and Lake Victoria, where transmission of P. falciparum is perennial and intense [13]. Nagongera Health Centre IV in located in Nagongera sub-county in Tororo district. Nagongera sub-county is predominantly a rural setting, with traditionally very high malaria transmission [14]. Between 2019 and 2020 the total Outpatient Department treatment (OPD) attendance in Masafu General Hospital was 40,471, whilst in Nagongera Health Centre IV 18,624 [15].

In both settings, hospital and health centre, malaria diagnosis is routinely performed by HRP-2 based rapid diagnostic test (Standard Q Malaria Pf Ag, SD Biosensor, South Korea). However, as part of a research study on P. falciparum, clinic technicians all received formal microscopy training and thick and thin blood films were used for malaria diagnosis, instead of RDTs. Haemoglobin measurements were performed by Hemocue™ (haemoglobinometer, Angelholm, Sweden).

Case-patient 1

On January 30th 2020, a 15-year-old male presented to the OPD with history of fever, headache and joint pain for 3 days with no evidence of severe malaria or danger signs. At initial presentation, he had an axillary temperature of 36.8 °C and his physical examination was within normal limits. Malaria parasites were reported on the thick and thin blood film. By thick blood smear, total parasite count was 896 parasites/μL; malaria schizonts were observed at 176 parasites/μL (and included in the total parasite density estimate). Haemoglobin concentration was 12.3 g/dL. The patient was diagnosed with P. falciparum malaria.

Case-patient 2

On February 19th 2020, a 20-year-old female presented to the OPD with history of fever for 3 days and headache, weakness and joint pain for 2 days. Her temperature on admission was 37.2 °C and her physical examination did not show abnormalities. After reading the thick blood film, malaria infection was diagnosed with a total of 2288 parasites/μL (2080 schizonts/μL). Mild anaemia was detected with haemoglobin of 10.8 g/dL. The patient was diagnosed with P. falciparum malaria.

Case-patient 3

On February 27th 2020, a 39-year-old female presented to the OPD with history of fever and joint pain for 2 days and headache, weakness and low back pain for 4 days. Her axillary temperature on admission was 37.0 °C and she was diagnosed with uncomplicated P. falciparum malaria based on a thick blood film with 336 parasites/μL (272 schizonts/μL). Mild anaemia was detected with haemoglobin of 9.5 g/dL.

Case-patient 4

On March 3rd 2020, a 2-year-old boy presented to the outpatient department with persistent vomiting, anorexia and headache. He had an axillary temperature of 38.3 °C. Microscopy slides were interpreted as P. falciparum/P. malariae mixed infection based on presence of 32 ring-stage parasites/μL and 520 schizonts/μL for P. falciparum and one trophozoite “band form” for P. malariae. Haemoglobin was 7.8 g/dL. The patient was diagnosed with uncomplicated mixed species malaria infection.

Case-patient 5

On April 29th 2020, a 14-year-old male with mild symptoms of fever, headache, joint pain and anorexia presented to Nagongera Health Center IV, Tororo district. He had an axillary temperature of 37.4 °C. Malaria was diagnosed by microscopy with 4016 parasites/μL (96 schizonts/μL). Haemoglobin was 11.1 g/dL. The patient was diagnosed with uncomplicated P. falciparum malaria.

All five cases were treated for uncomplicated P. falciparum infection according to national Ugandan treatment guidelines with artemether lumefantrine 20 mg/120 mg twice per day for 3 days and discharged home [16]. Although there are some reports of persisting P. malariae parasites after artemether-lumefantrine [5, 17], suggestive of reduced treatment efficacy for this species, most available data support the use of artemether-lumefantrine for P. malariae [18, 19]. Thick and thin smears were re-read by expert microscopists and representative images taken (Fig. 1). Parasite DNA was extracted from fixed Giemsa-stained thick and thin smears using a modified protocol for isolating genomic DNA from dried blood spots (QIAamp®DNA Micro kit, cat numb. 56304) [20]. Plasmodium speciation was performed using modified methods described by Snounou et al. [21]. Plasmodium malariae parasite DNA was detected in material from all slides; P. falciparum was also detected for case 4 (Table 1).

