{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T17:04:27Z","timestamp":1789578267129,"version":"build-2803163510"},"update-to":[{"DOI":"10.12688\/f1000research.173540.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000}},{"DOI":"10.12688\/f1000research.173540.2","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000}}],"reference-count":23,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns4:p>\n                    Background Malaria is a life-threatening vector-borne disease caused by five species of the genus\n                    <ns4:italic>Plasmodium (P.)<\/ns4:italic>\n                    It can present as mixed infections that remain underreported and pose diagnostic, therapeutic and prognostic challenges. The objective of the present study was to describe the epidemiological, clinical, and biological features of four cases of mixed\n                    <ns4:italic>Plasmodium<\/ns4:italic>\n                    infection. Methods This retrospective descriptive study included cases of mixed\n                    <ns4:italic>Plasmodium<\/ns4:italic>\n                    infections diagnosed at the Parasitology Laboratory of the Military Hospital of Tunis between 2022 and 2025. Diagnosis was established using May\u2013Gr\u00fcnwald\u2013Giemsa\u2013stained thin blood smears, Giemsa\u2013stained thick smears, and rapid diagnostic tests.\n                    <ns4:italic>Plasmodium<\/ns4:italic>\n                    species were identified according to standard microscopic morphological criteria. Results We report four cases of mixed\n                    <ns4:italic>Plasmodium<\/ns4:italic>\n                    infection diagnosed in military personnel returning from deployment in Central African Republic. The patients were aged 31\u201352 years (mean age 38.8 years) with sex ratio 3. They presented within 10\u201330 days post-return with fever (38\u201342\u00b0C) associated with headache, myalgia, abdominal pain, and\/or vomiting. All had adhered to malaria chemoprophylaxis, and three reported no prior malaria episodes, while one patient had a history of six previous attacks. Rapid diagnostic tests were positive for pan-Plasmodium pLDH in all cases; two patients were also positive for HRP2. Peripheral blood smears confirmed mixed infections. Parasitemia was generally low (&lt;1% in three cases, 1% in one case). Treatment consisted of artemether\u2013lumefantrine (Coartem\n                    <ns4:sup>\u00ae<\/ns4:sup>\n                    ) for all patients, combined with primaquine for those with\n                    <ns4:italic>P. ovale<\/ns4:italic>\n                    infection. Clinical evolution was favorable in all cases, and follow-up blood smears on days 3, 7, and 28 confirmed parasitological clearance, with only rare degenerated trophozoites observed in one patient on day 3. Conclusion Mixed\n                    <ns4:italic>Plasmodium<\/ns4:italic>\n                    infections can influence disease severity, treatment outcomes, and drug resistance. Accurate diagnosis and radical therapy are keys to preventing relapses.\n                  <\/ns4:p>","DOI":"10.12688\/f1000research.173540.3","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T06:59:08Z","timestamp":1789455548000},"page":"1389","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Mixed Plasmodium Infections: Report of Four Cases from the Military Hospital of Tunis"],"prefix":"10.12688","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1720-2381","authenticated-orcid":false,"given":"Latifa","family":"Mtibaa","sequence":"first","affiliation":[{"name":"Laboratory of Parasitology Tunis Tunis 1008 H\u00f4pital Militaire Principal d'Instruction de Tunis"},{"name":"Tunis Tunis University of Tunis El Manar Faculty of Medicine of Tunis"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-3358-4439","authenticated-orcid":false,"given":"Souha","family":"Hannachi","sequence":"additional","affiliation":[{"name":"Tunis Tunis University of Tunis El Manar Faculty of Medicine of Tunis"},{"name":"Infectious disease Tunis Tunis H\u00f4pital Militaire Principal d'Instruction de Tunis"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zeinab","family":"Denden","sequence":"additional","affiliation":[{"name":"Tunis Tunis University of Tunis El Manar Faculty of Medicine of Tunis"},{"name":"Infectious disease Tunis Tunis H\u00f4pital Militaire Principal d'Instruction de Tunis"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rym","family":"Abid","sequence":"additional","affiliation":[{"name":"Tunis Tunis University of Tunis El Manar Faculty of Medicine of Tunis"},{"name":"Infectious disease Tunis Tunis H\u00f4pital Militaire Principal d'Instruction de Tunis"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riadh","family":"Battikh","sequence":"additional","affiliation":[{"name":"Tunis Tunis University of Tunis El Manar Faculty of Medicine of Tunis"},{"name":"Infectious disease Tunis Tunis H\u00f4pital Militaire Principal d'Instruction de Tunis"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Boutheina","family":"Jemli","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitology Tunis Tunis 1008 H\u00f4pital Militaire Principal d'Instruction de Tunis"},{"name":"Monastir Monastir University of Monastir Faculty of Pharmacy of Monastir"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2026,9,8]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"43-44","DOI":"10.4103\/jacm.jacm_36_21","article-title":"Mixed infection of Plasmodium vivax and Plasmodium falciparum diagnosed by polymerase chain 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status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.191370.r448073, Manju Rahi, Indian Council of Medical Research\u2014Vector Control Research Centre (ICMR-VCRC), Puducherry, Puducherry, 04 Feb 2026, version 1, 1 approved with reservations","URL":"https:\/\/f1000research.com\/articles\/14-1389\/v1#referee-response-448073","order":0,"name":"referee-response-448073","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Latifa Mtibaa<\/b>; \n<i>Posted: 09 Mar 2026<\/i>; \n<b>Response to reviewer 1<\/b> We sincerely thank Dr. Manju Rahi for her careful review of our manuscript and for her constructive and insightful comments. We have revised the manuscript accordingly and respond point-by-point below. \n<b>Comment 1<\/b> \n<i>Why were these infections associated with low parasitaemia despite symptoms? Which species were dominant? What was the proportion of mono-infections among the remaining patients?<\/i> \n<b>Response:<\/b> We agree this point required clarification. Low parasitaemia despite symptoms may be explained by partial immunity related to repeated exposure in endemic areas, prior malaria episodes (notably in patient 3), and chemoprophylaxis use, which may attenuate parasite multiplication without preventing infection. In our series, \n<i>Plasmodium falciparum<\/i> was morphologically dominant in cases 1, 3, and 4, whereas \n<i>Plasmodium ovale<\/i> predominated in case 2. Among the remaining 171 imported malaria cases, species distribution was as follows:  \n<i>Plasmodium falciparum<\/i>: 40% \n<i>Plasmodium ovale<\/i>: 57% \n<i>Plasmodium malariae<\/i>: 3%  These data have now been added to the Results section. \n<b><b>Comment 2<\/b><\/b> \n<i>Treatment history of patient 3 (six prior relapses).<\/i> \n<b>Response:<\/b> We have added detailed treatment history for patient 3. Previous episodes were treated with artemisinin-based combination therapy without primaquine in earlier episodes, which may explain relapses related to dormant liver hypnozoites. This clarification has been incorporated into the Results section. \n<b><b>Comment 3<\/b><\/b> \n<i>Chemoprophylaxis details.<\/i> \n<b>Response:<\/b> All four patients received malaria chemoprophylaxis with doxycycline (100 mg once daily), initiated 1\u20132 days before travel to endemic areas, continued throughout deployment in Central Africa, and maintained for four weeks after return, in accordance with current WHO and Tunisian military health recommendations. Adherence was assessed through structured medical interviews upon return; compliance was self-reported and could not be objectively verified. In addition to chemoprophylaxis, preventive measures included the use of insecticide-treated bed nets, topical mosquito repellents (DEET-based), and protective clothing. These details have been added in the Methods section. \n<b><b>Comment 4<\/b><\/b> Typographical error corrected (\u201cBlack arrow\u201d). \n<b><b>Comment 5<\/b><\/b> Standardisation of drug formatting. \n<b>Response:<\/b> We have standardised the use of \u201cCoartem\n<sup>&reg;<\/sup>\u201d throughout the manuscript. \n<b><b>Comment 6<\/b><\/b> Diagnostic methods details and RDT brand. \n<b>Response:<\/b> We have expanded the Methods section to include:  Morphological criteria for species identification (size and shape of trophozoites, Sch&uuml;ffner\u2019s stippling, band forms, schizont morphology, gametocyte characteristics). Rapid diagnostic tests were performed using the \n<b>STANDARD\u2122 Q Malaria P.f\/Pan Ag (SD Biosensor&reg;)<\/b>\n<b>,<\/b> which detects histidine-rich protein 2 (HRP2) specific for \n<i>Plasmodium falciparum<\/i> and pan-\n<i>Plasmodium<\/i> lactate dehydrogenase (pLDH). Tests were conducted according to the manufacturer\u2019s instructions.  \n<b><b>Comment 7<\/b><\/b> IRB approval. \n<b>Response:<\/b> An ethics statement has now been added to the Methods section. As this was a retrospective study using fully anonymised patient data, formal Institutional Review Board approval was not required according to institutional regulations. The study was conducted in accordance with the principles of the Declaration of Helsinki. \n<b>Comment 8<\/b> Prevalence correction. \n<b>Response:<\/b> We thank the reviewer for noting this. The prevalence has been corrected to 2.3% (4\/175), and we clearly state that the denominator refers to imported malaria cases. \n<b><b>Comment 9<\/b><\/b> Sex ratio correction. \n<b>Response:<\/b> The sentence has been revised to: \u201cPatients were aged 31\u201352 years (mean age 38.8 years) with a sex ratio of 3 M: 1 F.\u201d \n<b><b>Comment 10<\/b><\/b> Primaquine safety and G6PD testing. \n<b>Response:<\/b> We have clarified that patients receiving primaquine were monitored by serial complete blood counts (CBC) to detect any signs of hemolysis. No quantitative G6PD testing was performed. During treatment, no hemolysis or significant changes in hemoglobin levels were observed. Safety monitoring details have been added to the Methods and Results sections. \n<b><b>Comment 11<\/b><\/b> WHO 2025 update. \n<b>Response:<\/b> We have updated the manuscript to reflect the latest WHO 2025 malaria treatment guidelines, particularly regarding the use of ACTs and radical cure recommendations for relapsing species \n<b><b>Comment 12<\/b><\/b> Strengthening epidemiological background. \n<b>Response:<\/b> Thank you for this comment. We have strengthened the epidemiological background in both the Introduction and Discussion sections. Updated WHO 2023 data were included, highlighting that the African Region accounts for 94% of global malaria cases and 95% of malaria-related deaths. We also provided specific context for the Central African Republic to situate the study population. Additionally, we reinforced the relevance of mixed \n<i>Plasmodium<\/i> infections by discussing their prevalence, diagnostic challenges, and potential clinical implications in high-burden regions. \n<b><b>Comment 13<\/b><\/b> Preventive measures and adherence details. \n<b>Response:<\/b> Additional details regarding chemoprophylaxis regimens, adherence assessment, and personal preventive measures have been added in the Methods section. \n<b><b>Comment 14\u201315<\/b><\/b> Overgeneralisation in conclusion. \n<b>Response:<\/b> We have revised the conclusion to avoid broad generalisations. The revised conclusion now emphasises that our findings are limited to a small case series and should be interpreted cautiously, without extrapolation beyond the presented cases. \n<b><b>Comment 16<\/b><\/b> Absence of molecular confirmation. \n<b>Response:<\/b> Thank you for this comment. We acknowledge that molecular methods could improve the confirmation of mixed \n<i>Plasmodium<\/i> infections. However, these techniques were not available in our laboratory during the study period. This limitation has now been clearly acknowledged in the Discussion section. We thank the reviewer again for her valuable comments, which have significantly strengthened our manuscript.","URL":"https:\/\/f1000research.com\/articles\/14-1389\/v3#referee-comment-15616","order":1,"name":"referee-comment-15616","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.209513.r519191, Felix Zoiku, University of Ghana, Accra, 16 Sep 2026, version 3, indexed","URL":"https:\/\/f1000research.com\/articles\/14-1389\/v3#referee-response-519191","order":2,"name":"referee-response-519191","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.209513.r519645, Loick P. Kojom Foko, Animal Organisms, The University of Douala, Douala, Littoral, 15 Sep 2026, version 3, indexed","URL":"https:\/\/f1000research.com\/articles\/14-1389\/v3#referee-response-519645","order":3,"name":"referee-response-519645","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"The author(s) declared that no grants were involved in supporting this work","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}