Plasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium.18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte.Whole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL.Overall, 87.5% of the 26 samples, previously diagnosed as P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.For the first time, a molecular detection of The online version contains supplementary material available at 10.1186/s12936-023-04601-7. According to the World Health Organization, in 2021 there were 247 million cases and 619,000 deaths from malaria in 85 endemic countries .Plasmodium species: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasmodium knowlesi is a primate malaria parasite that primarily affects long-tailed and pig-tailed macaques, which has emerged as an important human pathogen in recent years, responsible for over 70% of human cases in southeastern Asia [Plasmodium species that cause infection in non-human primates (NHP) are of great interest because they can be naturally-transmitted to humans, thus representing a challenge to malaria elimination.Most human malaria infections are caused by five ern Asia –7. PlasmP. knowlesi, at least other three species, which infect primarily NHP, are involved in the zoonotic transmission of malaria to humans, including Plasmodium cynomolgi in Asia [Plasmodium brasilianum and Plasmodium simium in the Americas [Plasmodium simium, a parasite of a small number of species of Neotropical monkeys, is restricted to the Atlantic Forest from Southeast and South regions of Brazil and it was recently shown to cause zoonotic infections in humans [P. vivax in the Atlantic Forest areas of the state of Rio de Janeiro have been shown to be, in fact, caused by P. simium [Plasmodium simium and P. vivax are genetically, morphologically, and immunologically similar, and the brown howler monkey (Alouatta guariba clamitans) has been suggested to be the main reservoir host of P. simium [P. simium infection occurs in this species, but their potential for natural transmission (i.e. infectivity to mosquitoes) in this setting is not yet known.Besides  in Asia  and PlasAmericas –12. Plas. simium , 13, 14.Plasmodium infection for humans presents a challenge for malaria elimination, as they contribute to the persistence of the parasite and act as source of re-introduction into human populations, in areas where infection has otherwise controlled [Plasmodium for mosquitoes, in reservoir hosts are important factors to estimate their potential for malaria transmission [Despite the importance of parasites with potential for zoonotic transmission, little is known about their epidemiological importance to infect and cause disease in humans. The potential for NHP to act as reservoirs of ntrolled –17. In tsmission –20. Furtsmission , 21.P. falciparum and P. vivax, respectively [Pvs25 gene is highly conserved among P. vivax isolates [Plasmodium is the P48/45 protein, which is expressed in both male and female sexual stages starting at stage II gametocytes and continuing until fertilization is complete and forms a complex with P230 [Plasmodium species, again exhibiting low levels of genetic diversity [P. vivax showed that they cluster in two groups of co-regulated genes, one includes Pvs25 and the other Pvs48/45, suggesting that the regulation of male and female genes is independent of each other [Among the proteins expressed in large quantities after gametocyte activation in the midgut of the mosquito are the P25 orthologues Pfs25 and Pvs25, of ectively . Althougisolates –25, makiisolates . Anotherith P230 –28. Despith P230 , and is ch other .P. simium, which were potentially infective to human beings. This finding can contribute to understanding the occurrence and intensity of malaria transmission from NHP reservoir hosts to mosquito vectors, and so help to define public policies for the control, prevention and eventual elimination of malaria.The aim of the current study was to assess the potential for malaria transmission by naturally-infected howler monkeys through identification of gametocyte transcripts and estimation of gametocyte density through reverse-transcription quantitative PCR. Consequently, it was possible to identify through molecular methods howler monkeys from the Brazilian Atlantic Forest infected with gametocytes of Capture, handling and blood sampling of free-living primate in the municipality of Joinville in the state of Santa Catarina, Brazil, was approved by the Ethical Committee on the Use of Animals of the Fundação Universidade Regional de Blumenau (FURB) under the protocol nº 012/15. The Brazilian government authorized this study, access to, and transport of, biological samples through the Sistema de Autorização e Informação em biodiversidade (SISBIO) no. 43375–4/2015 (for CPRJ samples) and nº 43375–6 (for Joinville samples).Alouatta guariba clamitans (the Atelidae family) (n = 33) and the black-headed uakari, Cacajao melanocephalus (the Pitheciidae family) (n = 2). The samples used here have been both previously diagnosed and published by our group using conventional PCR of 18S rRNA locus [cytochrome c oxidase I locus [P. simium-infected NHP, five P. brasilianum-infected NHP, and 13 NHP with mixed-infections (i. e. both P. simium and P. brasilianum), as well as six NHP diagnosed as non-infected. Aliquots of whole blood samples were stored in RNAprotect (Qiagen) at a 1:5 ratio and stored at −20 °C until RNA extraction.Whole blood samples were obtained from NHP from fragments of the Brazilian Atlantic Forest located in the states of Santa Catarina and Rio de Janeiro. The sampled animals were free-living NHP from Joinville n = 32) and captive primates from the Centro de Primatologia do Estado do Rio de Janeiro (CPRJ) (n = 3), comprising two species of the Neotropical primates, the brown howler monkey,  and captily) n =  and the  I locus . The sam® IV Reverse Transcriptase  and random primers , according to the manufacturer’s instructions. For a final volume of 20 μL, a reverse transcription reaction mix was made containing 1 μL of 50 μM random hexamers, 1 μL of 10 mM dNTPmix, up to 11 μL of template RNA (up to 500 ng mRNA), and up to 13 μL of nuclease-free water. The reaction was incubated at 65 °C for 5 min, and then on ice for at least 1 min. Next, 4 μL of 5 × SSIV Buffer, 1 μL of 100 mM DTT, 1 μL of RNaseOUT™ Recombinant RNase Inhibitor and 1 μL of SSIV were added. The reactions were performed on a Veriti 96-well Thermal Cycler (Thermo Fisher Scientific) at 23 °C for 10 min, 55 °C for 10 min, and then 80 °C for 10 min.RNA extraction was performed using different blood in RNAprotect volumes (100 to 1000 μL) using the commercial RNeasy Mini Kit (Qiagen), according to manufacturer’s protocol, which resulted in 30 μL of RNA. DNA removal was done immediately after RNA extraction using the Turbo DNA-free™ Kit . In order to synthesize the complementary DNA (cDNA), reverse transcription was performed using the enzyme SuperScriptPlasmodium infections and detection of P. simium gametocytes, three different quantitative PCR (qPCR) protocols were performed using the cDNA obtained as described above. For the 18S rRNA transcript, the primers used were those described by Wampfler et al. [Pss25  transcript, the primers used were designed for Pvs25 by the same authors, because of the high identity between P. simium and P. vivax  transcript, new primers were designed using the OligoAnalyzer software based on the sequence available in GenBank . The best of primer sequences identified were 5'-CTCTACCGGAACCATGTTGAAG-3' (forward) and 5'-GACGTACTTGACCTCTCCTTTG-3' (reverse), which generate a fragment of 109 base pairs. For 18S rRNA transcript amplification, the qPCR reaction was performed using a 10 μL final volume containing 900 nM of each primer, 5 μL GoTaq® qPCR Master Mix, and 1 μL cDNA. The Pss25 and Pss48/45 reactions were performed, separately, using 10 μL final volume containing 200 nM of each primer (forward and reverse), 5 μL GoTaq® qPCR Master Mix, and 1 μL cDNA. The qPCR assays were performed on an automatic thermocycler ViiA7 Real-Time PCR System (Thermo Fisher Scientific) with an initial denaturation at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. A final cycling for dissociation curve analysis of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s was used. A sample previously diagnosed as P. vivax by 18S RNA PCR described by Snounou et al. [18S rRNA, Pvs25 and Pvs48/45 was used as a positive control in all qPCR assays, as well as a negative control (without cDNA). The results of the qPCR were analysed using the QuantStudio Real Time PCR Software v1.3.7.For confirmation of r et al. . For theu et al. , and pos18S rRNA and Pvs25 fragments of interest were previously prepared by Salazar [P. simium gametocytes. Plasmid DNA concentration was obtained by fluorimetric quantification using a Qubit 4 (Invitrogen). The plasmid copy number (PCN) was calculated to determine the dilutions to be used for the standard curve, which was constructed based on seven ten-fold serial dilutions (ranging from 1 × 106 to 1 × 101). The number of gametocytes based on Pss25 transcript copy number were estimated using a previously published conversion factor  calculated by Koepfli et al., which was based on a random-effect model from log10-transformed quantities of gametocyte trend-lines [Plasmids containing the  Salazar  and werend-lines . For the18S rRNA, Pss25, and Pss48/45, while non-linear correlation was used to compare the copy number between the 18S rRNA and Pss25 transcripts, through the non-parametric Spearman’s rank correlation coefficient. The sample J14 was excluded from the linear correlation analysis. The significance level of 5% was considered for all analysis.The statistical analyses were performed using GraphPad Prism 8.0.2 . Linear regression was used to compare the Cq values obtained for Plasmodium infection by RT-qPCR of 18S rRNA, and gametocyte-specific identification through detection of the Pss25 and Pss48/45 transcripts , mostly in agreement with our previous molecular diagnosis .Thirty-five samples of NHP previously evaluated for simian malaria were used for detection of P. simium samples that were positive for the 18S rRNA transcript (n = 21), 13 samples were also positive for Pss25, indicating that 61.9% of the 18S rRNA-positive samples contained P. simium gametocytes, of which seven samples also amplified Pss48/45 transcripts (i. e. 53.8% of the Pss25 positive samples)  were negative for all three assayed loci. Another nine samples showed discordant results between our previously molecular diagnosis and the amplification reported here of 5.7% were18S rRNA, with an average of 24.6 (range 13.8 to 33.5), while those observed for Pss25 were higher with a Cq average of 32.1 (range 24.9 to 38.4)    . The non-linear Spearman's rank correlation coefficient revealed a positive correlation between the copy numbers of Pvs25 and 18S rRNA   was very low (less than 1 gametocyte/µL), except for one animal (J14) which showed a high density of gametocytes (5.8 gametocytes/µL)  have been suggested to be the main reservoir of malaria in Atlantic Forest areas. In the current study, more than 60% of Plasmodium-positive samples from this non-human primate species were positive for molecular markers consistent with the presence of gametocytes in their blood, demonstrating the potential of this vertebrate host species to act as a reservoir for malaria transmission. The failure to detect gametocytes in all P. simium-infected howler monkeys may be because the low densities of the gametocytes in the peripheral blood or because of their sequestration in the bone narrow, as showed for P. vivax gametocytes [P. brasilianum, had positive amplification using the primers to Pvs25 locus. This amplification was unexpected because of the many polymorphisms within the primers and probe binding sites in the Pvs25 orthologue of P. brasilianum  was positive for P. simium infection, but negative for both gametocyte transcripts. Since this species is an endemic of the Amazon, these results suggest that this monkey species might be a dead-end host for P. simium, without epidemiological importance for malaria transmission in the Atlantic Forest. However, because this animal was housed in captivity in the Atlantic Forest, it was not possible to exclude that the gametocytes were in low densities. This requires further investigation, since only two specimens of the black-headed uakari were included here, and large sample sizes are needed.In Brazil, the highest prevalence of malaria cases is in the Amazon region, with over 99% of the national notifications. However, autochthonous infections have also been described in the extra-Amazonian region, mainly in Atlantic Forest areas. The state of Rio de Janeiro recorded an average of four autochthonous malaria cases per year from 2006 to 2014 . Howevers region , 33. Broetocytes , 39. IntP. simium and P. brasilianum, all based on the detection of this stage through blood smears under an optical microscope [Plasmodium species, since gametocyte genes were not previously studied, and their sequences were only recently elucidated with the sequencing of the complete genomes of these two parasites [P. simium and P. vivax . Imwong et al. [P. vivax in malaria endemic areas and demonstrated that parasitaemia persists in humans at levels that optimize the probability of generating densities of transmissible gametocytes without causing illness. The authors reported that as gametocytes are eliminated more slowly than asexual stages from the peripheral blood, a significant proportion of the parasites detected by uPCR in asymptomatic individuals are likely to be gametocytes. Additionally, a recent study by Almeida et al. [P. vivax transmission to the vector Anopheles (Nyssorhynchus) darlingi, the main vector of P. vivax in the Brazilian Amazon. Although lower infectivity rates were observed for blood from asymptomatic individuals (2.5%) compared to symptomatic patients (43.4%), many asymptomatic carriers maintained parasitaemia for several weeks, indicating their potential role as an infectious reservoir. Symptomatic P. vivax infected individuals are able to infect mosquitoes at variable rates from 20–90% [P. vivax infections constitute the main infectious reservoir of this parasite [A deeper understanding of gametocyte carriers is essential to better understand their potential for malaria transmission. There are only a few studies of identifying gametocytes from croscope , 40, 41.arasites –44. Consy linked , 45. Mory linked . In Ethiinked [.  showed t9 to 92% . Kosasiho 92% [.  showed tg et al.  used an a et al.  using hum 20–90% , 55–57 am 20–90% , 49, 57.m 20–90% –61. Therparasite .. [Pvs25 transcripts correlates positively with the number of circulating mature gametocytes and can be used as an indirect estimate of gametocyte density in the sample. Koepfli et al. [Pvs25 transcripts into the number of gametocytes. Applying this conversion factor, which is defined for P. vivax, a high frequency of low gametocyte densities—less than one gametocyte/μL—was observed here. Therefore, as suggested for P. vivax human infections, the low densities of gametocytes could be compensated by the high frequency of infection. Nonetheless, one howler monkey showed higher levels of gametocytes, which may act as a potential “super-spreader” of P. simium infection. Historical studies have suggested around 10 gametocytes/μL is an infective density [Anopheles dirus and Anopheles stephensi [Anopheles from Kerteszia group has been incriminated as the main vector of malaria [P. simium transmission to mosquitoes. Previously, this density could not be identified, since the experimental infections performed using laboratory P. simium infection of monkeys only quantified infective gametocytes by microscopy and did not perform mosquito infections using a range of different gametocyte densities [Molecular methods have been developed to detect transcripts of gametocyte-specific genes, with a detection limit of 0.02–10 gametocytes per microlitre of blood , 64. Acc. , the numi et al.  has gone density , 66. Rectephensi , 67. In  malaria , 68. Hig malaria , togetheensities .Plasmodium life cycle in the Atlantic Forest.Interruption of malaria transmission is considered a priority task in the process of malaria elimination , 72. TheP. simium infected brown howler monkeys (Alouatta g. clamitans). This is strong evidence that howler monkeys are acting as Plasmodium reservoirs in the Atlantic Forest. Transmission could be maintained by the high frequency of low-level gametocyte carriers and the low frequency of high-level carriers. This finding will contribute towards the modelling of zoonotic malaria transmission and definition of public policies for malaria control, prevention and elimination.Gametocytes were detected in Additional file 1: Alignment of Pvs25 gene sequence and its orthologous from Plasmodium simium, P. malariae and P. brasilianum.


Plasmodium knowlesi is the major cause of zoonotic malaria in Southeast Asia. Rapid and accurate diagnosis enables effective clinical management. A novel malaria diagnostic tool, Gazelle  detects haemozoin, a by-product of haem metabolism found in all Plasmodium infections. A pilot phase refined the Gazelle haemozoin identification algorithm, with the algorithm then tested against reference PCR in a larger cohort of patients with P. knowlesi mono-infections and febrile malaria-negative controls. Limit-of-detection analysis was conducted on a subset of P. knowlesi samples serially diluted with non-infected whole blood. The pilot phase of 40 P. knowlesi samples demonstrated 92.5% test sensitivity. P. knowlesi-infected patients (n = 203) and febrile controls (n = 44) were subsequently enrolled. Sensitivity and specificity of the Gazelle against reference PCR were 94.6% (95% CI 90.5–97.3%) and 100% (95% CI 92.0–100%) respectively. Positive and negative predictive values were 100% and 98.8%, respectively. In those tested before antimalarial treatment (n = 143), test sensitivity was 96.5% (95% CI 92.0–98.9%). Sensitivity for samples with ≤ 200 parasites/µL (n = 26) was 84.6% (95% CI 65.1–95.6%), with the lowest parasitaemia detected at 18/µL. Limit-of-detection (n = 20) was 33 parasites/µL (95% CI 16–65%). The Gazelle device has the potential for rapid, sensitive detection of P. knowlesi infections in endemic areas. Plasmodium knowlesi infections across most Southeast Asia countries3 is exemplified by the significant ongoing burden of zoonotic malaria in Malaysia, with 17,125 cases and 48 deaths since 20174 despite near-elimination of other human Plasmodium species6. Rapid and accurate detection remains a crucial pillar of effective clinical management for zoonotic and human-only malaria as recommended by the WHO in all endemic settings7. Delays in treatment for P. knowlesi infections can lead to the development of severe disease and increased risk of fatal complications8.The public health implications of emerging P. knowlesi due to misidentification with other Plasmodium species10. Microscopy also requires trained laboratory technicians and equipment, necessitating ongoing quality assurance11. In Malaysia, the implementation of routine polymerase chain reaction (PCR) in Plasmodium species confirmation for all malaria cases since 201212 has enabled accurate public health reporting for P. knowlesi13. Although demonstrating an increasing P. knowlesi case incidence, particularly in East Malaysia14, routine PCR is not feasible for immediate clinical management and requires expensive laboratory infrastructure and trained personnel.Microscopy is the current standard for malaria point-of-care (POC) diagnosis in most endemic countries in Southeast Asia but is unreliable for P. knowlesi detection methods in 2017 in response to the increasing public health threat posed by zoonotic malaria transmission with an intractable monkey parasite reservoir15. One novel diagnostic approach utilizes rotating magneto-optical technology for the detection of haemozoin crystals, a paramagnetic by-product found in all malaria species16. The Gazelle™  is a battery-operated device. It functions by detecting changes in light signal intensity as it passes through magnetically-induced reorientated haemozoin crystals found in Plasmodium-infected blood samples collected from a finger prick or venous sampling. Previous studies have demonstrated the efficacy of the Gazelle™ in detecting P. falciparum17 and P. vivax18 when compared to nested PCR as the reference standard19. In addition to reporting detection limits for Plasmodium species infections as low as 40 parasites per microliter of blood20, the Gazelle™ was able to detect haemozoin in malaria-infected whole blood across early and late asexual stages of the parasite life-cycle21. Existing haemozoin-based detection assays have not been designed or evaluated for P. knowlesi human infections.The World Health Organization (WHO) Evidence Review group first recommended developing and improving POC P. knowlesi infections compared to reference PCR was assessed in a setting endemic for P. knowlesi.In this study, the performance of the Gazelle™ in detecting clinical 2), including 16 primary health clinics, with all cases of malaria admitted for in-patient management according to national guidelines12. Patients presenting at the hospital study site were included if they were positive for P. knowlesi by microscopy, were aged > 1 year, and they or their guardian provided