Fig. 1
figure 1

Features of Plasmodium malariae parasites in thin and thick blood smears. A typical band form mature trophozoite. × 10 ocular, × 100 magnification, thin smear. B young trophozoite. Pictures A, B taken with a smart phone camera. C, D: “daisy shape” schizonts. × 10 ocular, × 100 magnification, thin film. E, F: schizonts. × 10 ocular, × 100 magnification, thick smear. CF: Pictures taken with Axio Cam MRc-5, Zeiss, Germany. Blood smears were stained with 10% Giemsa solution

Table 1 Patient characteristics at presentation

Discussion and conclusions

In these five symptomatic malaria cases, P. malariae was the most likely cause of the clinical symptoms. All cases were classified as uncomplicated malaria, although they presented with anaemia, a condition that is often associated with chronic infection with P. malariae [3, 4]. In four cases, P. falciparum infection was initially diagnosed. Only after an expert microscopist reviewed the blood slides was P. malariae identified as the infecting species. The presence of symptomatic P. malariae mono-infections has implications for the use of HRP-2 RDTs. In both of the study settings, HRP-2 RDTs are routinely used to diagnose malaria. Since non-falciparum Plasmodium species do not express HRP-2 antigen, at least four of these cases would presumably have been missed in routine practice and would not have received malaria treatment.

he cases presented here all reported to the health facilities within a relatively short 13-week period. No effort is made to systematically investigate for non-falciparum malaria among individuals presenting with suspected malaria under standard care. This suggests that a considerable number of patients may present with uncomplicated non-falciparum malaria in this setting. The P. malariae cases reported here were older than typical P. falciparum cases in the area [22], which could be the consequence of a lower force of infection of non-falciparum malaria, older age at first infection, and higher average age at clinical presentation. However, other studies found P. malariae infections predominantly in children [23, 24], further highlighting the need for research into the clinical burden of non-falciparum malaria in African settings. Identifying the species of Plasmodium infections using molecular diagnostic techniques is needed to quantify the burden of malaria in areas where multiple species may be present.

Availability of data and materials

Not applicable. All data are included in the manuscript.



Histidine-rich protein 2


Outpatient department treatment


Polymerase chain reaction


Rapid diagnostic tests


  1. WHO. World malaria report 2020: 20 years of global progress and challenges. Geneva: World Health Organization; 2020.

    Google Scholar 

  2. Jamison DT, Feachem RG, Makgoba MW, Bos ER, Baingana FK, Hofman KJ, et al. Disease and mortality in sub-Saharan Africa. 2nd ed. Washington (DC): The International Bank for Reconstruction and Development/The World Bank; 2006.

    Google Scholar 

  3. Langford S, Douglas NM, Lampah DA, Simpson JA, Kenangalem E, Sugiarto P, et al. Plasmodium malariae infection associated with a high burden of anemia: a hospital-based surveillance study. PLoS Negl Trop Dis. 2015;9: e0004195.

    Article  Google Scholar 

  4. Vinetz JM, Li J, McCutchan TF, Kaslow DC. Plasmodium malariae infection in an asymptomatic 74-year-old Greek woman with splenomegaly. N Engl J Med. 1998;338:367–71.

    Article  CAS  Google Scholar 

  5. Betson M, Sousa-Figueiredo JC, Atuhaire A, Arinaitwe M, Adriko M, Mwesigwa G, et al. Detection of persistent Plasmodium spp. infections in Ugandan children after artemether-lumefantrine treatment. Parasitology. 2014;141:1880–90.

    Article  CAS  Google Scholar 

  6. Lubis IND, Wijaya H, Lubis M, Lubis CP, Beshir KB, Staedke SG, et al. Recurrence of Plasmodium malariae and P. falciparum following treatment of uncomplicated malaria in North Sumatera with dihydroartemisinin-piperaquine or artemether-lumefantrine. Open Forum Infect Dis. 2020;7: ofaa116.

    Article  Google Scholar 

  7. Oriero EC, Amenga-Etego L, Ishengoma DS, Amambua-Ngwa A. Plasmodium malariae, current knowledge and future research opportunities on a neglected malaria parasite species. Crit Rev Microbiol. 2021;47:44–56.

    Article  Google Scholar 

  8. Collins WE, Jeffery GM. Plasmodium malariae: parasite and disease. Clin Microbiol Rev. 2007;20:579–92.

    Article  Google Scholar 

  9. Bharti PK, Chand SK, Singh MP, Mishra S, Shukla MM, Singh R, et al. Emergence of a new focus of Plasmodium malariae in forest villages of district Balaghat, central India: implications for the diagnosis of malaria and its control. Trop Med Int Health. 2013;18:12–7.

    Article  Google Scholar 

  10. Savargaonkar D, Shah N, Das MK, Srivastava B, Valecha N. Plasmodium malariae infection: a case of missed diagnosis. J Vector Borne Dis. 2014;51:149–51.

    PubMed  Google Scholar 

  11. Noland GS, Graves PM, Sallau A, Eigege A, Emukah E, Patterson AE, et al. Malaria prevalence, anemia and baseline intervention coverage prior to mass net distributions in Abia and Plateau States, Nigeria. BMC Infect Dis. 2014;14:168.

