Malnutrition and malaria are common co-morbidities in low-income countries, especially among under-fives children. But the malnutrition situation in Masasi and Nanyumbu districts, its interaction with malaria infection and the influence of socioeconomic factors are not well understood.Children aged between 3–59 months in Masasi and Nanyumbu were screened for nutritional status and malaria infection in the community. Nutritional status was determined using age and anthropometric parameters. Z-scores (weight for age (WAZ), height for age (HAZ) and weight for height (WHZ)) were calculated based on the World Health Organisation (WHO) growth reference curves. Malaria infection was determined using malaria rapid diagnostic test and microscopy. Hemoglobin concentration was assessed using HemoCue spectrophotometer, and anemia was classified as hemoglobin concentration < 11.0g/dL. Structured questionnaire was used to collect socio- demographic information electronically.x2 = 5.045, p = 0.025). Prevalence of malnutrition was higher in boys (53.0%) than in girls (45.0%) . Stunting was the most prevalent component of undernutrition; it was slightly prevalent in Nanyumbu (46.5%) compared to Masasi (42.0%),  and in boys (48.2%) than in girls (39.1%), x2 = 17.44, p<0.001. Only 15.8% of the undernourished children had malaria infection. Sex, age group and anaemia were significantly associated with undernourishment (p<0.05), while district and malaria infection were marginally (p≤0.06) associated with undernourishment. None of the undernutrition indices was associated with malaria infection.A total of 2242 children, 1539 (68.6%) from Masasi and 1169 (52.1%) females were involved in the study. The mean z-scores (WAZ = -0.60 and HAZ = -1.56) were lower than the WHO reference population. The overall prevalence of malnutrition was 49%, and it was significantly higher in Nanyumbu (52.5%) than in Masasi (47.3%), (Undernutrition was highly prevalent in the study population and was influenced sex, age, anaemia and malaria infection. More emphasis is needed to address the malnutrition problem especially stunting in Masasi and Nanyumbu districts. Plasmodium parasites , medium [40th-79th] and high SES [80th-100th]) for easy interpretation of results [Briefly, a samples size of 120 children in each of 20 wards selected was anticipated to provide a power of 80% and an alpha (type one error) of 0.05 with an attrition rate of 20% in explaining the variation of malaria incidence between the two districts. All the households with child/children were invited to participate in the socioeconomic status (SES) survey. Principal component analysis was used to estimate the scores for the socio-economic status of the households using household characteristics and assets. The SES scores of the first principal component were categorized into three groups of percentiles (low [0-39 results .χ2-tests while continuous variables were summarised as medians with associated range of inter-quartile range (IQR), means with standard deviations and 95% confidence intervals (95%CI). For continuous variables which were non-normal distributed, medians were compared using Wilcoxon rank-sum test while for normal distributed continuous variables, means were compared using t-test. A χ2-test was also used to assess the trend of continuous and categorical variables. Logistic regression analysis was used to assess the association between binary response variables  and risk factors in a univariate and multivariate models. Only variables that had a p-value <0.1 were included in the multivariable model. Variance inflation factor (VIF) was used to assess for multi-collinearity of independent variables, where a VIF<5 was considered a moderate correlation not requiring intervention. R-squared was used to assess how best the regression model fitted the data. A p-value < 0.05 was considered statistically significant.Data were collected electronically in open data kit (ODK) software using tablets computers. Spatial data (point coordinates for households and other features from the study areas) were collected during the household visit using Global Positioning System (GPS). Quality control and assurance of the data were maintained at all stages of data collection to archiving. Data were cleaned and analysed using STATA version 13 statistical packages. Categorical variables were presented in proportions and compared using x2 = 5.045, p = 0.025).A total of 2242 children with median age of 27 months (range: 3.0–59.9) were involved in the study. 1539 (68.6%) of the study participants came from Masasi and 1169 (52.1%) of them were females. The baseline characteristics of the children are presented in The relationship between anthropometric characteristics and the sex of the children is presented in x2 = 21.6, <0.001), the prevalence of severe wasting  and severe underweight  was not significantly different between boys and girls.Conversely, severe stunting, severe wasting and severe underweight occurred in 18.8% (391/2077), 2.6% (49/1917), 2.9% (63/2157) of the children. With the exception of severe stunting , wasting , thinness  and obesity  was decreasing significantly with an increasing age, whereas that of underweight  was increasing with increasing age. It was interesting to note that underweight and obesity had linear relationship with age; the increase in age was associated with the increase in the risk of underweight and vice versa for the obesity, p = 0.06) and thin  had slightly higher risk