appropriate written informed consent. Appropriate written informed consent was obtained from febrile individuals aged more than 12 years with negative malaria microscopy at the same healthcare facility, prospectively enrolled as controls. Patient blood samples were collected, and their demographic and clinical information were recorded on standardized case record forms as part of an ongoing prospective observational malaria study. Severe knowlesi was defined using WHO 2014 research criteria22 for P. knowlesi, including hyperparasitaemia threshold of 100,000/μL, and jaundice defined as bilirubin > 50 μmol/L with parasite count > 20,000/μL and/or creatinine > 132 μmol/L24.Malaria patients were enrolled between July 2020 and November 2021 at Ranau District Hospital in Sabah, Malaysia  and Menzies School of Health Research, Northern Territory, Australia (HREC 10-1431). Written informed consent was obtained from all participants and/or their legal guardians. All methods were performed in accordance with relevant guidelines and regulations.P. knowlesi parasitaemia was conducted by an experienced research microscopist equivalent to WHO Level 1 competency. The final parasite count per microlitre was calculated from the number of parasites counted to 200 white blood cells on a thick blood film, multiplied by the individual patient’s total white cell count25 obtained from routine hospital laboratory automated counts. The percentage of each parasite developmental stage26 was determined based on the number of early rings, late rings, mature trophozoites, schizonts, and gametocytes in 40 high-powered fields  on thin blood films.Venous whole blood samples were collected in ethylenediaminetetraacetic acid (EDTA) vacutainers prior to antimalarial treatment where possible. Thick and thin blood films were generated to verify parasite count by microscopy. Microscopic quantification of 27. The remaining volume of EDTA whole blood samples were stored at − 80 °C for subsequent malaria PCR species confirmation.A 30 µL blood sample (as specified in the manufacturers’ instructions) was used to evaluate the Gazelle™Plasmodium species: P. knowlesi, P. falciparum, P. vivax, P. malariae and P. ovale spp. Genomic DNA was extracted from 200 μL of whole blood using QIAamp DNA Blood Mini Kits  according to the manufacturer’s manual, with a final elution volume of 200 μL. PCR was performed by laboratory research members blinded to the microscopy results. A real-time PCR assay, QuantiFast™ targeting the 18S SSU rRNA gene was conducted once for each sample as per the manufacturer’s and Sabah Public Health Laboratory protocols, using the Bio-Rad CFX96 Touch™ PCR machine . QuantiFast™ real-time PCR was carried out over two separate reactions. The first was for Plasmodium genus screening, and if positive, was followed by subsequent P. knowlesi and other human Plasmodium species-specific detection28.Malaria PCR was conducted on all microscopically diagnosed malaria cases and microscopy-negative controls for the following P. knowlesi. Whole blood samples of microscopy-identified positive P. knowlesi, which were later confirmed via PCR, were placed into separate test cartridges, consisting of 30 µL of whole blood sample and 65 µL of assay buffer, then loaded into the Gazelle™ device27. Test results were displayed on the device screen within a minute and systematically documented by a trained research staff member. Each sample was tested only once. In the event of an invalid test result, a new cartridge was prepared to repeat the test. Daily calibration of the Gazelle™ device was conducted prior to testing of clinical samples and comprised running a haemozoin positive control , negative control (ingredients: assay buffer and dye), and an empty cartridge.An initial pilot phase was conducted to refine the Gazelle™ detection algorithm for P. knowlesi against P. falciparum.Pilot data from the Gazelle™ were uploaded to Hemex servers in real-time. Algorithm refinement was conducted at the completion of this initial study phase by reducing background noise, updating algorithm threshold values used for positive or negative detection, and optimizing the speciation algorithm for P. knowlesi malaria as well as malaria-negative febrile controls. A single empty cartridge was run daily as a blank sample negative control prior to testing clinical samples. Similar to the pilot phase, all patient samples were tested only once on the device.The refined detection algorithm was then tested on a subsequent larger prospective cohort of patients diagnosed with P. knowlesi parasite density limit of detection for the Gazelle™ device. Whole blood samples in EDTA from P. knowlesi infected patients with research microscopy parasite quantification were diluted using ABO blood-group matched  whole blood from healthy donors at twofold serial dilutions. Sample testing was performed up to the dilution whereby two consecutive negative readings were obtained. The limit of detection was defined as parasite concentration of the dilution tested prior to consecutive negative results. To increase the number of samples in this analysis based on the availability of matched blood group donors, the limit of detection was also tested on a subset of P. knowlesi clinical samples taken from patients who had already received anti-malarial treatment. To confirm and improve the quality and reliability of the limit of detection findings, research microscopy readings were also carried out on individual samples at each of the LOD dilutions.The secondary study analysis evaluated the P. knowlesi clinical infections for both the pilot phase and primary analysis were compared against reference PCR results using STATA v16 . Wilcoxon rank-sum test compared differences between the sex of patients  with age (median) as a dependent variable. The distribution of microscopy parasite counts for infections within defined groups were summarised and compared using the geometric mean. As defined below using the number of true positive (TP), false negative (FN), false positive (FP) and true negative (TN) results, diagnostic tests for sensitivity (TP/TP + FN), specificity (TN/TN + FP), and positive and negative predictive values were calculated29. Exact binomial confidence intervals of 95% for each of the above diagnostic metrics were calculated and reported. Overall device performance was compared by testing equality of the receiver operating characteristic (ROC) areas.The diagnostic accuracy of the Gazelle™ to detect Independent comparisons between the sensitivity of the Gazelle™ device versus reference PCR were conducted using Fisher’s exact test for: (a) parasitaemia of above or below 200 parasites/µL, and (b) samples collected before versus after administration of antimalarial treatment. For the latter, logistic regression was used to compare the device sensitivity further when controlling for median time in hours post-treatment. Logistic regression was used to confirm log-transformed parasite counts influenced Gazelle™ test positivity. For the limit of detection analysis, geometric mean LOD with 95% confidence intervals were estimated and reported using GraphPad Prism v9.4.1 . A p-value of less than 0.05 for 2-sided tests was considered significant. P. knowlesi malaria using screening by microscopy. Of this number, 513 (95%) received species confirmation via real-time PCR, as required by the Malaysian Ministry of Health guidelines for malaria.From July 2020 to November 2021, 1757 febrile patients presented at the hospital study site, including 540 individuals diagnosed with P. knowlesi cases, and a validation cohort with 203 P. knowlesi cases and 44 malaria-negative controls. Overall, 45% (110/243) of P. knowlesi cases and 80% (32/44) negative controls presented directly at the hospital and were not referred from catchment area clinics as mandated for all cases of microscopically confirmed malaria. Extrapolating the proportion of PCR-confirmed P. knowlesi cases and malaria-negative controls directly presenting at Ranau hospital yielded a crude estimate of background malaria prevalence of 18.8% (95% CI 16.7–21.1%) for this febrile cohort.A total of 287 febrile patients were enrolled into this study Table , with a P. knowlesi-infected patients in the pilot phase had a geometric mean parasite count of 577/µL (95% CI 331–1006/µL), ranging from 34 to 14,888/µL   for P. knowlesi malaria cases and 44 were malaria-negative controls. All patients had their malaria status confirmed via reference PCR  years, similar to the febrile controls who had a median age of 40 (range 12–80) years. The majority of malaria patients and controls were male, 80% and 70%, respectively. A statistically significant age difference was observed between the sex of the malaria patients, with a median age of 33 (IQR 22–48) years for males and 44 (IQR 29–56) years for females (p = 0.018).The validation phase prospectively recruited a further 247 febrile patients; 203 (82%) were confirmed CR Table . The medP. knowlesi-infected patients was 837/µL (95% CI 638–1099/µL), ranging between 18 and 331,727/µL. There were 2 adults enrolled with severe malaria according to WHO criteria22: one patient had jaundice with a parasite count of 30,906/μL, the other had both jaundice and hyperparasitaemia with a parasite count of 331,727/μL. All other patients were admitted with uncomplicated malaria.The median duration of self-reported fever prior to presentation was 4 days (range 1–14 days and 2–21 days) for both malaria cases and negative controls respectively. Eighty-three malaria patients (41%) and 5 febrile controls (11%) had a self-reported history of malaria but none within the 2 months prior to study enrolment. The geometric mean parasitaemia of the 203 P. knowlesi-positive samples tested on the Gazelle™, 143 were collected from patients prior to anti-malarial treatment. There were 60 patients with blood samples collected after administration of anti-malarial treatment, from which 95% (57/60) received at least one dose of oral artemether-lumefantrine and another 5% (3/60) received intravenous artesunate.Of the P. knowlesi samples collected and tested before any anti-malarial treatment, the reported test sensitivity was 96.5%  . The Gazelle™ recorded a sensitivity of 92.0% (95% CI 84.3–96.7%) for the 81 P. knowlesi patients with no previous self-reported malaria history.From the 143 P. knowlesi-infected patient samples evaluated after the initial administration of weight-based antimalarial treatment. The median time between treatment and blood sampling for Gazelle™ testing was 2.6 h (IQR 1.5–4.2 h). The geometric mean parasitaemia of samples tested in this group was slightly lower than those tested prior to treatment at 567/µL (95% CI 334–963/µL). When