    Article  Google Scholar 

  12. Doctor SM, Liu Y, Anderson OG, Whitesell AN, Mwandagalirwa MK, Muwonga J, et al. Low prevalence of Plasmodium malariae and Plasmodium ovale mono-infections among children in the Democratic Republic of the Congo: a population-based, cross-sectional study. Malar J. 2016;15:350.

    Article  Google Scholar 

  13. Kajubi R, Ochieng T, Kakuru A, Jagannathan P, Nakalembe M, Ruel T, et al. Monthly sulfadoxine-pyrimethamine versus dihydroartemisinin-piperaquine for intermittent preventive treatment of malaria in pregnancy: a double-blind, randomised, controlled, superiority trial. Lancet. 2019;393:1428–39.

    Article  CAS  Google Scholar 

  14. Nankabirwa JI, Arinaitwe E, Rek J, Kilama M, Kizza T, Staedke SG, et al. Malaria transmission, infection, and disease following sustained indoor residual spraying of insecticide in Tororo, Uganda. Am J Trop Med Hyg. 2020;103:1525–33.

    Article  Google Scholar 

  15. Ministry of Health. Annual health sector performance report 2019-2020 FY-1. Kampala, Uganda.

  16. Ministry of Health. Uganda clinical guidelines 2016—National guidelines for management of common conditions. Kampala, Uganda. 2016.

  17. Calleri G, Balbiano R, Caramello P. Are artemisinin-based combination therapies effective against Plasmodium malariae? J Antimicrob Chemother. 2013;68:1447–8.

    Article  CAS  Google Scholar 

  18. Visser BJ, Wieten RW, Kroon D, Nagel IM, Belard S, van Vugt M, et al. Efficacy and safety of artemisinin combination therapy (ACT) for non-falciparum malaria: a systematic review. Malar J. 2014;13:463.

    Article  Google Scholar 

  19. Groger M, Veletzky L, Lalremruata A, Cattaneo C, Mischlinger J, Zoleko-Manego R, et al. Prospective clinical trial assessing species-specific efficacy of artemether-lumefantrine for the treatment of Plasmodium malariae, Plasmodium ovale, and mixed Plasmodium malaria in Gabon. Antimicrob Agents Chemother. 2018;62:e01758-e1817.

    Article  CAS  Google Scholar 

  20. Cnops L, Van Esbroeck M, Bottieau E, Jacobs J. Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR. Malar J. 2010;9:370.

    Article  CAS  Google Scholar 

  21. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, et al. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol. 1993;61:315–20.

    Article  CAS  Google Scholar 

  22. Zehner N, Adrama H, Kakuru A, Andra T, Kajubi R, Conrad M, et al. Age-related changes in malaria clinical phenotypes during infancy are modified by sickle cell trait. Clin Infect Dis. 2021.

    Article  PubMed  Google Scholar 

  23. Lo E, Nguyen K, Nguyen J, Hemming-Schroeder E, Xu J, Etemesi H, et al. Plasmodium malariae prevalence and csp gene diversity, Kenya, 2014 and 2015. Emerg Infect Dis. 2017;23:601–10.

    Article  Google Scholar 

  24. Roucher C, Rogier C, Sokhna C, Tall A, Trape JF. A 20-year longitudinal study of Plasmodium ovale and Plasmodium malariae prevalence and morbidity in a West African population. PLoS ONE. 2014;9: e87169.

    Article  Google Scholar 

Download references


We thank the patients who participated in this study. We further thank Kjerstin Lanke for molecular assays and Marga de Vegte-Bolmer for support in microscopy.


This is supported by the National Institute of Allergy and Infectious Diseases (NIAID) as part of the International Centers of Excellence in Malaria Research (ICEMR) program (U19AI089674) and the Bill & Melinda Gates Foundation (INDIE OPP1173572). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. TB and CA are further supported by a European Research Council-Consolidator Grant to TB (ERC-CoG 864180; QUANTUM).

Author information

Authors and Affiliations



DA, BO, JO read the blood smears and counted the parasites; OM, DA, CA wrote the draft of the manuscript; SGS and TB conceived and prepared the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Teun Bousema.

Ethics declarations

Ethics approval and consent to participate

Data was collected under a protocol that was approved by the School of Biomedical Sciences Research and Ethics Committee Makerere University College of Health Sciences (SOM-REC IRB; Protocol # SBS-363), UNCST (Number HS-2110), the Human Research Protection Program Institutional Review Board of the University of California-San Francisco (IRB # 16-2056), the London School of Hygiene & Tropical Medicine and the Liverpool School of Tropical Medicine (15-025).

Consent for publication

All authors have given their consent for this publication.

Competing interests

The authors have declared that no competing interests exist.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ayo, D., Odongo, B., Omara, J. et al. Plasmodium malariae infections as a cause of febrile disease in an area of high Plasmodium falciparum transmission intensity in Eastern Uganda. Malar J 20, 425 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: