Plasmodium pitheci and Plasmodium silvaticum, have been rarely described and reportedly cause relatively benign infections. Orang-utans at Rescue Rehabilitation Centres (RRC) across the orang-utan natural range suffer from malaria illness. However, the species involved and clinical pathology of this illness have not been described in a systematic manner. The objective of the present study was to identify the Plasmodium species infecting orang-utans under our care, define the frequency and character of malaria illness among the infected, and establish criteria for successful diagnosis and treatment.Plasmodial species naturally infecting orang-utans, During the period 2017–2021, prospective active surveillance of malaria among 131 orang-utans resident in a forested RRC in West Kalimantan (Indonesia) was conducted. A total of 1783 blood samples were analysed by microscopy and 219 by nucleic acid based (PCR) diagnostic testing. Medical records of inpatient orang-utans at the centre from 2010 to 2016 were also retrospectively analysed for instances of symptomatic malaria.Active surveillance revealed 89 of 131 orang-utans were positive for malaria at least once between 2017 and 2021 . During that period, 14 cases (affecting 13 orang-utans) developed clinical malaria (0.027 attacks/orang-utan-year). Three other cases were found to have occurred from 2010–2016. Sick individuals presented predominantly with fever, anaemia, thrombocytopenia, and leukopenia. All had parasitaemias in excess of 4000/μL and as high as 105,000/μL, with severity of illness correlating with parasitaemia. Illness and parasitaemia quickly resolved following administration of artemisinin-combined therapies. High levels of parasitaemia also sometimes occurred in asymptomatic cases, in which case, parasitaemia cleared spontaneously.P. pitheci very often infected orang-utans at this RRC. In about 14% of infected orang-utans, malaria illness occurred and ranged from moderate to severe in nature. The successful clinical management of acute pitheci malaria is described. Concerns are raised about this infection potentially posing a threat to this endangered species in the wild.This study demonstrated that  Human plasmodial parasites responsible for clinical malaria infect hundreds of millions of people annually, causing more than half a million deaths in areas of endemicity , 2. MalaPlasmodium are known to naturally infect some mammals, birds, and reptiles [Plasmodium knowlesi, a non-human primate (NHP) Plasmodium that occurs in macaques, but also infects humans [Plasmodium knowlesi malaria in humans is sometimes lethal, and in Malaysia today it is the overwhelmingly dominant cause of human malaria [Many dozens of species in the genus reptiles –13. Justreptiles , 15, a rs humans , 17. Pla malaria , 17–21.Plasmodium in their natural host is controversial, and questions remain regarding their potential pathogenicity [Plasmodium, sometimes with high prevalence, are widely reported in wild as well as captive populations of great apes within their range and are commonly considered harmless in their natural host with allegedly negligible clinical implications for infected individuals [P. pitheci at a zoo in Sydney [The health effects of malaria in humans are well-known and have serious implications for human health among malaria-endemic nations , 11, 22.genicity , 26–33. ividuals , 33–38. ividuals , 39 and ividuals . Similarividuals , 33, 34,n Sydney , 33. Morn Sydney , 29, 41 P. pitheci [P. silvaticum, was described [Plasmodium pathogens from non-invasive samples from African great apes [Plasmodium species [P. silvaticum and P. pitheci which were morphologically described as species long before the advent of molecular techniques, have yet to be genetically characterized. Consequently, there are no validated molecular probes for PCR-based identification of these two species.The novel discovery of a malaria parasite in NHP occurred over 100 years ago when Halberstaedter and Prowazek at a Berlin zoo housing an orang-utan originating from Borneo first described  pitheci , 23, 33. pitheci , 40, andeat apes , 43. As  species , 40. Mea species , 44, 45.Plasmodium vivax) and a macaque (Plasmodium cynomolgi) Plasmodium species [Plasmodium species involved were neither P. vivax nor P. cynomolgi. As no morphological characterization was made in that study, the species involved remains unknown [P. pitheci or P. silvaticum are considered candidate pathogens in this case [A previous study on orang-utans at an RRC in Borneo , 29 attr species . This wa species , 40, 47. species , who dem unknown , 46, buthis case . Neverthhis case .Plasmodium spp. infection and potentially threatening illness in orang-utans. In doing so, in an attempt to optimize and validate the clinical management of infected orang-utans thresholds of parasitaemia, sickness and regimens of clinical management and chemotherapy leading to safe and full recovery were quantified.Rescued wild orang-utans displaced from their forest habitat due to habitat fragmentation or the demand for illicit wildlife pet trade, are cared for at RRCs. The number of orang-utans at these RRCs represents an important proportion of the remaining wild population, and therefore, this illness could have serious implications for the conservation of the species. A large body of laboratory and clinical evidence was explored to examine possible causal relationships between Inisiasi Alam Rehabilitasi Indonesia Foundation (IAR Indonesia) works under the Directorate of Biodiversity Conservation of the Ministry of Environment and Forestry (MoEF) of the Republic of Indonesia to operate a rescue and rehabilitation centre (RRC) for Bornean orang-utans in Ketapang, West Kalimantan, Indonesia, the IAR RRC. This centre began operations in 2009 and has since rescued over 250 orang-utans. The observational study reported herein was reviewed and approved by the Ethics in Research Commission of the National Research and Innovation Agency of the Republic of Indonesia .The The data used in this study was collected retrospectively (2010–2016) and prospectively (2017–2021). The retrospective study consisted entirely of a review of medical records for instances of symptomatic confirmed malaria illness. During the prospective study, a total of 131 orang-utans  were observed. Among those, 49 were lost to observation—48 by being reintroduced into the wild, and one orang-utan that died—and 29 newly arrived at IAR RRC.All orang-utans arriving at IAR RRC spend a minimum of 60 days in quarantine. During this time, all undergo medical checks as part of their medical quarantine procedure. Upon completion of the quarantine period, healthy orang-utans are transferred to the rehabilitation areas or socialization cages, where they join with conspecifics of approximately the same age and size. Some orang-utans are deemed unsuitable for rehabilitation and release, and they become permanent residents. These include: older juveniles or adult orang-utans that matured in captivity (with private owners); those who spent longer than eight to nine years in that condition; or those with disabilities or diseases that make survival in the wild unlikely. All other orang-utans undergo a ‘rehabilitation’ process that ends with reintroduction into suitable wild protected forested areas.The rehabilitation activities take place in semi-natural secondary forested areas, delimited by artificial canals and electric fences. All the orang-utans temporarily housed on these islands are grouped by size and/or age. Each area, depending on its size (approximately 10–20 ha), can accommodate between 10 and 20 orang-utans where they have contact with wild animals such as macaques, leaf monkeys, proboscis monkeys, and a variety of other mammals, birds and reptiles, as well as non-vertebrate fauna. After rehabilitation, orang-utans are reintroduced into a protected natural habitat designated for this purpose. Intense post-reintroduction monitoring continues for as long as possible to evaluate their natural behaviours and their capacity to adapt to the environment.The health of all orang-utans managed by IAR RRC is closely monitored daily by a team of veterinarians, veterinary assistants and animal keepers. Biological samples are collected only for medical purposes from healthy animals during routine medical check-ups or when orang-utans show any signs of illness. Medical records are kept for each orang-utan and every medical procedure is recorded both in a hard copy format (paper files) and soft copy format (using FileMaker® database).(i)Routine medical health checks: (a) annual health checks; (b) quarantine procedures on arrival and prior release; or (c) health check-ups conducted to monitor the health of the population ;(ii)Health monitoring for diagnostic purposes of inpatients during any illness  and during recumbence periods;(iii)Any medical or handling procedure requiring anaesthesia(iv)Monitoring the health of patients presenting Plasmodium spp. patent infections: routine consecutive screening in those individuals known to present patent malaria infection either symptomatically or asymptomatically, for the monitoring of haematology values and/or parasitaemia levels.As part of routine medical checks conducted by the staff veterinarians, a total of 1783 blood samples from a cohort of 131 orang-utans housed at IAR RRC were collected and analysed at the in-house laboratory facility between January 2017 and December 2021. Blood samples were collected from orang-utans during either manual restraint or incident to clinically indicated anaesthesia. Samples were classified per sampling purpose of each screening event:Plasmodium spp. by microscopic examination. If one or more sexual or asexual forms of Plasmodium spp. parasite was detected, the sample was classified as positive.Fresh blood samples were collected by venipuncture and immediately prepared in thick and thin blood smears on glass slides. Smears were dried, thin smears were dipped in absolute methanol, and both thin and thick smears then stained for 15–20 min using Giemsa’s solution at 10%. The slides were then examined by trained personnel using a light microscope (Olympus® CX22LED) set at 1000× oil immersion optics and analysed for the detection of DNA was extracted directly from fresh blood in EDTA, or from frozen whole blood in EDTA stored at − 80 °C using a commercially available kit: PureLink® Genomic DNA Mini Kit , following the protocol provided with the kit. Extracted DNA was used directly for PCR analysis, or stored at − 80 °C.Plasmodium  Genesig® Easy kit  following the standard protocol provided with the kit. This kit targets the 18S ribosomal gene achieving a broad-based detection profile for several species of Plasmodium.Blood samples were tested by q-PCR on a Genesig q16® Real Time-PCR machine  using a commercially available kit: Plasmodium spp. published primers targeting highly conserved regions of the 18S small subunit ribosomal RNA genes were used (Table Fresh and stored blood samples (frozen at − 80 °C) were analysed on a conventional gel-based nested PCR. For the detection of ed Table  followinSpecies identification was conducted by morphologic characterization of parasites infecting intact red blood cells on thin blood films using light microscopy , 21. IdePlasmodium  and the annealing temperatures for species specific primers which was as follows: for 5 cycles The number of sexual and asexual parasites in red blood cells (RBC) was counted by examining Giemsa-stained thick smears by 1000× light microscopy using oil immersion until a total of 200 leukocytes (WBC) had been counted , 52. Thi9/L, and thrombocytopenia as PLT lower than 70*109/L of blood.Analysis of haematology values including red blood cell count (RBC), leukocytes count (WBC), haemoglobin (HGB), haematocrit (HCT) and platelets (PLT) was conducted by collecting fresh blood samples by venepuncture and transferring them to EDTA coated tubes. These blood samples were directly analysed using an in-house Abaxis VetScan® HM5 automated haematology analyser. Haematology ranges were obtained by analysing 758 samples corresponding to 150 healthy orang-utans at the IAR RRC. The sampled population comprised rehabilitant, wild and pet orang-utans in the range of ages from infant, juvenile, sub-adult and adult  Table 44. For thThe prospective epidemiological analysis of orang-utans at the IAR RRC commenced in 2017. The initiation of surveillance and follow-up in each individual occurred opportunistically, i.e., when permitted by accessibility of a blood sample for analysis and at random routine tests conducted in the population. Among the 131 orang-utans involved, the number of malaria independent examinations  ranged from 1 to 23 with a median of 8. As there were newly arrived rescued orang-utans added and others that were removed from the study population during the surveillance period, the initiation and finalization of testing varied widely for each individual. The results of these examinations were classified as negative, positive asymptomatic, or positive symptomatic.Incidence rate (IR) per 100 orang-utan—year was obtained reviewing data from inpatient medical records between 2010 and 2021 and was defined as the number of new cases of clinical malaria in a year divided by the total orang-utan-time at risk. IR was calculated using an approximate denominator based on the total number of disease-free animals at the start of the time period, from which ½ of the withdrawn animals were subtracted and ½ of the new additions were added .Asymptomatic malaria defined as the presence of Plasmodium spp. parasites in blood (patent infection) in the absence of fever [of fever , 56–59 aClinical malaria (symptomatic) cases were categorized as follows:The following definitions were developed based on published literature for human malaria , 52, 56 “Acute uncomplicated malaria” defined as symptomatic parasitaemia in the presence of fever and other symptoms and in the absence of signs of severity and/or evidence of vital organ dysfunction or the presence of any sequela post-infection ;“Chronic uncomplicated malaria” in cases when parasitaemia and anaemia persist for prolonged periods;Severe malaria was defined as malaria infection in which the patient presents vital organ impairment [pairment , 53, inc“Mild malaria” as clinical cases of uncomplicated self-limiting illness  with spontaneous decrease in parasitaemia and with mild symptoms that resolve without antimalarial treatment.A retrospective review of all medical histories of febrile illnesses recorded as acute malaria between January 2010 and December 2021 was conducted. Medical history files of orang-utan patients at IAR RRC included data on anamnesis, results of all diagnostic procedures conducted, daily evolution notes, treatment administered, prognosis, differential and final diagnosis (when known). During the review, case reports in which microscopy-demonstrated malaria was confirmed as the diagnosis were selected for further investigation of the course of disease and treatment. The criteria for case selection were as follows: (1) axillary temperature > 38 °C with other symptoms recorded; (2) haematology values recorded; (3) malaria confirmed with numbers of parasites in blood recorded; and (4) responses to anti-malarial therapy recorded.Evaluation of the response to treatment was attempted using information on the evolution of the severity of symptoms, recording the length of the course of the illness, and by comparison of pre- and post-treatment clinical variables (haematology and parasitaemia) for all the medical records for which this data was available. Any possible correlation or association with level of parasitaemia and signs of illness was examined, i.e., fever, anaemia, thrombocytopenia, and leukopenia. The presence of any sequela was evaluated and when possible, any known recurrence of illness was recorded .Oral administration of quinine sulphate at 10 mg of salt/kg of body weight 3 times a day (Q8H) in combination with doxycycline 2.2 mg/kg in a single dose per day (Q24H) for 7 days;The anti-malarial treatment selected to treat malaria in orang-utans at IAR RRC were based on guidance for therapies used in human malaria , 57:OralOral administration (PO) of artesunate (4 mg/kg BW) combined with amodiaquine (10 mg/kg BW) once a day (Q24H) for 3 daysIntramuscular administration of artemether at 3.2 mg/kg on an initial dose on the first day, followed by 1.6 mg/kg single dose a day (Q24H) for 6 days combined with doxycycline at 5 mg/kg (Q24H) for 7 days.And artemisinin-based combination therapy (ACT):In human malaria, a correlation between parasitaemia loads and morbidity and severity of clinical malaria illness has been demonstrated , 15, 60.Statistical analysis was conducted with the R statistical package (R Core Team 2020). For comparison of pre and post treatment clinical values, the within-subject post-treatment value was subtracted from the pre-treatment value, and this was tested for a non-zero mean with a one-sample t-test. For reported correlations , the statistical significance was computed as the p-value for a least-squares linear regression . Due to the small sample sizes, p values were not adjusted for multiple comparisons.Plasmodium spp. in this cohort; 13.04% of microscopy-negative samples were found to be positive by conventional nested PCR  and 21.83% positive by Real-Time PCR . Approximately 20% of PCR-confirmed infections were not detected by light microscopy. In the analyses that follow, only microscopic diagnostics were considered because these were complete across orang-utan examinations over the years of observation. Routine PCR diagnosis of malaria is not practical at this RRC.The sensitivity and specificity of the microscopy testing was tested obtaining a 100% specificity of the microscopy test when compared to nested or Real-Time PCR. Sensitivity of the malaria microscopy test was 78.17%  compared to Real-Time PCR and 83.96%  compared to nested PCR. Microscopy diagnostics were compared to conventional PCR and Real-Time PCR for the detection of P. falciparum, P. vivax, P. malariae, P. ovale, P. knowlesi, P. cynomolgi, and P. inui were applied P. pithecInpatient medical records data were used to identify all clinical malaria cases detected since the beginning of the operations of the centre, between January 2010 and December 2021 following the criteria defined in the methods. A total of 17 cases of clinical malaria (symptomatic) cases were recorded comprising an overall incidence rate (IR) of 3.25 cases per 100 orang-utan-years for the entire period. Figure Plasmodium spp. infection during the surveillance period between January 2017 and December 2021 including those samples collected from orang-utans suffering clinical malaria. For the epidemiology analysis, only the first sample tested of each screening event was considered (defined by the screening purpose) for each individual. A total of 1255 blood samples from 131 orang-utans that were assessed for malaria during this period, and among them, 466 samples (37%) and 89 individuals (68%) were found positive for Plasmodium, all of which presented morphologic features consistent with P. pitheci. Figure A total of 1783 samples were collected and microscopically examined for During 2017–2021, 76 of the 89 (85%) orang-utans found positive for malaria experienced no signs of illness despite weeks, months, and in some cases years of intermittent or sustained positivity Fig. . 27 blooP. pitheci > 32,000/µL. He also presented normocytic normochromic anaemia and thrombocytopenia, but no other specific finding was reported. He received antipyretics and remained in observation. The following hours he was reported bright, alert, and with good appetite, fever remitted within 48 h and parasite numbers in blood trended downwards without the need for any anti-malarial treatment. No further signs of illness were reported.Case 1 (Udin): A juvenile male orang-utan (approximately 5 years old) under rehabilitation was reported feverish and lethargic. He was brought to the clinic for further examination. His temperature was 39 °C. Microscopic examination of a blood sample revealed patent parasitaemia of P. pitheci parasites at a density of 16,600/µL. During observation fever remitted with the use of antipyretics alone, and the parasites in blood spontaneously decreased in number , and her condition returned to normal within 48 h.Case 2 (Kandi): A juvenile (4–5 years old) female orang-utan presented with fever (39.2 °C), normocytic normochromic anaemia and thrombocytopenia, but she remained active and with good appetite, and no other symptoms. Blood examination revealed Balantidium spp. infection, therefore the patient was administered a course of metronidazole. He also received supplements to help resolve persistent anaemia. After the course of antibiotics, diarrhoea had resolved, however, his blood profile still revealed persisting anaemia and leukopenia, and a blood film examination revealed P. pitheci parasites > 12,000/µL. ACT was administered and improvement of the clinical and laboratory picture was nearly complete within five days. After this clinical episode, and during the whole period of surveillance (for over 4 years), no further anaemia or any other related medical problem was reported in this orang-utan.Case 3 (Boy): A juvenile male orang-utan of approximately 4 years of age while undergoing rehabilitation at the RRC, had presented a history of persistent weight loss, frequent illness and chronic microcytic hypo-chromic regenerative anaemia that did not respond to iron or vitamin supplements. He was presented to clinic suffering appetite loss, inactivity, and “looking pale” according to his keeper. Further examination revealed a fever (39 °C), diarrhoea and dehydration, and a blood profile of anaemia and leukopenia. Microscopic faecal examination revealed Plasmodium spp. infection. She was administered IV fluids and other supportive therapy, and quinine/doxycycline combination. She fully recovered within seven days of treatment administration.Case 4 (Karmila): A juvenile (approximately 4–5 years) female orang-utan undergoing rehabilitation presented at clinic with a fever (39.6ºC), lethargy, anorexia and a patent Case 5 (Gakkum): A juvenile male was brought to the clinic experiencing onset of fever (38.6 °C). He was active, had a good appetite and no other symptom was recorded. His blood sample was positive plasmodia at 7623/µL blood. He remained under observation. During the ensuing 5 days, he would peak a fever in the late afternoon/early evening hours (up to 39.9 °C) for which he received antipyretics. Although his general condition remained stable, his blood haematology values (HGB and PLT) started dropping. On day 6 after the onset of the fever, a normocytic normochromic anaemia was diagnosed while PLT values continue dropping and parasite numbers in blood climbed above 30,000/µL. His condition began deteriorating with conspicuous lethargy and inactivity. ACT was then administered and his condition began improving within 24 h, and after 4 days he had fully recovered.P. pitheci at 17,500/µL. He was monitored for 3 days while his blood values  steadily dropped and his fever would spike in the afternoon. Nonetheless, he remained active and with good appetite. He was only administered antipyretics. On the 4th day after the onset of fever, his blood values remained dropping down, his temperature reached 39.8ºC and he presented more lethargic and unresponsive. He was administered ACT therapy. Within 24 h fever remitted and his condition started