comparing test performance in this group to pre-treatment samples, a trend towards lower test sensitivity of 90.0%  was observed (p = 0.062). The median parasitaemia of six post-treatment P. knowlesi clinical samples with a false-negative result was 40 parasites/µL , with no statistically significant difference in the distribution of parasite counts between pre-and post-treatment groups.There were 60 additional P. knowlesi detection including both pre-treatment and post-treatment samples was 94.6% . The improvement in sensitivity for the validation cohort after refinement of the detection algorithm was not statistically significant compared to the overall pilot phase sensitivity of 92.5%.The combined sensitivity for The specificity of the Gazelle™ was 100% (95% CI 92.0–100%) for malaria diagnosis, as demonstrated by negative test results for all malaria-negative febrile controls. A high diagnostic accuracy was observed with a ROC of 0.97 (95% CI 0.96–0.99), a positive predictive value (PPV) of 100% (95% CI 55.0–100%) and a negative predictive value (NPV) of 98.8% (95% CI 97.7–99.3%).P. knowlesi-infected patients (23%) had parasite counts less than 200/µL, with a median parasitaemia of 60 (IQR 40–120) parasites/µL. The Gazelle™ achieved a diagnostic sensitivity of 80.9% (95% CI 66.7–90.9%) in this group .Against reference PCR results, sensitivity of the Gazelle™ (96.5%) was comparable to that of microscopy (97.2%) for P. knowlesi samples. The mean proportions of early trophozoite (rings), late trophozoite and schizont asexual life-stages in a single infection were 2.2% (95% CI 1.4–3.4%), 92.9% (95% CI 90.5–95.2%) and 5.1% (95% CI 3.3–7.8%), respectively. The mean proportion of rings was 1.5% in those positive by the Gazelle™ compared to 6.6% for those that were negative, with no statistically significant difference between groups. Gazelle™ test positivity correlated with parasitaemia , however it did not correlate with the proportion of ring, trophozoite or schizont life-stages within individual P. knowlesi infections.Microscopic evaluation of parasite life-stages was carried out on the 147 pre-treatment P. knowlesi-positive patient samples was included in the LOD analysis with serial parasitaemia dilutions. Of the 20 clinical samples, 8 (40%) were obtained from patients who had anti-malarial treatment prior to blood sampling . The LOD geometric mean was calculated to be 33 parasites/µL (95% CI 16–65 parasites/µL)  reported sensitivities ranging from 72 to 82% compared to PCR, out-performing current lateral flow malaria RDTs34. The utility of the Gazelle™ as a diagnostic screening tool previously demonstrated 100% specificity for malaria from a survey of 440 febrile individuals in Sri Lanka, although in comparison to light microscopy was unable to differentiate between the small number of P. falciparum, P. ovale, P. vivax and P. malariae cases included in the study34. Improved sensitivity of the Gazelle™ for P. vivax and P. falciparum detection has also been reported in patients with no prior history of malaria or treatment17, potentially indicating a greater or more rapid degree of haemozoin phagocytosis in individuals with previous malaria exposure. However, this phenomenon was not observed in the current evaluation for P. knowlesi, although the small numbers of those with previous P. knowlesi infections limited this analysis.Previous studies evaluating the clinical performance of the Gazelle™ for malaria diagnosis  targets, or cross-reactivity with epitopes for P. vivax-specific pLDH37. However, adapting technology for P. knowlesi detection designed for more common human malaria species requires trade-offs between test accuracy, complexity, and cost. The Gazelle™ was tested on a similar parasite density distribution sample set of clinical P. knowlesi infections as a previously published large evaluation of 10 pLDH-based malaria rapid diagnostic tests37. The best performing Plasmodium species pan-pLDH and P. vivax-pLDH tests for P. knowlesi detection reported sensitivities of 87.0% and 92.0%, respectively37, comparable to the overall Gazelle™ performance in the current study. Other antigen- and antibody-based novel diagnostic assays are being designed to detect multiple infectious causes of acute febrile illness, including the DPP® Fever Panel II Asia for diagnosis of falciparum and non-falciparum malaria, dengue and melioidosis38. Automated microscopy technology utilising machine learning software to visualize malaria blood films is being developed to detect Plasmodium species39. Although these devices demonstrate promising alternatives to conventional first-line malaria diagnostics, they have not been evaluated for clinical P. knowlesi infections to date. They would ideally require validation to either encompass P. knowlesi detection or confirm initial reported specificity for other Plasmodium species before field deployment31.Recent improvements in lateral flow point-of-care diagnostics for A limitation of this study was that the background prevalence of malaria could only be a crude estimation based on patients who presented directly at the referral hospital site. Despite this, a prevalence estimate that is lower by 5% would not significantly affect the NPV (99.1% [95% CI 98.5–99.6%]).Plasmodium species differentiation and quantification. This study provides important additional data on the first systematic evaluation of a haemozoin-based detection tool for detection of one zoonotic malaria species in an endemic clinical setting. The Gazelle™ device is not able to differentiate between Plasmodium species, which is crucial for targeted management in areas where multiple endemic Plasmodium species are present. In its current form, the Gazelle™ only allows for rapid screening to differentiate malaria due to any Plasmodium species infection from other causes of acute febrile illness for those presenting to health facilities with laboratory capacity, as demonstrated by the consistent 100% specificity from febrile malaria-negative controls observed in this study and other previous evaluations40. Improvements to the Gazelle™ are ongoing, with subsequent versions expected to distinguish between clinically important Plasmodium spp. via algorithms developed based on species-specific differences in haemozoin morphology41.The current Gazelle™ version evaluated in this study provides qualitative malaria diagnosis without the current ability for 42 using cellulose acetate-based cartridges on a microchip electrophoresis platform. This device has been evaluated for point-of-care screening for other blood-related diseases and hemoglobinopathies such as sickle cell44 and thalassemia44 traits. In malaria endemic areas with a high prevalence of thalassaemia, a single strategically placed device may be able to perform routine screening of thalassemia in addition to rapid detection of clinical malaria infections, thus maximizing its cost-effectiveness in the field.In addition to detection of malaria based on paramagnetic properties of hemozoin crystals, the current iteration of the Gazelle™ has a separate slot for identifying common haemoglobin variantsPlasmodium species infection in many organs, including the liver, spleen and bone marrow46. Haemozoin isolated from cultured P. falciparum isolates showed evidence of extreme resistance to phagocytic degradation and was not easily digested by a single process. This suggests haemozoin may be actively redistributed in the body for long periods of time through repeated phagocytosis, not only at the time of liberation from erythrocytes47. To our knowledge, the duration by which haemozoin crystals persist in successfully treated P. knowlesi infected patients has not been reported. The potential persistence of haemozoin in P. knowlesi-infected humans may include retention within the circulatory system or intravascularly in extracellular fluids. It remains unclear what possible immune-modulatory effects they illicit over multiple rounds of phagocytosis, or if the forms in which they persist may be detectable by currently available tools such as the Gazelle™. Despite this theoretical possibility, the current study did show that the Gazelle™ gave 100% specificity for P. knowlesi mono-infections; this high specificity may not be achievable in settings of high malaria transmission.In terms of diagnostic limitations, there remains the possibility that haemozoin detection methods may give false positive results in cases where circulating haemozoin persist despite the complete clearance of parasites from a previously resolved infection. Murine models have demonstrated the persistent presence of haemozoin for an extended period of 196–270 days post-48. The Gazelle™ device also provides a comparably quick result compared to that of a typical RDT, which takes about 15 min after loading the blood sample. An economic limitation would be the cost involved in supplying devices to many small clinics. The optimal utility of the Gazelle™ for most low-income countries would be limited to district referral hospitals frequented by many febrile patients daily.The Gazelle™ offers several major advantages in point-of-care functional utility for malaria diagnosis. This includes minimal time-to-detection of about 1–2 min for the actual blood analysis test component on all study participants. Peripheral blood samples are easily obtainable, particularly given the low blood volume required for testing. This is possible through more acceptable and better tolerated finger-prick sampling of patients compared to more invasive venepuncture. Improving end-user utility would be aided by future designs allowing, i.e., self-loading of 30 µL whole blood directly from finger pricks into cartridge containing the buffer solution, as opposed to requiring pipettes in a laboratory setting. The device is potentially suitable for remote primary health care settings where power supply and laboratory consumable cold chain processes may not be available. In addition, the Gazelle™ requires no pre-requisite manual sample preparation such as Giemsa-staining which may lower overall sensitivity and specificity of testP. knowlesi detection. The most relevant context would be in countries where other human Plasmodium species are approaching elimination and high-quality microscopy is not used as the first-line diagnostic tool. P. knowlesi parasitaemia limit of detection of the Gazelle™ was comparable to expert microscopy. Further development of the Gazelle™ is required to accurately differentiate between Plasmodium species, including validation for other zoonotic malaria, and to guide appropriate treatment.The Gazelle™ shows potential for use as a non-species-specific tool for malaria diagnosis encompassing Supplementary Information.


Plasmodium vivax malaria are now becoming a common place, but respiratory complications are described in less than 3% of global literature on severe vivax malaria.Malaria is endemic and represents an important public health issue in Brazil. Knowledge of risk factors for disease progression represents an important step in preventing and controlling malaria-related complications. Reports of severe forms of A severe respiratory case of imported vivax malaria in a previously healthy 40-year-old woman has been reported. The patient died after the fifth day of treatment with chloroquine and primaquine due to acute respiratory distress syndrome.Respiratory symptoms started 48 h after the initiation of anti-malarial drugs, raising the hypothesis that the drugs may have been involved in the genesis of the complication. The concept that vivax malaria is a benign disease that can sometimes result in the development of serious complications must be disseminated. This report highlights, once more, the crucial importance of malaria early diagnosis, a true challenge in non-endemic areas, where health personnel are not familiar with the disease and do not consider its diagnosis promptly. Plasmodium vivax is the most prevalent causing species, implicated in over 80% locally diagnosed cases. Plasmodium vivax infection is usually associated with benign presentations of malaria. However, serious manifestations with potentially kidney injury, acute respiratory distress syndrome (ARDS), splenic rupture, and even cerebral malaria have been reported. The underlying mechanisms of such severe manifestations are not well understood [P. vivax can cause severe forms of malaria that are potentially fatal [Malaria is an endemic disease in Brazil, and derstood . Therefoderstood . It is wly fatal –3.A healthy 40-year-old female Brazilian patient residing in the state of Rio de Janeiro reported a trip to São Gabriel da Cachoeira (Amazon State) between October 18, 2019 and November 26, 2019. On November 30, 2019, she presented to a general hospital in Rio de Janeiro with malaise, myalgia, and arthralgia. On December 2nd, 2019 she presented high fever, accompanied by chills and sweating. The patient sought medical care and was medicated with analgesics and antithermics for treatment support of a possible nonspecific viral infection. The case evolved with periods of recurrent fever.On December 6, 2019 (6 days after the onset of symptoms), she was admitted to the Emergency Department with a severe headache, anaemia (haemoglobin: 7.8 g/dL), heavy vaginal bleeding, and high fever. Vaginal bleeding continued for 5 days and severe thrombocytopenia was reported observed . The patient was not pregnant as she had previously undergone a uterus tubal ligation. She had no history of using contraceptives. She left the hospital on December 12, 2019, without bleeding and with improved thrombocytopenia .Fundação Oswaldo Cruz, with moderate fever, headache and malaise, where the diagnosis of vivax malaria primary infection was made (3720 parasites/mm3). Treatment with chloroquine for 3 days (600 mg—first day/450 mg—second and third days) and primaquine (30 mg/day—7 days) was started for the patient who weighed 79.4 kg. Vaginal bleeding stopped without recurrence and platelets increased to 144,000/µL. On the same day , the patient was discharged from the outpatient clinic. On discharge, she still had fever, but did not present any sign of severe illness. It was recommended that she return for a follow-up consultation for clinical and laboratory evaluation.On December 13, 2019 (13 days after the onset of symptoms), the patient was admitted to the Infectious Diseases ward of the Three days after beginning treatment, the patient reported myalgia, high fever, severe headache, and progressive tachypnea (54 breaths per minute). A blood smear exam and reverse transcriptase polymerase chain reaction (RT-PCR), were performed, both of which were found to be negative for the presence of malaria parasites on December 16, 2019.Physical examination  showed moderate hepatosplenomegaly, lungs with bilateral crackling rales, and severe anaemia. Blood gas tests revealed progressive hypoxia (oxygen saturation: 62%). A chest X-ray showed bilateral interstitial infiltrates. A diagnosis of severe ARDS was confirmed  the following day, and the patient was admitted to the Intensive Care Unit. Special care was initiated and intravenous artesunate (2.4 mg/kg) was prescribed because the possibility of recurrence was considered, despite negative parasitaemia. The patient developed progressive worsening of tachypnea and low blood oxygenation, requiring orotracheal intubation. Antibiotic therapy with ceftriaxone and azithromycin was started to treat possible nosocomial bacterial pneumonia. Whole blood collected prior to antibiotic therapy was culture negative. Intensive care was not enough to reverse the metabolic changes, however, and the patient died on December 17, 2019 following three episodes of cardiac arrest.Diagnostic tests for HIV infection were negative. Serum samples from the 18th day after the onset of symptoms had negative RT-qPCR results for Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Serological test performed for CHIKV IgM and IgG antibodies, were positive in the serum from the 18th day after the onset of symptoms. Non-reactive anti-DENV-IgM and IgG serological test were confirmed.A progressive increase in the patient’s leukocytes and C-Reactive Protein suggested worsening of the inflammatory and infectious injury. Laboratory tests performed during treatment showed severe anaemia, mild thrombocytopenia, and a moderate increase in blood creatinine. Liver function was normal throughout the treatment follow-up Table . Figure It is troubling that the diagnosis of malaria was made a full 13 days after symptom onset, and 17 days after the traveller returned from the Amazon, which is the main area of transmission in Brazil that is responsible for 99% of malaria cases annually. Health professionals outside malaria endemic areas should be trained to investigate patients’ travel history, to optimize early diagnosis and adequate treatment of malaria and prevent morbidity and fatal outcomes .According to a review by Val et al. , the preAs expected, the RT-PCR results for a panel of arboviruses made 18 days after the onset of symptoms were negative, since viraemia tends to be lower after the acute phase of the disease. On the same day serological tests performed for IgM and IgG antibodies to CHIKV were positive in the patient’s serum (low reactivity). Previous CHIKV infection could not be confirmed as the first and only serum sample did not allow the comparison of titers of immunoglobulin from a single sample, a well-known limitation of serological tests , it was The pathogenic mechanisms associated with the respiratory complications of vivax malaria are still unclear. In a study that followed 30 patients with respiratory complications, respiratory symptoms were observed after the initiation of anti-malarial drugs  performeIn a study with 79 patients diagnosed with falciparum malaria with respiratory complications, 42% had the complication within the first 48 h after starting treatment and 46% after 48 h, when parasitaemia was falling . Our patP. vivax to cause cytoadherence of red blood cells, which could explain the potential to develop severe manifestations, but this characteristic is by far more observed in Plasmodium falciparum infections [Some studies point to the ability of fections , 14. TheP. vivax infection. The patient in the case report did not have any risk factors associated with progression to severe disease. However, the delay in diagnosis and in starting specific treatment might have contributed to the poor outcome.For endemic malarial regions, some risk factors may be implicated as disease severity criteria, such as patient age (age extremes), pregnancy, presence of comorbidities, and disease relapses . The patRespiratory complications should be considered in vivax malaria, especially after delayed diagnosis and treatment. Further studies are needed to elucidate the mechanisms underlying such severe complications, to avoid fatal outcomes. Daily monitoring during treatment is mandatory for early identification of complications to facilitate rapid clinical support. Travel history to malaria-endemic areas should be rigorously investigated, with the aim of early diagnosis and adequate treatment of malaria.