to improve, eventually resulting in an uneventful full recovery.Case 6 (Asoka): a lethargic juvenile male orang-utan presented to the clinic with onset fever (38.3 °C). He was otherwise bright, responsive and with good appetite. His blood showed normocytic normochromic anaemia and parasitaemia of P. pitheci . She was administered ACT and supportive therapy (including IV fluid therapy). Her fever remitted after 48 h and she had completely recovered by the end of the treatment.Case 7 (Lady): A sub-adult female orang-utan of approximately 9 years of age undergoing rehabilitation, presented with a high fever (40 °C), anaemia, thrombocytopenia and leukopenia. Microscopic examination of a blood sample revealed an infection of P. pitheci parasitaemia at 11,080/µL of blood, although she remained active and bright. She was started on ACT and provided supportive therapy. Signs of recovery were evident within 24 h and no further sign of the illness was reported.Case 8 (Lady): 14 months after her first malaria episode in 2017, this sub-adult female orang-utan (10 years old) was again reported to be anorexic and lethargic. She presented to clinic with fever (39.7ºC), anaemia, thrombocytopenia and a P. pitheci parasitaemia at 20,740/µL. She was diagnosed with acute malaria and administered ACT and other supportive treatments. Although she at first improved, five days after the start of the treatment her condition worsened with fever, lethargy and anorexia. Her haematology values remained low. An in-house blood culture revealed infection with Pseudomonas spp. She was administered a course of antibiotics and she fully recovered.Case 9 (Bunga): A sub-adult female orang-utan around 11 years of age was being monitored in the wild after having been reintroduced 6 weeks earlier. She was found to be inactive, anorexic and feverish. She was evacuated to the clinic at RRC and presented with a high fever (40 °C), lethargy, dehydration, and findings of normocytic normochromic anaemia, leukopenia, thrombocytopenia and P. pitheci parasitaemia at approximately 12,000/µL of blood. Blood biochemistry analysis also revealed hyperkalaemia and elevated creatinine levels. A possible renal disorder was suspected and since there was no clear sign of clinical malaria  the primary cause of his illness was unclear. He remained under observation and supportive therapy. The following day, his condition had not improved and new symptoms were detected such as fever (39.5 °C) and thrombocytopenia. The parasite load in blood had increased to 42,600/µL. Based on the new symptoms (fever and thrombocytopenia) and the increasing levels of Plasmodium in his blood, anti-malarial treatment (ACT) was initiated. Symptoms persisted for 48 h, with haemoglobin values dropping during this time. On day three after the initiation of the treatment, however, the fever remitted and he appeared fully recovered. By the end of the ACT treatment course, he had no parasitaemia and a normal blood profile.Case 10 (Melky): A sub-adult male orang-utan (approx. 12 years old) released into the wild 3 weeks earlier was found inactive, anorexic and febrile (39 °C). He was administered analgesics (paracetamol) and transported to the RRC for further medical attention. On arrival at the clinic, he was lethargic and vomiting with a productive cough. He was administered IV fluids and other supportive medication. No specific diagnosis was made but he remained under observation and supportive IV fluid-therapy. Four days after the onset of symptoms, blood film examination revealed Case 11 (Ceria): a sub-adult female orang-utan (approx. 12 years old) had experienced a minor surgery and was in the process of recovery. In the days post-surgery, she was lethargic and peaked a fever of 39.4 °C. Examination of her blood showed thrombocytopenia and a high parasitaemia load . She remained in observation. On the following day, her condition deteriorated, becoming more lethargic. Her fever peaked again (38.7 °C) and her blood sample revealed hyperparasitaemia . She was then administered supportive IV therapy  and she was started on ACT. Fever remitted within 24 h post treatment and full recovery was reported in 72 h.P. pitheci load of 3400/µL. Differential diagnoses included typhoid fever, malaria, and dengue fever, but all diagnostic tests conducted were inconclusive with regard to the cause of illness, so no final diagnosis was made. She was administered IV fluids and other supportive therapy, including analgesics/antipyretics, and remained in observation for 48 h. A new blood sample revealed that the parasite load had increased to 4680 pars/µL of blood. The increase in parasites and in severity of symptoms indicated malaria as the most probable cause of illness. ACT was administered together with other supportive therapy. After completing the course of antimalarial treatment, her condition and her appetite had improved and blood haematology values had increased, but she remained lethargic. At this stage, a positive Salmonella spp. test indicated a possible typhoid fever infection and she was placed on a course of antibiotics. At the end of the treatment, her condition as well as her haematology values were back to normal.Case 12 (Maili): A sub-adult (approximately 12 years old) female orang-utan and mother to a 1-year-old infant, was being monitored in the wild after having been released 5 weeks prior to the onset of symptoms. She was found inactive and lethargic and was evacuated to the clinic at RRC. At physical examination, the patient presented a fever (38.6 °C) and slight dehydration. Further tests revealed normocytic normochromic anaemia, leukopenia and thrombocytopenia, and microscopic blood examination revealed a P. pitheci in blood. ACT was administered together with antipyretics. Symptoms improved immediately after initial dose and a full recovery, including haematology values, occurred by the end of the treatment course (7 days).Case 13 (Susi): A sub-adult female orang-utan (approximately 13 years old), released into the wild 3 weeks before the onset of symptoms, was found lethargic, feverish and anorexic. After evacuation to the RRC, she was found to present a fever (38.8 °C), severe normocytic normochromic anaemia, leukopenia and thrombocytopenia, and a blood sample examination revealed a high level of P. pitheci parasites in her blood . The following day the patient remained anorexic, lethargic and severely dehydrated. Further examination under sedation revealed a high fever (40.3 °C), a deepening thrombocytopenia, leukopenia, and a parasite concentration in her blood of 23,000/µL. She was started on ACT and administered supportive therapy. Fever remitted within the first 48 h after treatment. Four days after the initial antimalarial treatment, she presented active and with a normal appetite, and fully recovered before the end of the antimalarial treatment.Case 14 (Cinta): An adult female orang-utan over 15 years was reported lethargic, anorexic and feverish. Blood analysis revealed severe thrombocytopenia and P. pitheci parasites at 9300/µL and blood analysis revealed severe anaemia, leukopenia and thrombocytopenia. She was administered supportive therapy and started on ACT. Her condition started improving within 24 h. She was considered fully recovered within a week.Case 15 (Nicky): An adult female orang-utan of approximately 15–17 years of age was found lethargic, febrile and anorexic. She could not be handled without sedation, so she was administered analgesics (paracetamol) and monitored for the following 48 h. She did not improve and so was sedated for a closer physical examination. She was shivering but did not present a fever. Microscopic examination of a blood sample revealed P. pitheci at a density of 14,000/µL. Haematology values also showed normocytic normochromic anaemia, thrombocytopenia and leukopenia. She was started on anti-malarial medication (ACT) and was administered IV fluids and other supportive medication. Potential early signs of pregnancy were identified. 48 h after the start of the treatment she still appeared lethargic and anorexic. She was sedated and administered supportive therapy again and started on a course of antibiotics. Two weeks after the first onset of the illness, her condition worsened. She was moved to intensive care and administered antibiotics and other IV therapies. 48 h later, her condition started to improve. After the course of antibiotics, she showed obvious improvement. Since no further signs of pregnancy were detected, a miscarriage possibly related to acute malaria was considered. She fully recovered within a month of the onset of symptoms.Case 16 (Neng): An adult female orang-utan of about 17 years of age and suspected to be pregnant was found inactive, lethargic, anorexic and feverish. She was handled under sedation for further examination and her blood showed the presence of Plasmodium spp. infection. The course of illness included fever (38.7 °C), multiple convulsions, nystagmus and other neurological impairments such as blindness and hearing loss, along with a mild anaemia. Treatment included intravenous (IV) fluids, anti-malarial treatment, analgesics, antibiotics, anti-convulsive and other supportive therapies. The first anti-malarial treatment consisted of oral quinine and doxycycline combination, but did not result in improvement within 48 h, so anti-malarial medication was switched to ACT. The most severe neurological signs remitted within 48-72 h after the start of the ACT, and hearing improved in the weeks after the severe clinical episode. Vision impairment did not improve and cortical blindness and strabismus remained as permanent sequelae of the infection : An infant female orang-utan (age between 2 and 3 years old) was rescued by the authorities from a forest clearance area. She presented to the RRC febrile and prostrated with a low level of consciousness  and 19 days with an average of 9 days . The maximum period required for HGB values to return to normal values ranged between 5 and 16 days . In the case of PLT, the mean value of PLT went up 7 days later as compared to the value at the start of treatment by a mean difference of 17.6 (*10^10/L) (p < 0.001). In the case of parasites in blood, the mean density of parasites went down 7 days later as compared to the value at the start of treatment by a mean difference of 27,795 parasite/µL of blood (p < 0.05). In the case of HCT (4.8% p < 0.05) and HGB (1.48 g/dL p < 0.05), the values went up 7 days later  since prospective active case detection started, and very probably since the beginning of operations over a decade earlier. The majority of instances of confirmed parasitaemia were not associated with any signs or symptoms of illness , apart from occasional mild anaemia. Nonetheless, moderate to severe and potentially life-threatening illness attributable to P. pitheci infection occurred among 13 of the 89 (14%) orang-utans known to have been infected. Three more clinical cases were detected between 2010 and 2016. These attacks involved febrile syndromes with anaemia, thrombocytopenia, leukopenia, anorexia, and lethargy. In one instance, cerebral malaria with permanent sequelae occurred in an infant. Although in the cerebral malaria case morphological identification of the species was not conclusive, it was not diagnosed as a human malaria parasite. A validated means of diagnosis and treatment of this apparently common illness in this endangered species would serve the cause of its conservation. This study strived to contribute to that goal.Stable endemic transmission of 9/L) and/or leukopenia (WBC below 4.7*109/L), accompanied by other general symptoms including lethargy and/or anorexia. Potentially more severe syndromes involving vital organs such as kidney or brain may also occur. Some studies on human malaria estimated blood parasite density thresholds to define clinical malaria [Based on the experience reported here, orang-utan clinical pitheci malaria diagnostic criteria were developed. Clinical malaria may be affirmed by finding an increasing density of parasitaemia with an axillary temperature > 38 °C, moderate to severe normocytic normochromic anaemia (HGB below 8.2 mg/dL), and/or thrombocytopenia . There was fever, anaemia and signs of neurological impairment , along with cortical blindness and strabismus remaining as sequelae of the illness. The macular whitening of the retina which was diagnosed in this patient a decade after the acute illness also occurs in human patients surviving cerebral malaria –65 and iOne adult female orang-utan case 16) may have suffered a miscarriage during her acute malaria attack. In humans, placental malaria can lead to miscarriage, low birth weight, peri-partum haemorrhage or increased maternal and neonatal mortality, amongst others 6 may hav, 58. WhiP. pitheci infections as inconsequential, especially when wild orang-utans, alike the captive population, would go untreated for malarial symptoms. In one of the patients (case 3), a chronic microcytic and hypochromic regenerative anaemia which had not responded to iron supplementation, had been diagnosed prior this malaria episode. This patient had also been found to present high morbidity due to different illnesses and had poor general health before the confirmed malaria episode. Following anti-malarial treatment his anaemic condition resolved and no further episodes of sickness were reported post treatment and for the following five years. The relationship between such poor general health and chronic malaria in otherwise asymptomatic plasmodial infections may be confounded by many factors [It may be overly optimistic to dismiss the health impact of the majority of  factors , 58, 67. factors , 58 and P. pitheci  cannot be determined from this data. However, a patent P. pitheci infection was observed in an orang-utan upon rescue from a captive situation on another island who had been living in a malaria-free urban area without contact with other orang-utans for over 10 years. Peters et al. reported ‘persistent’ infections with P. pitheci in orang-utans lasting at least seven years [Many of the orang-utans were followed for up to 5 years and were repeatedly positive for eci Fig. . Whetheren years . Very loPlasmodium reichenowi, a species known to naturally infect these apes, have been reported [Plasmodium spp. infection of great apes could indeed be harmful in their natural host [P. pitheci in orang-utans, i.e., a non-sterilizing immunity that sometimes fails to curb progression of the infection and illness, especially in the very young. Since malaria cases diagnosed at RRCs are likely to be clinically managed, estimating potential mortality rates of this illness is not possible. However, infant mortality rates in wild orang-utan populations are reportedly higher than those of other great ape species [The study findings confirm deleterious effects of natural malaria in orang-utans. In chimpanzees, two clinical cases of malaria caused by reported , 39, 40.reported , these fral host . The mosral host , 4, 60. ral host , only a  species . PitheciP. pitheci on that preserve and the onset of moderate to severe illness in 14% of infected orang-utans. Acute malaria typically presented with fever, anaemia, thrombocytopenia, leukopenia, and lethargy with sometimes anorexia in association with relatively high parasitaemia. A case of cerebral malaria with permanent neurological sequelae is also reported. The successful diagnosis, clinical management, and anti-malarial chemotherapy of acute pitheci malaria in orang-utans are described. Contrary to the conventional view of natural P. pitheci infection as benign and inconsequential to the health of Pongo pygmaeus, this study confirms this parasite as the cause of sometimes serious illness at the IAR RRC. This endemic infection did not infect humans despite prolonged high risk of exposure among IAR RRC staff. As no human malaria occurred at this site, it is not confirmed if anthroponotic infections of orang-utan may occur. In summary, P. pitheci poses an endemic threat to the health of orang-utans in rescue and rehabilitation settings, as may also occur in the wild.This study spans a decade of observations in caring for orang-utans in rescue and rehabilitation at a forested IAR RRC preserve in West Kalimantan, Indonesia. The study findings confirm the stable endemic presence of


P. falciparum or P. vivax in Colombia in its gestational malaria (GM), placental malaria (PM), and congenital malaria (CM) forms as well as to compare its accuracy in different subgroups of pregnant women according to the presence of fever, anemia and a history of malaria. This was a diagnostic evaluation of 829 pregnant women, 579 placentas, 381 umbilical cord samples, and 221 neonatal peripheral blood samples. Accuracy was evaluated based on the parameters of sensitivity, specificity, predictive values, likelihood ratios, and validity index, with their 95% confidence intervals. The frequency of GM was 36% (n = 297/829), PM 27% (n = 159/579), and CM 16.5% (n = 63/381) in umbilical cord samples and 2% (n = 5/221) in neonatal peripheral blood samples. For GM, the sensitivity was 55%, with higher rates in those infected with P. vivax (68%), with a history of malaria (69%), and with fever (96%). These three subgroups presented the best results in terms of the negative likelihood ratio and validity index. For PM, sensitivity was 8%; in subgroup analyses in terms of species, symptomatology (anemia and fever), and history of malaria, it was 1–18%, and the negative likelihood ratio was >0.80 in all subgroups. No false positives were recorded in any of the subgroups. The TBS did not detect any cases of CM. This study found the TBS yielded satisfactory results in terms of diagnosing GM for P. vivax, pregnant women with previous malaria and febrile. It also showed that the TBS is not useful for diagnosing PM and CM. It is necessary to conduct surveillance of MAP with molecular methods in in groups where TBS is deficient  to optimize the timely treatment of PM and CM, avoid the deleterious effects of MAP and achieve the malaria elimination goals in Colombia.This study aimed to evaluate the accuracy of the thick blood smear (TBS) versus quantitative polymerase chain reaction (qPCR) for the diagnosis of malaria associated with pregnancy (MAP) caused by  The World Health Organization (WHO) reported 247 million new cases of malaria in 2021 and 619,000 deaths . In ColoPlasmodium spp. infection in maternal peripheral blood, placental malaria (PM) or the presence of the parasite or hemozoin in placental tissue, and congenital malaria (CM) in which the parasite is found in the newborn’s umbilical cord blood or peripheral blood discarding vector bite ) in GM, PM, and CM ; they arpossible ,24,25,26P. falciparum PM even in low (less than 1000 parasites/µL) and moderate  parasitemia, with sensitivity ranging between 76% and 78% , specificity [true negative {TN}/], positive predictive value [TP/(TP + FP)], negative predictive value [TN/(TN + FN)], the negative likelihood ratio , the Youden index [(S + E) − 1], and the validity index or proportion of patients correctly diagnosed [(TP + TN)/Total]. All of these parameters were calculated with 95% CI, and the positive likelihood ratio was not calculated given the absence of FP results with the TBS. These parameters were calculated for the entire group and the following subgroups: infected with The guidelines of the Declaration of Helsinki and Resolution 8430 of Colombia for research with pregnant women were followed. The study was classified as minimal risk and was approved by the Ethics Committee of the Sede de Investigación Universitaria, Universidad de Antioquia (Minutes # 21-101-961) and the Instituto de Investigaciones Médicas (Minutes # 008). All pregnant women signed the informed consent  or assent (for women under 18 years of age) form, which was obtained in writing and signed by the pregnant woman and a witness . According to Colombian law, adolescents (over 10 years of age) may sign the informed consent form without the consent of their parents or guardians. All data were coded (anonymized) by someone outside the research team. To ensure the confidentiality of the data, the authors of this manuscript had access to a database that did not contain any information that could reveal the identity of the pregnant woman.n = 347) having malaria in the last year and 25.1% (n = 265) in the current pregnancy. Among the 661 pregnant women, 19.1% (n = 126) reported having fever during the current malaria infection, and among the 792 patients with hemoglobin records, 31.3% (n = 248) had anemia (hemoglobin level < 11.0 g/dL) during gestation or at delivery.Information was retrieved from the medical records of 1057 pregnant women with a history of malaria, with 32.8% (n = 297) tested positive with the following results in terms of species: P. vivax, 63% (n = 187); P. falciparum, 34% (n = 102); and both species , 3.0% (n = 8). The same 829 underwent TBS, 19.7% (n = 163) were positive for malaria   .n = 159) were positive, while the TBS revealed that 2.2% (n = 13) were positive. The species identified with qPCR were P. falciparum, 45.3% (n = 72); P. vivax, 40.9% (n = 65); and mixed, 13.8% (n = 22), while with the TBS, no mixed species were identified and the respective frequencies of P. vivax and P. falciparum were 69.2% (n = 9) and 30.8% (n = 4)  .P. vivax (n = 3) and P. falciparum (n = 2). According to the umbilical cord blood samples, 16.5% (n = 63) were positive, P. falciparum 63.5% (n = 40), P. vivax 30.2% (n = 19), and mixed malaria 6.3% (n = 4)  and peripheral blood . Only the qPCR data are presented because none of the children tested positive in the TBS. According to the neonatal peripheral blood samples, there were no mixed malaria, and there was a low frequency of  (n = 4) .P. falciparum (30%); pregnant women without fever (29%), without anemia (38.5%), or mild anemia (33%); and those without a history of malaria in the previous year (13%) or in the current pregnancy (20%). On the contrary, the best sensitivity results were found in those infected with P. vivax (68%), with a history of malaria (69%), and with febrile symptoms (96%). The lowest negative predictive values were recorded in pregnant women with anemia (66% in mild anemia and 57.5% in moderate anemia) and with a history of malaria , while the highest values were found independent of diagnosis of species  or febrile state (89% in pregnant women without fever and 91% in febrile symptomatic women)  .P. falciparum infection, no fever, and no history of malaria had the worst results in the negative likelihood ratio and Youden index. However, the best results in these two parameters were shown in pregnant women with fever, a history of malaria, and a P. vivax infection. The proportion of correctly diagnosed patients was higher in febrile symptomatic women and those infected with P. vivax  of 83.8%. In the subgroup analyses, pregnant women with P. vivax .The diagnostic accuracy parameters for these cases could not be calculated because of the small number of mixed malaria results. It was also not possible to perform calculations according to the parasitemia reported in the TBS because parasite density was only obtained in 152 samples, with 35 pregnant women having less than 1000 asexual parasites/µL, 26 having 1000–2500 asexual parasites/µL, 30 having 2501–5000 asexual parasites/µL, and 61 having more than 5000 asexual parasites/µL. Both tests revealed that all of these samples were positive .P. falciparum by TBS was a P. vivax infection on qPCR, one case of P. falciparum with TBS was a mixed malaria on qPCR, four cases of P. vivax on TBS were mixed malarias on qPCR, and one mixed malaria with TBS was a P. falciparum infection on qPCR.The diagnostic errors in terms of species were as follows: one case of P. vivax showed better results in terms of sensitivity, negative likelihood ratio, and in the Youden index. The negative predictive value for P. falciparum and P. vivax did not differ in any of the subgroups analyzed according to symptomatology and history of malaria. This parameter yielded the lowest results in pregnant women with anemia  and without malaria in the current pregnancy . The only subgroups with satisfactory diagnostic accuracy results were pregnant women with fever, regardless of the species, and those with a history of P. vivax malaria (in the last year or in the current pregnancy) (egnancy) .For PM, the sensitivity of the TBS was 8.2%; in subgroup analyses by causal species, symptomatology, and history of malaria, it ranged from 1.2% to 17.6%. The negative predictive value ranged from 49.3% to 93.9%, while the proportion of patients correctly diagnosed ranged from 52.0% to 94.0%, with the latter being explained by the absence of FPs in the TBS . SpecifiIn 17 negative TBSs, mixed PM was diagnosed by qPCR. There was no analysis of diagnostic evaluation parameters in the subgroup of febrile pregnant women because only 33 cases were found, 29 of which were negative with both tests and four of which were negative with the TBS but positive with qPCR . Finally, the deficiencies of TBS in detecting PM cases in all subgroups analyzed according to species, symptomatology, and history of malaria were maintained in the estimation of diagnostic accuracy parameters for the interaction of these variables.In the study population, 19.1% of patients had febrile symptoms and 31.3% had anemia. These findings are consistent with the Health Situation Analysis of Colombia 2019, which reported a frequency of anemia of 28%, with anemia being 40% more frequent in the lowest wealth quintile , as wellP. vivax predominates, causing 63% of cases of GM , which is novel in this field of research, where evaluations of diagnostic accuracy predominate in areas with a higher frequency of P. falciparum [These findings apply to territories where lciparum ,29,30.P. vivax predominated in GM as a reflection of the study region’s parasitological and epidemiological situations. However, in PM, both species had a similar proportion , whereas in CM, the proportion of P. falciparum was higher (62% vs. 32% for P. vivax), which would be supported by specific clinical, physiological, and pathological aspects of these species. Therefore, the similar proportion of P. vivax and P. falciparum in PM would contradict several assumptions about P. vivax’s lower capacity for clinical damage [P. falciparum would reflect greater virulence, pathogenicity, and the ability to cross the placenta of this species [l damage ,21,22,48 species .The frequency of GM was 36%, PM 27%, and CM 2% in neonatal peripheral blood samples and 16.5% in umbilical cord samples, which are higher (except for CM) than those reported in a meta-analysis in the Colombian literature, with frequencies of 17%, 11%, and 16%, respectively . DespiteP. vivax infections  in pregnant women with a history of malaria  and in febrile symptomatic women . In this last case, this research makes no new contributions; rather, it confirms the excellent diagnostic capacity of the TBS in cases of advanced infection or parasitemia levels that cause obvious signs and symptoms, as indicated by WHO guidelines, the Colombian CPG, and some studies on MAP [For GM, TBS presented satisfactory diagnostic accuracy parameters for s on MAP ,17,29.P. vivax and in pregnant women with a history of malaria. Therefore, sensitivity and specificity demonstrate the test’s ability to detect positives and negatives without being affected by disease prevalence, implying that the results could be easily applied to settings with a different prevalence. These parameters are only affected by the disease spectrum, so patients with clearly manifested disease have better outcomes. In contrast, disease prevalence can influence the overall validity index or correctly classified subjects because accuracy increases as disease prevalence decreases [This study provides new evidence of the good diagnostic performance of the TBS for GM caused by ecreases .The negative likelihood ratio (1 − S/E) is the ratio of the probability of a negative result in subjects with the disease to the probability of the same result in subjects without the disease . This is the most recommended measure for diagnostic accuracy because its sensitivity and specificity are independent of disease prevalence and can be used to calculate predictive values .The lowest negative predictive values were found in pregnant women with anemia and a history of malaria, indicating that a negative TBS result in this type of pregnant woman is not very reliable for the clinician or treatment staff and that other diagnostic options should be explored when malaria is highly suspected and the TBS result is negative in order to prevent the infection from progressing. In clinical practice, a common line of reasoning is to know how good the test is at predicting disease or health. Therefore, predictive values are more commonly used, although they depend on disease prevalence .P. falciparum cases and asymptomatic pregnant women or those with no history of malaria), TBS performed poorly. Furthermore, in these cases, other diagnostic options should be explored if progress is to be made in achieving the goals of pre-elimination, elimination, or prevention in the reintroduction of cases [In the other subgroups analyzed  of seven cases of diagnostic mismatch by causal species were related to mixed malaria, which would generate errors in epidemiological and clinical profile.P. falciparum infections detected by qPCR in peripheral blood and placenta. Undetected infections are associated with increased clinical risks, highlighting the need for improved MAP diagnosis and to avoid the consequences of hidden infections during pregnancy [For PM, the sensitivity was 8%; in subgroup analyses according to species, symptoms, and history of malaria, it was 1–18%. The negative likelihood ratio (1 − S/E) was greater than 0.8 in all subgroups, indicating that with the TBS, the probability of a FN result (1 − S) is nearly equal to that of getting a TN result (E); this indicates that this test is not useful in any case or subgroup for PM . This isregnancy .P. falciparum-infected erythrocytes in the placenta or the presence of Plasmodium spp. or malarial pigment in this tissue, which can lead to reduced parasite circulation in the maternal peripheral blood and transmission to the fetus. PM is a difficult clinical form to diagnose , with several studies demonstrating high variability in the sensitivity and specificity of qPCR and rapid diagnostic tests. Histopathology is the gold standard for PM diagnosis because it can detect both parasites and hemozoin. Histopathology can also detect immune cell infiltrates and classify placental tissue into acute active infection (presence of parasites with little or no pigment), chronic active infection (presence of parasites and abundant pigment), or past infection (only presence of pigment) based on the inflammatory process, which PCR cannot detect because it does not identify hemozoin [In pathophysiological terms, a factor for the control of MAP and its clinical consequences in the mother, fetus, and newborn revolves around PM, which is defined as the sequestration of hemozoin .However, this method is not without challenges because its results depend on the quality of the sample, and the formalin pigment can be confused with hemozoin, leading to diagnostic errors ,52,53,54It is worth noting that a study conducted in the same region of this research compared the TBS, nested PCR, and histopathology for the diagnosis of MM, discovering a sensitivity of 33% and a specificity of 95% for the TBS, whereas in nested PCR, these parameters were 47% and 77%, respectively, when compared to histopathology .The TBS did not detect any cases, demonstrating the need for molecular diagnosis for this clinical form. Other studies have indicated that the diagnosis of CM is more complicated because it is an infection with low parasitic density in umbilical cord blood or peripheral blood of the newborn. Therefore, tests with better sensitivity, such as molecular techniques, are required . In geneGiven the low number of cases, one of the study’s limitations is the lack of diagnostic accuracy analyses for mixed malaria. Furthermore, comprehensive analyses based on parasitemia levels were not conducted. Nevertheless, determination of parasitemia and classification are difficult and far from perfect; inaccurate diagnosis and classification have caused a high mortality rate, especially in children ,55. Alth(i)in other models, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cycle threshold (Ct) of qPCR  and its correlation with clinical data have been used to suggest reference ranges for Ct . Similar studies could be suggested for MAP , and(ii)based on the outcomes previously mentioned for submicroscopic MAP cases, it can be assumed that the possibility of detecting asymptomatic carriage of malaria is low in the case of the molecular diagnosis of MAP. However, there is a clear need for further research and an increase in the health budget allocated to the molecular diagnosis of this disease.In terms of qPCR, one of its limitations is its inability to determine the viability of the microorganism because it only detects genetic material and does not identify individuals with the ability to transmit the agent and cause new infections. Therefore, two final considerations should be highlighted:(i)It is Colombia’s first study with a large sample size for the mother–placenta–neonate trinomial.(ii)It is one of the few studies in the world that presents the thorough diagnostic parameters for clinical and epidemiological programs in a comprehensive manner.(iii)It assesses diagnostic ability in key subgroups according to symptoms, malaria history, and causal species and discusses the effect of the interaction of these factors on the accuracy of the TBS.(iv)P. vivax and in pregnant women with previous malaria, expanding the evidence in favor of the use of this test in various clinical and epidemiological situations (aspects for which there are no previous studies and demonstrate the novelty of this research). Moreover, there was no false positive in PM with TBS which is relevant to avoid subsequent resistance problems due to poor prescription of antimalarials. However, in GM, classification errors occurred in 0.8% mainly in cases of mixed GM; despite being a very low number, it shows the importance of carrying out more diagnostic accuracy studies for mixed malaria.Although TBS was historically used in people with fever and PCR was developed to identify parasite components (DNA) in asymptomatic people or those with very low parasitemia, this study showed satisfactory results for cases of GM caused by (v)Evidence was gathered to support the importance of active surveillance of MAP in Colombia, which requires more effort compared to passive detection because health workers must search for patients in the community. This type of search is crucial for the pre-elimination and elimination phases of malaria because it allows for the detection of undiagnosed symptomatic cases with passive surveillance, as well as asymptomatic patients using focus studies.The following are the strengths of this study:P. vivax infection, previous malaria and fever (or symptoms). It also demonstrated that the TBS performed poorly to detect PM and CM because of its low case-detection capacity. It is necessary to conduct MAP surveillance, follow-up, and control with molecular diagnosis in order to improve the detection of asymptomatic GM and P. falciparum in pregnant women with no history of malaria, optimize the timely treatment of PM and CM, avoid the clinical consequences of MAP.The TBS performed well in this study for the diagnosis of GM in pregnant women with


Plasmodium parasite is heterogenous and stage-specific both in the human host and mosquito vector. The Plasmodium parasite virulence is predominantly associated with its ability to evade the host’s immune response. Despite the availability of drug-based therapies, Plasmodium parasites can acquire drug resistance due to high antigenic variations and allelic polymorphisms. The lack of licensed vaccines against Plasmodium infection necessitates the development of effective, safe and successful therapeutics. To design an effective vaccine, it is important to study the immune evasion strategies and stage-specific Plasmodium proteins, which are targets of the host immune response. This review provides an overview of the host immune defense mechanisms and parasite immune evasion strategies during Plasmodium infection. Furthermore, we also summarize and discuss the current progress in various anti-malarial vaccine approaches, along with antibody-based therapy involving monoclonal antibodies, and research advancements in host-directed therapy, which can together open new avenues for developing novel immunotherapies against malaria infection and transmission.Malaria is a global infectious disease that remains a leading cause of morbidity and mortality in the developing world. Multiple environmental and host and parasite factors govern the clinical outcomes of malaria. The host immune response against the  Protozoan parasites belonging to the genus Plasmodium, mainly cause malaria, which is prevalent mainly in tropical and subtropical regions, and is a major global health problem  have long been recognized to infect humans and cause illness  and secondary host (human). Plasmodium completes asexual development inside human hepatocytes and erythrocytes. Inside hepatocytes, the parasite undergoes differentiation into trophozoite and schizont stages to form first generation of merozoites  cytokine. MIF inhibits the migration and activation of phagocytes. It can also manipulate T-cell differentiation resulting in reduced anti-Plasmodium CD4+ T-cell response  . Althouger cells . CSP proer cells , 22. Furer cells . Plasmodresponse , 25.via upregulation of host heme oxygenase-1 protein (HO-1) (+ T-cells (Antibodies against free sporozoites and CSP are the first line of defence to prevent the invasion of hepatocytes . Antibodn (HO-1) . Furthern (HO-1) . After hn (HO-1) . A parasn (HO-1) . Hepaticn (HO-1) . These i T-cells .2.1.2Plasmodium employs various immune evasion strategies to evade the host’s immune response. Plasmodium manipulates the NF-kB and Type 1 interferon pathway to drive inflammation responsible for malaria pathogenesis  proteins promote immune evasion. Both MSPs and EBLs are present as multiple alleles, thereby showing a high degree of polymorphism . VSAs consist primarily of an immunodominant molecule known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by the var multigene family (PfEMP1 protein expression on infected RBCs (iRBCs) is responsible for adhesion to endothelial cells   and STEVOR (mature trophozoite), belonging to other multigene families (rif and stevor) also contribute to the adherence of iRBCs to endothelial cells, leading to their sequestration in the microvascular system of host organs, preventing splenic elimination  . Regulatnscribed . Anti-separasite . Recentlar genes . Althougpression . In a stiability . Howeverpression . Further PfSir2b , 52. Vargulation , 54. In ar genes . Future ar genes . Apart fmination , 56. Botne cells , 58. Rec, LILRB1 , 60. Of of iRBCs . Moreovenfection .2.2Plasmodium zygote matures into an ookinete. Physical barriers such as peritrophic membrane (PM) of the midgut, acts as a first line of defense of Anopheles mosquito against ookinetes  expressed on the surface of ookinete and sporozoites is required for parasite evasion from mosquito complement-like response  as an entry receptor for invading RBCs using parasite ligand PfRh4  at every stage of their life cycle. DCs can phagocytose sporozoites and prime antigen-specific T-cell responses (Plasmodium inhibits DC activation and functioning which interferes with the development of protective immune responses (Plasmodium infection can lead to reduced DC numbers due to increased DC apoptosis (via their cell surface receptors can recognize parasite-generated epitopes which interact with MHC molecules present on the cell surface of antigen-presenting cells (APCs). P. falciparum has been shown to inhibit the maturation of APCs, resulting in impaired T-cell responses  from the mosquito to escape from the immune system. Silencing of CTL4 and CTLMA2 in susceptible mosquitoes triggered melanization and reduced oocyst formation  expression  is the major protein involved in the humoral immune response against asmodium . Plasmodormation . Recentlocytosis . The priasmodium . Hemocytasmodium . Furtherlciparum . Mosquitpression . Inside  oocysts . Oocystsormation . Sporozoormation . Sporozo4Plasmodium expresses a variety of surface antigens during its developmental stages- pre-erythrocytic stage, erythrocytic stage, gametocyte/sexual stage and mosquito stage. Over the past few decades, various anti-malaria vaccine candidates have been assessed from different parasite stages  and secondary human host (pre-erythrocytic stage). CSP protein of P. falciparum sporozoites contains highly conserved protein domains structures which have been characterized by repeating amino acid, asparagine-alanine-asparagine-proline (NANP) motifs  along with poly (I:C) adjuvant. Vaccination with PfTRAP induced Th1 immune response and high titers of protective IgG antibodies  has been shown to improve immunogenicity and enhance protection. R21 is an emerging vaccine formulation which is under phase II field trials and needs further investigations (PfCSP (AAV8-PfCSP) generated protective humoral and cellular immune responses by inducing high antibody titres and recruiting liver-resident memory CD8+ T (TRM) cells in a mice model  motifs . CSP has) motifs . Current protein . However protein . RTS,S/A protein . Anothertibodies . In anoty titers . Recent y titers , necessiigations . In anotce model . In addiresponse , 115.Plasmodium-specific TRM cells which confer protection in mice against wild-type sporozoite challenge. RAS vaccination strategy has been improved by prime and trap strategy which involves epidermal priming of CSP antigen. A single intravenous dose of RAS aid in the activation of TRM in the spleen, along with trapping and expansion of CD8+ T-cells in the liver region of BALB/c mice  vaccines induces B/c mice . Furtherhallenge . Ribosom in mice .®PfSPZ-GA1 is a genetically attenuated whole sporozoite vaccine. It was generated by knocking out B9 and SLARP genes to halt the development of sporozoites in the early liver stages  GAP was generated against the human malaria parasite. Specifically, a LARC GAP for P. falciparum was generated by deleting the Mei2 (Meiosis inhibited 2) gene. The Mei2 gene is expressed by the late liver-stage parasite. -PfMei2 liver stages failed to complete their intra-hepatic development and do not form infectious exoerythrocytic merozoites . In this approach, human volunteers are immunized with cryopreserved PfSPZ along with a 10 mg/kg chloroquine base. PfSPZ-CVac immunization conferred protection in malaria-naive volunteers by inducing high levels of anti-PfCSP antibodies (PfSPZ-CVac (CQ) was safe and conferred protection to malaria-naïve participants in controlled human malaria infection, this vaccine was unable to protect against P. falciparum infection in a very high transmission setting , utilize genetic attenuation/deletion of genes essential for the completion of liver stage development . Sanariar stages . Anotheric stage . The PfGhallenge . Anothernfection . In addinfection . Recentlrozoites . Anothertibodies . While P setting .P. berghei-based vaccination (PbVac) confers cross-species protection against P. falciparum malaria (P. berghei is highly amenable to a genetic modification that enables the gene insertion of other human Plasmodium species antigens (such as CSP) into its genome loci, which may aid in the expression of heterologous Plasmodium antigens  protein sequences were designed. Administration of PfAMA1-DiCo along with Alhydrogel to malaria-exposed adults resulted in a significantly higher antibody response against DiCo variants  based vaccination strategies are considered an efficacious vaccine delivery platform for multiple antigens. Three VLPs, MSP8, MSP9 and RAP1 (Rhoptry-associated protein) were complexed with influenza virus matrix protein. Mice were immunized with a mixture of these VLPs and challenged with on later . VLP vacal blood . Based onfection . Anothervariants . Althougnfection . In a reproaches .4.3Plasmodium. Several parasite proteins are expressed exclusively by gametocytes and constitute targets for malaria transmission-blocking vaccines (TBVs)  . These c PfMDV-1 . Among Tandidate . The admith time . The effw months .Pfs230 or Pfs25 antigens with EPA along with Alhydrogel. As compared to Pfs25, the Pfs230 vaccine induced a much greater complement-dependent transmission-blocking activity in humans  induce antibodies which show transmission-reducing activity against Plasmodium. Both Pf77 and PfMDV-1 display less antigenic polymorphism and are known to induce naturally occurring antibodies in individuals living in endemic areas of Africa. These antigens are highly immunogenic and can induce transmission-reducing antibodies which may aid in the reduction of oocyst counts in Anopheles mosquito midgut  and innate immune responses of the mosquito midgut. There are some protein antigens such as PM4 (aspartic protease plasmepsin 4) and CHT1/CHT2 (chitinase) which may prove to be transmission-blocking targets of Plasmodium ookinete. Antibodies against both PM4 and CHT1 block the passage of ookinetes through ECM, thereby reducing oocyst counts and infectivity of malaria  along with TLR-based adjuvant, elicited specific anti-PfCelTOS antibody-mediated immune response, which has been shown to induce transmission-reducing activity in mosquito  is a merozoite surface antigen involved in RBC invasion. In one pre-clinical study, it was found that vaccine formulation containing CyRPA along with Alhydrogel elicit neutralizing antibody and anti-parasite cytokine response in mice. Therefore, it could be a potential vaccine candidate against blood stages of P. falciparum infection  protein. A total of 10 epitopes (5 B and 5 HTL epitopes) were linked by suitable linkers along with flagellin adjuvant to enhance the immunogenicity of the vaccine construct . Anotheproteins . Since tvaccines . A multionstruct . While iluations .Pfs48/45-6C protein with GMZ2 (a fusion protein of GLURP and MSP-3). GMZ2.6c vaccine efficacy can be enhanced by using TLR4 agonists which have been reported to induce parasite-specific antibodies and T-cell-mediated immunity in mice models  present on the surface of the syncytiotrophoblast of the placenta  of miRNA are more advanced, fast and effective methods for studying novel malaria vaccine antigens. The miRNA-based translational repression can be monitored within a few days. It can be used for the characterization and identification of malaria vaccine antigens from different developmental stages which may contribute to the development of effective subunit vaccines (P. vivax merozoites contain Duffy binding protein (PvDBP) which is involved in reticulocyte invasion via interaction with DARC (Duffy antigen receptor for chemokines) receptors present on host reticulocytes (PvDBP (PvDBPII) serves as an attractive target. However, the generation of a DBP-based vaccine is still a distant dream and further investigations are required to prove its efficacy against P. vivax malaria  is a whole sporozoite based vaccination approach. CPS immunization has been shown to induce sterile immunity in human volunteers against pre erythrocytic stage of P. falciparum  isolated from an RTS, S-immunized individuals, imparted sterilizing immunity , isolated from the whole sporozoite immunized mice  showed inhibition of liver infection when mice were challenged with sporozoites (PfCSP), was intravenously administered to patients which protected against controlled malaria challenge  and erythrocyte-binding antigens (EBA) are also targets of antibody response , and some of these antibodies were seen to exhibit anti-parasitic activity in vitro (P. vivax (PvMSP1P) can also reduce parasite invasion  can inhibit parasite invasion into reticulocytes  exhibited both the growth inhibitory and neutralizing activity against the Plasmodium parasite  in naturally acquired immunity to malaria is still unclear  have been linked to protection against malaria  (expressed in schizont-infected red blood cells), decreased parasite replication by arresting schizont rupture, and maternal antibodies to PfSEA-1 protected infants from severe malaria  by inducing complement-mediated lysis or promoting phagocytosis  markedly reduced early Plasmodium infection in a murine model  can block malaria transmission. Membrane-associated erythrocytic binding protein (MAEBL) is expressed in the liver stages. It is required for sporozoite infection of mosquito salivary glands and antibodies against MAEBL partially inhibit hepatocyte invasion by sporozoites and/or liver-stage development  was found to slow blood-stage malarial parasite growth. It may protect against dual-stage, species and strain-transcending malaria  family proteins can result in enhanced apoptosis of infected hepatocytes   and CD55 facilitate merozoite invasion to erythrocytes , 276. Thecule-4) . Treatmectivated . Blood sctivated , MEK1 inin vitro . Therefoin vitro . Plasmodnfection . One recnfection .6.3PfGPI-induced host inflammatory responses play an important role in the pathogenesis of severe cerebral malaria. PfGPI stimulates host macrophages and induces TNF-α secretion via activating MAPK pathways, including JNK2. Therefore, treatment with JNK2 inhibitors can decrease TNF-α secretion, thereby reducing inflammation in mice models of cerebral malaria (Aptamers are ss-oligonucleotides (ssDNA or RNA) which can recognize, bind and alter the activity of targeted molecules. It has been suggested that aptamers targeted against host cell-matrix receptors could be used in blocking the interactions between parasites and host cells . A combi malaria . Interes malaria . The bra malaria .P. vivax and P. falciparum for their development during blood stages, therefore, AQP3 inhibitors may contribute to pan anti-malaria activity (Furthermore, AQP3 is required by both activity , 290. Alactivity , 290. Anactivity .7The parasite immune evasion strategies contribute to parasite survival and are considered a big obstacle in developing effective therapeutics against malaria. Research gaps yet remain in our understanding of host-parasite interactions and insights into these mechanisms are of utmost importance for developing effective vaccines and immunotherapies that can overcome immune evasion mechanisms and induce long-lasting immunity against malaria.in vitro production of P. falciparum sporozoites warrants more research for malaria vaccine development. Moreover, most of the vaccination studies are based on P. falciparum and vaccine research on the second most malaria-causing strain P. vivax is lagging far behind. Furthermore, research on P. vivax candidates is limited due to difficulties associated with in vitro continuous culturing of P. vivax and only a few P. vivax vaccine candidates have reached to clinical development stages. Therefore, there is an emergent need to expand the repository of P. vivax vaccine candidates for demonstrating heterologous protection (in silico-based approaches, a combination of different vaccine antigens from multiple stages of the Plasmodium life cycle may prove beneficial for developing a multi-antigen or multi-stage vaccine against malaria. Additionally, improving vaccine protection utilizing a staggered, segmented dosage regimen and other alternative adjuvants along with novel delivery systems must be explored. Development of a variety of vaccine platforms including VLP-based, multi-stage chimeric, GAPs, LARC GAP, mRNA vector-based, CPS-based and nanoparticle-based vaccines along with immunogenic adjuvants that can elicit robust immune responses are currently underway (RTS,S is the most promising anti-malaria vaccine to date which has completed phase III clinical trials. However, its limited efficacy and geographically regional effect have been seen in many studies. Compared to RTS,S and other subunit vaccines, the whole sporozoites-based vaccine has had more success . Despiteotection . Althougunderway Figure 1P. falciparum and P. vivax helps to identify conserved epitopes that aid in vaccine development. Such approaches can also be extended to various stage-specific antigens of Plasmodium. Combination therapy with bispecific antibodies or cocktails of antibodies can be prepared that can target different antigens of parasite stages. Various antibody effector functions such as promoting neutrophil burst, NK cell-mediated killing, phagocytosis, agglutination, schizont egress inhibition, rosette inhibition, complement fixation, antibody-dependent complement-mediated lysis, inhibition of adhesion of infected erythrocyte and inhibition of merozoite invasion, have been investigated in various studies (Since humoral immunity contributes to immune defence mechanisms against malaria, antibody-based therapeutics may prove beneficial in the prevention or treatment of malaria. Monoclonal antibody-based therapy is of particular interest in containment and/or outbreak zones where active malaria transmission is confined to a particular area, season and travellers. Development of human monoclonal antibodies against key vaccine targets of  studies Figure 2Host-directed therapy, which is another major area of therapeutics, has emerged recently. Malaria parasite relies on a network of host pathways which contribute to its development. A wide variety of host factors that are involved in the development of the parasite present novel opportunities for host-directed therapies in malaria. Host-directed therapy particularly targets the host factors which makes parasites deprived of these essential factors needed for parasite invasion, multiplication, and survival inside host cells. Host-directed therapy acts synergistically with anti-malarial drugs and could also be used as novel adjunctive therapy in treating malaria. Furthermore, combining antimalarial drugs with vaccine/s or antibodies and using them as an adjunct therapy show potential to reduce the prevalence and transmission of malaria. It is expected that these combined approaches including antibody-based therapy, host-directed therapy and the development of novel and efficacious vaccines can contribute to the current goal of WHO for a malaria-free world. In summary, a thorough comprehension of the equilibrium existing between the host immune system and parasite immune evasion mechanisms is extremely important for the development of efficient immunological therapeutics.PC: original draft preparation, reference collection, and manuscript writing; RR: manuscript editing; SK: manuscript editing; SR: conceptualization, supervision, manuscript editing, and proofreading. All authors contributed to the article and approved the submitted version.


Acute undifferentiated febrile illness (AUFI) is the presenting symptom of various tropical and infectious diseases. Viral infection is generally the most common cause of AUFI, accounting for 8–11.8% of cases; thus, antibiotics might be unnecessary. Dengue and malaria are common tropical infectious diseases requiring effective supportive treatment and antimalarial agents, respectively. The uncertainty of early diagnosis results in widespread empirical antimicrobial treatment in high -income as well as in low-and middle-income countries. Although rapid diagnostic tests (RDTs) have been shown to limit antibiotic prescriptions in dengue and malaria, we observed a wide range of antibiotic prescriptions for 13–92.7% of cases in previous literature, particularly in RDT-negative malaria cases. Given several RDT limitations, antimicrobial stewardship (AMS) appears to be an effective strategy for controlling unnecessary antibiotic use and antimicrobial resistance (AMR) prevention. This program should be endorsed by a multidisciplinary team in tropical diseases to control collateral damage of inappropriate antimicrobial use. Empirical antibiotic treatment should be administered based on clinical judgement, microbiological evidence, and local epidemiological data. Rapid termination of antibiotic therapy, including disease control or elimination, is the mainstay of AMS in tropical diseases. Local and international sectors should implement an AMS programme to reduce AMR in the Tropics. Acute undifferentiated febrile illness (AUFI) is a common presentation of tropical infections. Dengue is hyperendemic in tropical and subtropical countries , predomiAUFI is a common presentation of tropical infectious diseases or bacterial infections without organ specific infection. This presentation is caused by a diverse range of pathogens and can vary between regions. A review in Southeast and South Asia found that viral etiologies were most common, followed by bacterial. Of the viral causes, dengue fever was the most common, accounting for 11.8% of cases, followed by leptospirosis, typhoid, scrub typhus and influenza . As dengStaphylococcus aureus followed by Salmonella Typhi as the most commonly identified pathogens.In a review study conducted in Sub-Saharan Africa, Marks et al. found that malaria was the most common cause of AUFI in children in Burkina Faso and Sudan . AnotherSpecific rapid diagnostic tests (RDTs) are required in malaria and dengue, both of which do not need empirical antibiotic therapy. Furthermore, this strategy may directly affect patients and indirectly introduce selective pressure, leading to AMR. The lack of a decision algorithm following negative RDTs in healthcare facilities can increase unnecessary antimicrobial use . The comMonitoring antibiotic use in AUFI is important to improve prescribing practices in the future. Previous literature on the types of antibiotics used in both malaria- and non-malaria- endemic regions was reviewed.A study conducted in Pune, India explored the use of antibiotics in suspected and diagnosed mosquito-borne illnesses among children and adults with acute febrile illnesses. Robinson et al. demonstrated, that on admission, 82% of adults and 94% of children received empirical antibiotics , i.e., tAnand Paramadhas et al. conducted a point prevalence study on antimicrobial use in public and private hospitals in Botswana, where human immunodeficiency virus (HIV) infections, tuberculosis (TB), and malaria were endemic. Cefotaxime followed by parenteral metronidazole were the most commonly prescribed antimicrobial agents in all public hospitals, whereas ceftriaxone was the second highest antimicrobial prescribed in specialist private hospitals . Of 711 A point prevalence survey conducted by Guterres et al. in Indonesia identified