Subgroup analysis showed a significant difference in copper levels between patients with malaria and uninfected controls among studies conducted in Asia  and studies employing plasma blood samples . The qualitative synthesis of the reviewed studies suggests a complex relationship between copper levels and malaria. The meta-analysis results showed no significant difference in copper levels between patients with malaria and uninfected controls overall. However, subgroup analyses based on various factors, including continent and blood sample type, showed copper level variations. These findings highlight the need for further research to better understand the role of copper in malaria pathogenesis by considering geographical factors and the blood sample type used for copper level measurement.Micronutrient insufficiency has been implicated in malaria pathogenesis. However, the role of copper in malaria remains inconclusive. This study aimed to investigate the association between copper levels and malaria pathogenesis, providing a deeper understanding of copper’s role in the disease. A systematic review was conducted following the registered protocol in PROSPERO (CRD42023439732). Multiple databases, including Embase, MEDLINE, Ovid, PubMed, Scopus, and Google Scholar, were searched for relevant studies reporting blood copper levels in patients with malaria. The Joanna Briggs Institute critical appraisal checklist was used for assessing methodological quality. Qualitative and quantitative syntheses were employed, organizing, and summarizing the findings of the included studies. To calculate the standardized mean difference (Hedge’s g) and 95% confidence intervals (CIs), a random-effects model was used. After screening the databases, 16 studies were included. Most studies (52.9%) reported that individuals with malaria had significantly higher copper levels than uninfected controls. The meta-analysis, based on 16 studies, showed no significant difference in copper levels between patients with malaria and uninfected controls overall ( Plasmodium species, which are parasitic protozoan organisms transmitted to humans through the bites of infected female Anopheles mosquitoes. Plasmodium falciparum , Plasmodium vivax (P. vivax), Plasmodium malariae , Plasmodium ovale , and Plasmodium knowlesi (P. knowlesi) are the five main Plasmodium species causing malaria in humans [P. falciparum is responsible for most malaria-related deaths, particularly in Sub-Saharan Africa, and is associated with severe complications. Conversely, P. vivax has a broader distribution and can cause recurring malaria episodes as it can lie dormant in the liver [P. malariae and P. ovale can still lead to persistent infections and relapses [P. knowlesi, traditionally considered a monkey malaria parasite, can infect humans and cause severe malaria, primarily in Southeast Asia [The global prevalence of malaria remains a significant public health concern, with the highest disease burden observed in regions such as Sub-Saharan Africa . Accordin humans . P. falche liver . Althougrelapses ,5. Additast Asia . Malariaast Asia ,8. Moreoast Asia , magnetoast Asia , and micast Asia , offer tMicronutrient insufficiency, including deficiencies of iron, zinc, vitamin A, and other micronutrients, has been reported to be associated with malaria pathogenesis ,13,14. SPlasmodium infection is crucial. Therefore, this study aimed to investigate the association between copper levels and malaria pathogenesis, aiming to enhance our understanding of the role of copper in the pathogenesis of malaria. The results of this study will contribute to bridging the knowledge gap in this area. Moreover, gaining insights into the role of copper in malaria pathogenesis may have implications for managing patients with malaria, potentially including the consideration of copper supplementation as a therapeutic approach.Considering the inconsistent findings regarding the role of copper in malaria pathogenesis, investigating copper levels in individuals with The systematic review protocol was registered with the PROSPERO number CRD42023439732. This systematic review was reported according to the updated guidelines for reporting systematic reviews, the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 statement [The main outcome of this systematic review was the difference in blood copper levels between individuals with malaria and uninfected controls. The secondary outcome focused on blood copper level variations in relation to parasite density.Plasmodium” OR “Plasmodium Infection“ OR “Remittent Fever“ OR “Marsh Fever“ OR “Paludism)”  terms and keywords such as “copper” and “malaria” was employed: “copper AND ” . AdditioStudies were included if they met the following criteria: (1) original research articles; (2) case–control, cohort, cross-sectional studies, or other designs; (3) assessed copper levels in humans with and without malaria; and (4) provided sufficient data for qualitative synthesis and effect size calculation. Exclusion criteria encompassed review articles, case reports, letters to editors, studies not conducted in humans, and studies that did not directly assess copper levels in relation to malaria.Two authors independently performed the study selection and data extraction. Initially, titles and abstracts of the articles retrieved through the search were screened to assess their relevance. At this stage, clearly irrelevant studies were excluded. Next, full texts of the articles that passed the initial screening were reviewed. At this stage, the articles were assessed against the inclusion and exclusion criteria. Those that did not meet these criteria were excluded, and the reason for exclusion was often documented. The extracted information included the first author’s name, publication year, study design, study location, participants’ age, assessment methods for copper levels and malaria parasites, parasite density, and types of blood samples for copper level measurement. Any discrepancies between the two authors were resolved through discussion or consultation with a third author if necessary.The Joanna Briggs Institute (JBI) critical appraisal checklist for observational studies was used for assessing the methodological quality of the included studies . For casI2 statistic was employed to assess the heterogeneity among the studies, with an I2 of 0–25%, 25–50%, 50–75%, and 75–100% indicating low, moderate, substantial, and considerable heterogeneity, respectively [A qualitative synthesis approach was employed, wherein the findings of the included studies were systematically organized and summarized. The results were structured around copper levels in relation to malaria, and any associations or correlations reported. For the meta-analysis, the effect sizes  along with their 95% confidence intervals (CIs) were computed from the included studies. To account for potential heterogeneity across studies, a random-effects model was used . The I2 ectively . To explTo assess the robustness of the meta-analysis findings, a sensitivity analysis was conducted by sequentially excluding one study at a time and examining the influence of each study on the overall effect size .n = 6) [n = 2) [Database searches identified a total of 1260 records. Specifically, 356, 161, 227, 152, and 364 records were identified from Embase, MEDLINE, Ovid, PubMed, and Scopus, respectively. After the initial search yielded 1260 records, 602 duplicates were removed, leaving 658 for screening. Of these, 534 were excluded (296 for not being related to the participants of interest and 238 for not being related to the outcome), and 124 were earmarked for retrieval but 4 could not be retrieved. Of the 120 records assessed for eligibility, 111 were excluded owing to various criteria, including being in vitro studies or reviews, or lacking pertinent information. After excluding the aforementioned records, eight studies met the eligibility criteria ,32,33,34n = 6) ,40,41 an [n = 2) ,43. Fina [n = 2) ,43  did not specify the participants’ age groups. Regarding methods for malaria detection, microscopy was predominantly used (93.8%), whereas one study (6.25%) used both microscopy and RDT. Regarding copper measurement, atomic absorption spectrophotometry was used in 81.3% of the studies, and spectrophotometry in 18.7%. Furthermore, serum was used in 75.0% of the studies for copper measurement, whereas plasma was used in 25.0%. Details of the studies are given in The characteristics of the 16 studies included in this review are presented in Using the JBI critical appraisal checklist for cross-sectional studies, it was observed that most studies , effMost of the studies (50.0%) ,38,42,43p = 0.39; Hedges’ g, 0.38; 95% CI, −0.48 to 1.25; I2, 98.73%; 16 studies, p < 0.05), whereas the remaining covariates showed no significant effects on the pooled estimate, including country, age group, Plasmodium species, diagnostic method for malaria, methods for measuring copper levels, and blood samples for copper level measurement (p > 0.05).The difference in copper levels between patients with malaria and uninfected controls was calculated using data from 16 studies ,41,42,43p = 0.54; Hedges’ g, 0.31; 95% CI, −0.68 to 1.29; I2, 98.89%; 13 studies). Similarly, no significant difference in copper levels was noted between patients with malaria and uninfected controls based on the publication year from 2000 to 2009 . Conversely, in the subgroup analysis comprising case–control studies, a significant difference in copper levels was noted between patients with malaria and uninfected controls .The subgroup analysis performed based on the publication year from 2010 to 2023 showed no significant difference in copper levels between patients with malaria and uninfected controls . Interestingly, the subgroup analysis of studies conducted in Asia demonstrated a significant difference in copper levels between patients with malaria and uninfected controls . In the age-based subgroup analysis, no significant difference in copper levels was observed between children  and adults .When the studies conducted in Africa were analyzed, no significant difference in copper levels was observed between patients with malaria and uninfected controls . For the subgroup that utilized serum blood samples, no significant difference in copper levels was noted between patients with malaria and uninfected controls , whereas a significant difference was detected in the subgroup employing plasma blood samples . When analyzing the subgroup of studies that utilized atomic absorption spectrophotometry for copper measurement, no significant difference in copper levels was observed between patients with malaria and uninfected controls . When analyzing the subgroup of studies from different databases, no significant difference in copper levels was observed between patients with malaria and uninfected controls among studies from the main databases  and other sources . To further investigate potential publication bias, a non-parametric trim-and-fill analysis was performed. The results indicated no significant difference in copper levels between patients with malaria and uninfected controls . These findings suggest that the observed asymmetry in the funnel plot is attributed to other factors, such as heterogeneity, rather than the absence of small studies in the meta-analysis.A funnel plot and Egger’s test were used to assess the presence of publication bias. The funnel plot exhibited asymmetry ; howeverp > 0.05, To assess the influence of individual studies on the pooled effect estimate, a leave-one-out meta-analysis was conducted. The results demonstrated that upon excluding each study from the re-run meta-analyses, none of the 15 re-run analyses showed a non-significant difference in copper levels between patients with malaria and uninfected controls . Of note, two studies within this majority group, Jimoh et al. [This systematic review showed that most of the included studies reported that individuals with malaria had significantly higher copper levels than uninfected controls. Nevertheless, a meta-analysis using data from 16 studies indicated no significant difference in copper levels between the two participant groups. The overall findings suggested that h et al.  and Saadh et al. , reporteh et al. ,30,39,41h et al.  reportedh et al. ,37. ThesI2 value, was explored using meta-regression and subgroup analyses. To investigate various factors that could potentially affect the results, subgroup analyses were performed. The results showed that publication years, age ranges, Plasmodium species, and the method of copper measurement did not affect the meta-analysis results. These findings suggest that these factors are not significant in determining copper levels in individuals with malaria. In contrast, when the studies were stratified by continent, the analysis showed interesting results. In studies conducted in Africa, no significant difference in copper levels was observed between patients with malaria and uninfected controls. However, in studies conducted in Asia, a significant difference was noted, suggesting potential geographical variation in copper levels among malaria cases. The observed higher copper levels in patients with malaria than those in uninfected controls, as reported by studies conducted in Asia, were not fully elucidated. Several potential explanations are proposed. One possibility is that individuals with malaria may concurrently have other conditions, such as Wilson’s disease. This disease, which is more prevalent in the Asian population than in other populations, is associated with elevated copper levels [P. falciparum malaria [Plasmodium infections in Asia occur in adults. Consequently, this population may have a stronger immune response against malaria than African children, potentially leading to elevated copper levels. Another possibility is that the Asian population may have higher incidences of conditions such as gestational diabetes mellitus [The high heterogeneity among the included studies in this meta-analysis, as evidenced by the high r levels ,45,46. A malaria . This im malaria . In Afrimellitus , preeclamellitus , and heamellitus , all of Furthermore, the blood samples used for copper level measurement yielded interesting results. The subgroup analysis based on blood sample type showed no significant difference when serum samples were utilized. However, a significant difference was observed when plasma samples were employed, suggesting that the choice of blood sample type influences the measured copper levels. Copper in the blood is very essential as it contributes to the immune response of the host to the antigenic challenge by malaria parasites since both elements are important for normal immune function . The incPlasmodium infection. A decrease in copper levels in patients with malaria may be observed more frequently in males than that in females, as males normally have low copper levels regardless of the presence of malaria infection [The inconsistency regarding copper level alterations in malaria may be because of its dual role, including elevated copper levels resulting in increased oxidative damage to lipids, proteins, and DNA, and copper deficiency that is associated with oxidative stress. For the first role, copper plays a crucial role in stimulating the immune system to combat infections, support tissue repair, and promote healing. Furthermore, the significant increase in copper levels may be a result of the body’s attempt to counteract free radical production, as malaria is characterized by inflammation and oxidative stress. The higher copper levels observed may be a response that occurs to enhance the activity of antioxidant enzymes, thereby addressing the increased free radical generation occurring during malaria infection. Saad et al. reported an increase in serum copper levels, which is attributed to its role as a component of ceruloplasmin, an acute phase reactant and antioxidant . For thenfection .I2 value. These limitations highlight the need for more comprehensive and standardized studies to elucidate the role of copper in malaria. Addressing the heterogeneity, considering geographical factors and the blood samples used for measuring copper levels will contribute to a better understanding of the mechanisms underlying copper alterations in malaria.The findings regarding copper level alterations in malaria demonstrate certain limitations and considerations. While most of the reviewed studies reported significantly higher copper levels in individuals with malaria than those in uninfected controls, the meta-analysis results showed no significant overall difference. This inconsistency may be attributed to the high heterogeneity among the included studies, as indicated by the high The qualitative synthesis of the reviewed studies suggests a complex relationship between copper levels and malaria. While some studies noted higher copper levels in individuals with malaria, others observed lower levels. The meta-analysis results showed no significant difference in copper levels between patients with malaria and uninfected controls overall. These findings highlight the need for further studies to better understand the role of copper in malaria pathogenesis by considering geographical factors and the blood sample type used for copper level measurement.


Recent advances in malaria genetics and genomics have transformed many aspects of malaria research in areas of molecular evolution, epidemiology, transmission, host–parasite interaction, drug resistance, pathogenicity, and vaccine development. Here, in addition to introducing some background information on malaria parasite biology, parasite genetics/genomics, and genotyping methods, we discuss some applications of genetic and genomic approaches in vaccine development and in studying interactions with microbiota. Genetic and genomic data can be used to search for novel vaccine targets, design an effective vaccine strategy, identify protective antigens in a whole-organism vaccine, and evaluate the efficacy of a vaccine. Microbiota has been shown to influence disease outcomes and vaccine efficacy; studying the effects of microbiota in pathogenicity and immunity may provide information for disease control. Malaria genetics and genomics will continue to contribute greatly to many fields of malaria research. Plasmodium parasites, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Whereas P. vivax is the most widespread species, P. falciparum causes the majority of the mortality, with an estimated 619,000 malaria deaths globally in 2021 [Plasmodium parasites generally have a limited host range, e.g., human malaria parasites do not infect rodent hosts and vice versa, although some parasites can infect both humans and non-human primates . Malaria parasites are single-cell eukaryotic organisms with complex life cycles involving the development of asexual and sexual stages (gametocytes) in vertebrate hosts  and sexual differentiation and fertilization in Anopheles mosquitoes [Human malaria is a disease caused by Genomics is defined as “the study of the functions and interactions of all the genes in the genome” . In infeP. falciparum isolates [Plasmodium c. chabaudi parasites [P. falciparum genome, PCR amplification of DNA with a small-size polymorphism (microsatellite) was established to determine the type of P. falciparum isolates [P. falciparum and P. yoelii parasites [Based on the types of genetic variations, various genotyping methods have been developed to study malaria parasite populations, transmission, evolution, and gene function through linkage and association mapping. Electrophoretic variants of enzymes were first introduced to study polymorphisms in rodent malaria parasites . Pulsed-isolates ,7. Basedisolates . Later, arasites ,10,11. Tarasites . Based oisolates ,13 and tisolates . With thisolates ,16,17. Barasites ,19,20,21arasites ,23.P. falciparum genome, sequencing using the isotope methods was very challenging initially. The second-generation sequencing used a luminescent method that measured pyrophosphate synthesis during amplification. This decreased the cost per base sequenced and allowed for higher throughput [DNA sequencing has been categorized into generations. DNA sequencing began using low levels of radioactive isotopes in DNA polymerization reactions ,25. The roughput ,27. Lighroughput . The thiPlasmodium species infecting humans, nonhuman primates, rodents, and birds have been sequenced. The first Plasmodium genome to be sequenced was the genome of the 3D7 clone of P. falciparum in 2002 [Plasmodium parasites. In contrast, the genome sequences of Plasmodium parasites can be very different in GC content, genome size, and the number of multigene families. For example, the P. falciparum genome is highly AT-rich (~80% AT), whereas the P. vivax genome is relatively more GC-balanced (~58% AT) [Plasmodium parasites have a long and complex evolutionary history [Plasmodium species, particularly for P. falciparum and P. vivax, have been sequenced, revealing diverse genomes with large numbers of SNPs and indels within a single parasite species [The genomes of many  in 2002 . The seq in 2002 ,39,40,41 in 2002 ,43,44. S~58% AT) ,45, whic history . The gen species ,48. Like species . The subPlasmodium species, such as P. falciparum, P. vivax, and rodent malaria parasites, through the genome sequencing of parasite isolates or strains [P. vivax isolates collected from 88 worldwide locations [P. falciparum, a total of 3,125,721 SNPs and 2,742,938 indels were reported from genome sequences of ~20,000 parasite isolates after excluding variants in subtelomeric and internal hypervariable regions, as well as the mitochondrial and apicoplast genomes [P. falciparum genome, there is approximately one polymorphic site per four bp sequences. However, the polymorphisms are not evenly distributed throughout the genome. Additionally, most of the polymorphic minor alleles are low-frequency (<5%), occurring in one or several parasites and thus suggesting recent mutations. Genes encoding immune and drug targets are likely to have more SNPs due to immune and drug selections [P. falciparum multidrug resistance 1 (PfMDR-1) can increase its copy number under mefloquine pressure [Genetic polymorphisms within individual malaria parasite species include chromosome-size polymorphisms, variations in gene copy number, SNPs, indels (insertion or deletion of nucleotides), and nucleotide modifications. Chromosome-size polymorphisms were recognized in the 1980s after the separation of parasite chromosomes in PFGE ,50,51. C strains ,40,48,60 genomes ,61. For lections ,63, and lections ,12. Copypressure ,65. Comppressure ,68,69,70Genetic and genomic tools have been employed to study malaria drug resistance extensively. The studies can be classified into three major directions: first, the identification of drug-resistant genes using linkage and association mappings, including genome-wide association study (GWAS); second, tracking and monitoring the origins and spread of drug-resistant parasites (drug-selective sweeps) using samples collected from patients; and third, drug selection in vitro or in vivo and genome sequencing or mapping of new mutations under drug pressure . These fP. vivax [Plasmodium parasites placed selective pressure on the human host, resulting in mutations in the human genome.Genetic variation in parasites leads to different disease outcomes, but host genetics can also impact the severity of the infection. For example, mutations in hemoglobin—leading to sickle cell amenia—or the absence of the Duffy antigen receptor give the host better survival outcomes. Sickle cell amenia is caused by a single amino acid mutation in hemoglobin, which leads to red blood cells having long crescent shapes ,80. SickP. vivax ,85,86. IP. falciparum parasites developed so far is the RTS,S/AS01 which consists of 18 copies of the central repeat and the C-terminal domain of PfCSP fused to the hepatitis B virus surface antigen (HBsAg) [There are many malaria vaccines under various stages of development. Malaria vaccines can be divided into three major categories on the parasite developmental stages, including pre-erythrocytic vaccines (anti-liver stages), blood-stage vaccines, and transmission-blocking vaccines  . Among t (HBsAg) ,94. The  (HBsAg) . HoweverNeisseria meningitidis using this approach [Genomic information and tools can be employed to identify candidate vaccine targets and predict T/B cell epitopes ,97. Compapproach . Recombiapproach ,100 and P. falciparum genes for polymorphisms identified some highly polymorphic loci and candidate genes. This included some genes of unknown function that were confirmed to encode antigens recognized by the human immune sera [P. falciparum isolates from West Africa identified 2853 genes that contained three or more SNPs [clag, PfMC-2TM, surfin, and msp3-like gene families. The genes under balancing selection can be prioritized for functional study and testing for use in potential vaccines. Unfortunately, highly polymorphic proteins may not be ideal vaccine candidates because of parasite-strain-specific immunity and immune evasion of parasites with variant alleles. However, it may be possible to search for protective antigens that are relatively conserved in parasite populations across geographic regions. Indeed, antigen candidates that had low naturally occurring sequence variation and high coverage across diverse bacterial populations were found after sequencing 2083 group A Streptococcus pyogenes bacterial isolates [P. falciparum genome. With genome sequences from over 20,000 P. falciparum isolates available, it is now possible to search for candidate vaccine targets that have relatively conserved motifs in most of the strains and are expressed in important parasite stages such as merozoite or sporozoite [Most of the malaria antigen genes are highly polymorphic due to balancing selection, and searches for hotspots of genome diversity may lead to potential unknown immune targets. A survey of 3539 une sera . In anotore SNPs . Genes wisolates . The mosisolates . The useorozoite ,61. Plasmodium chabaudi chabaudi genetic crosses, Cheeseman et al. developed a genetic approach called linkage group selection to identify target antigens of strain-specific protective immunity (SSPI) against malaria [P. falciparum merozoite, known as PfRh5, was identified through genetic mapping by the ability of the parasite to invade Aotus monkey erythrocytes as a phenotype [In addition to diversity-based search, experimental genetic crosses of parasites and genome-wide analyses have also been used to identify genes that encode targets of host immunity. In one study using progenies of  malaria . The ana malaria . The res malaria . In anothenotype . PfRH5 phenotype ,107.Plasmodium species using genomic tools [P. falciparum erythrocyte membrane protein 1), MSPs, EBA (erythrocyte-binding antigen), and CSP proteins [In addition to candidate antigens, large numbers of linear and discontinuous epitopes of T and B cells and MHCII have been detected in the genomes of ic tools . Many exproteins . AdditioP. falciparum isolates collected from Amazon basin in Loreto, Peru [P. falciparum populations in South America are relatively homogeneous due to drug-selective sweeps [A difficulty in developing an effective malaria vaccine is the high