ceftriaxone (15%), levofloxacin (9.2%) and ampicillin sulbactam (7.9%) as the top three antibiotics administered, mostly for the treatment of community-acquired infections, hospital-acquired infections, and prophylaxis, among inpatients in a national referral hospital . A pointThe aforementioned studies indicated that third-generation cephalosporin was the most commonly used antibiotic for febrile illnesses in both in- and outpatient settings. Prompt definite diagnosis remains problematic, resulting in the high rate of empirical antimicrobial agent prescriptionClostridioides difficile diarrhea [Distinguishing AUFI etiologies on presentation has been difficult, particularly when the mainstay of diagnosis is clinical history and examinations. The non-specificity of symptoms and signs and the lack of availability of diagnostic tests often lead to empirical and irrational use of antibiotics ,7,8. Nevdiarrhea . COVID-1diarrhea . HoweverSalmonella spp. ranged between 40% and 80%, and in dengue or rickettsiosis it is impossible to obtain both acute and convalescent sera, which results in delayed diagnosis [In addition, several limitations have been reported in various diagnostic tests; for example, blood culture sensitivity for iagnosis . Improveiagnosis .The aforementioned limitation of diagnostic tests remains problematic. To help clinicians in their decision, biomarkers such as C-reactive protein (CRP) and procalcitonin play some role in distinguishing bacterial and viral infections . In a stAlthough procalcitonin has been found to be specific to bacterial infections, it can be applied to tropical infectious diseases, such as dengue, to predict the severity. Thanachartwet et al. showed that procalcitonin and peripheral venous lactate levels were independently associated with dengue shock and/or organ failure . AdditioRDTs have been shown to influence antibiotic prescription. In a study conducted on the impact of dengue RDTs on antibiotic and anti-inflammatory prescriptions in Colombia, Tello-Cajiao et al. found that antibiotics were prescribed in 3% of RDT-positive patients and 14% of RDT-negative patients, resulting in a decreased risk of prescribing antibiotics with a positive test . HoweverThe treatment for malaria involves the use of antimalarials rather than antibiotics. Like antibiotics, inappropriate use of antimalarials can lead to resistance. The barriers to definite diagnosis can result in misdiagnosis and subsequently overtreatment, as displayed in Leslie et al.’s observational study in Afghanistan, where 413 of 414 patients had a negative malaria smear but 412 (99%) were prescribed an antimalarial drug . The WHOIn a different study in rural Tanzania, Njozi et al. found that predictors associated with the risk of antibiotic co-prescription with antimalarials were age groups and types of diagnosis . ChildreAnother factor to consider in malaria resistance is the implementation of over-the-counter antimalarial prescriptions in some countries, such as Kenya, which eventually result in overuse, as highlighted by its popularity even in areas with low and seasonal malaria transmission . Abuya eWe reviewed antibiotic use in malaria and dengue, as summarised in Figure 1. We classified previous literature into the following five scenarios: the proportion of empirical antimicrobial treatment, antimicrobial prescription in positive RDTs or blood smear, antimicrobial prescription in negative RDTs or blood smear, the proportion of inappropriate antimicrobial use, and the proportion of bacterial co- or concurrent infection. This review demonstrated that the proportion of antibiotic prescriptions in dengue and malaria greatly varies throughout the literature, especially in dengue. In addition, when malaria RDTs or blood smears were negative, the proportion was higher than when they were positive.9/L, hematocrit < 40%, serum sodium < 135 mmol/L, and serum urea ≥ 5 mmol/L. The DDIS showed the satisfied validation set area and good specificity to differentiate dengue patients for empirical antibiotics [Antibiotic use in dengue or malaria would be appropriate for bacterial co-infection. The incidence of bacterial co-infection in dengue appears to be low, although the exact numbers have not been extensively elucidated. Studies report the incidence to be as low as 7% and as high as 25% ,44. Concibiotics .Bacterial co-infection in malaria can result in higher morbidity and mortality and most commonly occur in falciparum malaria. Although both antibiotics and antimalarials are recommended in children with severe malaria, the aforementioned guidelines suggest that adults with severe malaria should not receive empirical antibiotics . BacteriTo our knowledge, there is no definite study on the causal relationship between inappropriate use of antibiotic and subsequently multidrug resistant (MDR) bacteria emergence in dengue or malaria. Adrizain et al. reviewed that the consequences of broad-spectrum antibiotics were the same as non-dengue patients .Although RDTs were generally used for the early diagnosis of dengue and malaria, several limitations have been recognised. For example, the capability of malarial RDTs for parasite detection and the intervention of temperature and humidity to the test accuracy had been observed ,52. DengAMS refers to a coordinated programme that promotes the appropriate use of antimicrobials to improve patient outcomes, reduce AMR and decrease the spread of infections caused by multidrug-resistant organisms . This teAMS can vary in LMICs and HICs. Initially, most research and evidence of AMS was restricted mostly to HICs but has now been redefined on a global scale . LMICs, AMS programmes require support and collaboration from all healthcare workers. A knowledge and perception study by Adegbite et al. in Gabon found that 64% of prescribers reported awareness that AMR is an issue in their country; however, only 30% considered it an issue in their health facilities . This coRDTs affected the antibiotic consumption burden, as our review showed that the highest proportion of antibiotic prescription was observed among RDT-negative cases. Unfortunately, antibiotic consumption remained high in RDT-positive cases, emphasising that AMS implementation is urgently needed in tropical diseases. Although empirical antibiotic treatment in early AUFI cases is mandatory to reduce the mortality rate, we encourage its immediate termination whenever RDTs show positive results without evidence of bacterial infection. Bacterial co-infections or concurrent infections vary among previous literature because of different epidemiological data at the country or hospital level. Accordingly, doctors in charge should decide based on their clinical judgement, microbiological results and local epidemiological data regarding antibiotic initiation and cessation.The evaluation of antimicrobial appropriateness of tropical diseases in previous literature is limited. Forty-two per cent of patients with malaria treated with antibiotics had no evidence of bacterial infection, whereas >10% of patients with malaria do not receive them . TherefoPrevious studies showed a wide range of antibiotic use in tropical diseases, particularly in dengue and malaria. RDTs are the supporting tools for the definite diagnosis in early AUFI to limit the overuse of empirical antibiotics. However, RDTs have several limitations; thus, an AMS programme with a multidisciplinary team is another strategy to control antimicrobial inappropriateness. Clinical judgement, microbiological evidence and local epidemiological data are the factors used to determine appropriate empirical treatment. Rapid antibiotic cessation, including disease control or elimination, is the mainstay of AMS in tropical diseases. We encourage local and international sectors to foster regular antimicrobial use evaluation in tropical diseases since an AMS program is useful to AMR reduction.


Plasmodium falciparum is responsible for the most severe and deadliest human malaria infection. The most serious complication of this infection is cerebral malaria. Among the proposed hypotheses that seek to explain the manifestation of the neurological syndrome in cerebral malaria is the vascular occlusion/sequestration/mechanic hypothesis, the cytokine storm or inflammatory theory, or a combination of both. Unfortunately, despite the increasing volume of scientific information on cerebral malaria, our understanding of its pathophysiologic mechanism(s) is still very limited. In a bid to maintain its survival and development, P. falciparum exports a large number of proteins into the cytosol of the infected host red blood cell. Prominent among these are the P. falciparum erythrocytes membrane protein 1 (PfEMP1), P. falciparum histidine-rich protein II (PfHRP2), and P. falciparum heat shock proteins 70-x (PfHsp70-x). Functional activities and interaction of these proteins with one another and with recruited host resident proteins are critical factors in the pathology of malaria in general and cerebral malaria in particular. Furthermore, several neurological impairments, including cognitive, behavioral, and motor dysfunctions, are known to be associated with cerebral malaria. Also, the available evidence has implicated glutamate and glutamatergic pathways, coupled with a resultant alteration in serotonin, dopamine, norepinephrine, and histamine production. While seeking to improve our understanding of the pathophysiology of cerebral malaria, this article seeks to explore the possible links between host/parasite chaperones, and neurotransmitters, in relation to other molecular players in the pathology of cerebral malaria, to explore such links in antimalarial drug discovery. Plasmodium species, five of which are known to infect human, namely, Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, with P. vivax and P. falciparum known to cause the most lethal infection  is a deadly complicating manifestation of severe n Africa . Cerebra 15%–25%  leaving  15%–25% . These m 15%–25% . For ins 15%–25% . There a 15%–25% . While d 15%–25% . Months  15%–25% . Meanwhi 15%–25% . However 15%–25% , necessi 15%–25% . Neverth 15%–25% . In addi 15%–25% . TherefoP. falciparum exports a large number of proteins into the cytosol of the infected host red blood cell (iRBC)  . Prominel (iRBC) . Detailel (iRBC) . Furtherl (iRBC) . Availabl (iRBC) . While svia immune evasion used by the parasites to avoid the removal of the cells by the spleen , ensuring an increased affinity of iRBCs to various receptors, notably, cytokine-activated endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1) on the brain endothelial cells , and that of the release of copious amounts of pro-inflammatory cytokines, have been put forward for the pathogenesis of CM, they do not fully account for disease progression, prognosis, and outcome . The vase spleen . Moreovee spleen . Human Ce spleen , and plae spleen . Sequeste spleen , which ce spleen . Meanwhial cells . This isal cells , and eveal cells .P. falciparum, causing an imbalance in systemic inflammatory responses, which are exacerbated due to sequestration and cytoadherence of iRBC , ring erythrocyte surface antigen (RESA) in iRBCs and increase the formulation of adhesion molecules on endothelial cells to aid binding of surface proteins, thereby proliferating vascular permeability in several organs . The PfHRP2 is known to be transported into the cytosol of infected erythrocytes . Interestingly, this suppression can be reversed in the presence of glutamine, which upregulates human Hsp70, thereby promoting the activation of NF-κB, and attenuation of overexpression of proinflammatory cytokines , norepinephrine, histamine, and serotonin (5-HT) . For ins—crystallin , requires further investigations.Cerebral malaria is a deadly complication of severe