level of polymorphism in candidate antigens, which may result in immune escape due to strain-specific immunity. Genomic information can be used to inform the design of malaria vaccines. A molecular vaccine can include multiple alleles of a target antigen to generate antibodies against different parasite strains circulating in a local endemic region as this will allow it overcome immune escape by parasite variants. For example, limited allelic variants  were found in genes encoding CSP, MSP-1, AMA-1, LAS-1 (liver stage antigen 1), and TRAP (thrombospondin-related anonymous protein) in 139 to, Peru . A multie sweeps ,111. Pare sweeps . SequencIn vaccine trials, it is often necessary to know whether the parasites from patients who received a vaccine have the same genotype as the ones used in vaccine development. For a molecular vaccine based on a single-parasite antigen, it is relatively easy to sequence the antigen coding gene in parasite isolates collected from vaccinated individuals. Several studies investigated the RTS,S vaccine efficacy through sequencing or genotyping the vaccine targets from vaccine recipients and compared the frequencies of matched (3D7 strain) or mismatched sequences ,115,116.P. falciparum parasites in Africa [For a whole-parasite vaccine such as attenuated sporozoite vaccines ,117, detn Africa .P. falciparum sporozoite (PfSPZ) vaccines. For a whole-organism vaccine, the protective epitopes or antigens are generally unknown. An approach called ‘sieve analysis’ has been used to compare viral or parasite genetic sequences between vaccine and placebo recipients in order to study breakthrough populations, vaccine efficacy, and potentially identifying vaccine targets [Comparison of the genome sequences of the vaccine strain and the strains before and after vaccination may help to identify some key protective epitopes or antigens in whole-organism vaccines such as  targets ,120,121. targets ,124,125. targets .P. falciparum or P. vivax circulating in a human population have different levels of virulence and cause different disease pathologies. For example, the carriage of the msp1-MAD20 or msp2-FC27 allele family was associated with increased susceptibility to severe malaria [Breakthrough infections in fully vaccinated individuals have been reported for vaccines against SARS-CoV-2 due to ineffective immune responses, waning immunity, or escape from immune recognition by viral evolution . Malaria malaria . VaccineMonoclonal antibodies (mAbs) produced by a single B cell population can be used as prophylactic or therapeutic agents against infecting microbes, and exciting results have been reported when using monoclonal antibodies against malaria ,135,136.An emerging field taking advantage of high-throughput genomic technologies and biocomputational analyses is studying the association of and interaction between microbiomes and various diseases. Microbial communities that inhabit different body parts such as the intestine and skin have been studied intensively for their association with various diseases in recent years ,139,140,Lactobacillus and Bifidobacterium microbes and an elevated humoral immune response compared with susceptible mice. Additionally, the use of antibiotic treatment and fecal microbiota transplant to change the gut microbiota in outbred Swiss Webster mice also altered the susceptibility to P. c. chabaudi infection and pregnancy outcomes [Plasmodium parasites can induce alterations in the composition of the gut microbiota [Plasmodium berghei (PbA) infection decreased the numbers of Bacteroidetes and Verrucomicrobia species in the mouse gut but increased the numbers of Firmicutes and Proteobacteria species [Although malaria parasite transmission typically requires and interacts with a vertebrate host and a mosquito vector, microbiota within the vertebrate host  and mosquito can also influence host metabolism and immunity, thereby altering the outcomes of parasite growth and transmission . Elucidaoutcomes . On the crobiota . Plasmod species .P. falciparum infection [Plasmodium infection can also lead to gut microbiota-dependent increases in splenic germinal center (GC)-associated immune cells in mice and in the titer of parasite-specific antibodies [Enterobacteriaceae disrupts P. relictum development within midguts and salivary glands of the vector Culex quinquefasciatus [Enterobacteriaceae to Bacteroides species was also observed in the responders of a vaccine against rotavirus in a study in Ghana [Increasing evidence from clinical studies and animal models has also shown that the composition of the gut and skin microbiota plays a critical role in modulating immune responses, disease severity, and vaccine efficacy ,149,150.nfection . Similarnfection . Childrenfection . Plasmodtibodies . Malariatibodies . Interesasciatus . An incrin Ghana . The difAn. stephensi had Proteobacteria as the most dominant population among the three laboratory-reared strains of An. Stephensi, and that antibiotic treatment increased susceptibility to PbA infection [Anopheles gambiae mosquitoes to malaria infection by disturbing their gut microbiota [P. falciparum infection load and mosquito lifespan, whereas doxycycline increased the parasite load at high concentrations. Additionally, gut bacteria may impair the effectiveness of mosquito control approaches using insecticide bacteria such as Bacillus thuringiensis subspecies israelensis (Bti) or Wolbachia to alter the fitness or inhibit insect reproductive organs of infected insects [Interestingly, the microbiota of the mosquito vector can also affect the transmission of the malaria parasite. One study found that the midguts of nfection . Similarcrobiota . In a focrobiota . Azithro insects . TherefoP. falciparum clones were frequently detected in Africa [Malaria genetics and genomics are important approaches for studying disease mechanisms, host–parasite interaction, gene function, drug resistance, parasite population structure and transmission, and for developing better vaccines. Although great progress has been made in malaria genomics and the applications of genomics in studying various aspects of the disease, there are some areas where exciting discoveries may be obtained by applying genomic methods. In particular, analysis of parasite populations before and after vaccination (vaccine and placebo recipients) using a whole-organism vaccine may lead to the detection of immune targets if the vaccinated individuals are infected with multiple parasite strains. Multiple infections with two to eight n Africa , and divn Africa . If a van Africa ,111. Aft


Malaria and typhoid fever remain the major cause of morbidity and mortality in tropical and subtropical countries. It is common today to see patients being concurrently infected or treated for the two diseases. Methodology. A cross-sectional and descriptive study was conducted on 208 febrile patients coming for medical consultation at Ngaoundéré Regional Hospital and suspected for malaria and/or typhoid fever from September to November 2019. After receiving the consent of each patient, clinical and sociodemographic information were collected using a pretested semistructured questionnaire. Thereafter, blood samples were collected and subjected to (i) blood film examination for malaria detection and (ii) qualitative and semiquantitative Widal assay for typhoid fever detection.  The aim of the present study was to determine the prevalence of malaria, typhoid fever, and their coinfection among febrile patients at Ngaoundéré Regional Hospital, Adamawa, Cameroon. Plasmodium falciparum was the only species identified, and the prevalence of malaria was 50.0% (n = 104). The seroprevalence of typhoid fever was 64.3% (n = 133). The malaria and typhoid fever coinfection was 30.3% (n = 63). The coinfection between S. paratyphi  and malaria was also observed with the prevalence ranging from 32.2 to 63.9%. Female group and children from 2 to 10 years old were the most affected groups by the two infectious agents.  Typhoid fever and malaria were more prevalent in Ngaoundéré City, particularly in children and women. Further studies should be done on the risk factors of malaria and typhoid fever coinfection in different study areas. Plasmodium that are transmitted through the bites of infected female Anopheles mosquitoes  ina Faso  and in Nmales 15.%; N = 33ina Faso –24. It wina Faso . In contina Faso . The medina Faso .P < 0.05) with education levels and profession with the high prevalence among the primary (64.2%) and secondary (20.3%) levels on the one hand and student (49.5%) on the other hand. These observations are in agreement with those reported by literature [N = 76) than males , but not statistically significant (P > 0.05). This result did not corroborate the report of [Salmonella sp. infection [The prevalence of malaria and typhoid coinfection was 30.3%. Despite the low coinfection prevalence, there are important implications for the coinfected individuals. There was no significant association with the marital status, age groups, and residence area. However, we noted a significant association (terature . We founnfection .P < 0.05)  responded positively for fever, 78 (37%) for chills, and 77 (37%) for vomiting. Also, 12 (5.8%) responded positively for headaches and 41 (19.7%) for fatigue. The similar clinical features have been mentioned in the literature . Concomi < 0.05) . These s% respondMalaria and typhoid fever are two leading infections of poverty with serious health and socioeconomic impacts, and due to their geographical overlap, coinfections are very common. Coinfection rate was higher in females than in males, age group of 0–10 years recorded the highest prevalence, and some other risk factors such as profession and level of education were significantly affected by the diseases studied. Since they both have similar symptoms, treatment is based on adequate laboratory diagnosis. The data obtained in this study should be used in order to reduce mortality and morbidity caused by these infections in Ngaoundéré City.