of malaria infection.Fourteen percent (312/2179) of the children had malaria infection. Only 15.8% (168/1064) of the undernourished children had malaria infection. The relationship between nutritional status and malaria infection is presented in In a univariate analysis district, sex of the children, age group anaemia and malaria were the factors found to be associated with undernourishment. The multivariate analysis on the other hand indicated sex, age group and anaemia were significantly associated with undernourishment (p<0.05), while district and malaria infection were marginally (p≤0.06) associated with undernourishment. The relationship between socio-demographic factors and undernourishment is presented in Malnutrition and malaria are among the major public health challenges in Tanzania. This study assessed the situation of malnutrition among underfive children in Masasi and Nanyumbu districts, its interaction with malaria infection and how it is influenced by socioeconomic factors. Nearly half of the recruited children were undernourished. Similar high prevalence of undernutrition has been reported in Cameroon ,38. The Stunting was highest in 1 year old age group followed by 2 years old group, whereas wasting, thinness and obesity were highest among children aged less than 1 year group, and underweight was highest among children in 4 years age group. Similar findings have been reported in other countries ,24 HowevOnly about 16% of the undernourished children had malaria infection. Furthermore, all the malnutrition indices including stunting, wasting, underweight and obesity had no association with malaria infection. Similar to the findings in this study, other studies have also found stunting ,16,22, uIn the study districts especially Nanyumbu, male gender, age group of children below 3 years and anaemia were the only other factors associated with malnutrition. Other studies have also found male gender ,26,27 loUndernutrition was highly prevalent in the study population and was influenced by sex, age, anaemia and malaria infection. More emphasis is needed to address the malnutrition problem especially stunting in Masasi and Nanyumbu districts.S1 File(XLS)Click here for additional data file.


HIV/AIDS is a global public health crisis that is yet to be contained. Effective management of HIV drug resistance (HIVDR) supported by close resistance monitoring is essential in achieving the WHO 95-95-95 targets, aiming to end the AIDS epidemic by 2030. Point-of-care tests (POCT) enable decentralized HIVDR testing with a short turnaround time and minimal instrumental requirement, allowing timely initiation of effective antiretroviral therapy (ART) and regimen adjustment as needed. HIVDR POCT is of particular significance in an era when ART access is scaling up at a global level and enhanced HIVDR monitoring is urgently needed, especially for low-to-middle-income countries. This article provides an overview of the currently available technologies that have been applied or potentially used in HIVDR POCT. It may also benefit the continued research and development efforts toward more innovative HIVDR diagnostics. The HIV/AIDS epidemic has spread to all populated continents in the past four decades, with no sign of ending in the foreseeable future. HIV has infected 79.3 million people since it was identified in the early 1980s, and approximately 36.3 million people have died from AIDS-related illnesses thus far ,3. HowevHIV infections are treated with drugs that target viral proteins essential for their replication, such as protease (PR), reverse transcriptase (RT), and integrase (IN). Nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTIs) prevent the reverse transcription of HIV RNA to proviral DNA. In contrast, protease inhibitors (PIs) prevent the cleavage of HIV polyproteins, and integrase inhibitors (INIs) interrupt viral integration into the host genome. The rise of HIV drug resistance mutations (HIVDRMs) may render these drugs inefficient in virological suppression for all available ART agents. As such, genotypic HIVDR typing aims to examine the presence of known HIVDRMs, qualitatively or semi-quantitatively . HIV RNAAffordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, and Deliverable) criteria endorsed by WHO  [To minimize the impacts of HIVDR variants, the WHO recommends routine surveillance of HIVDR to monitor ART and pre-exposure prophylaxis (PrEP) distribution . The infzerland) .POCT has been of great interest to HIVDR professionals for decades. It is acknowledged that the topic of HIVDR POCT had previously been reviewed by others ,9,10. WhConventional genotypic HIVDR typing relies on Sanger sequencing of target HIV genes and examines the presence of all known HIVDRMs within collectively. In contrast, while the mechanisms vary, all attempted HIVDR POCTs thus far target single or multiple selected known HIVDRMs only. Described below are several near-POCTs attempted for HIVDR testing thus far.pol gene with colorimetry or spectrophotometry [OLA is a point mutation test initially developed by Landegren et al. to detect mutations associated with sickle cell anemia ,12. The otometry ,15,16. Ootometry ,18,19,20otometry ,22.The latest version of the OLA-Simple is capable of detecting HIVDRMs across multiple HIV-1 subtypes  using different specimen types  . There aOLA-Simple has been successfully applied in detecting HIVDRMs across multiple major HIV-1 subtypes using specimens from Kenya, South Africa, Peru, Thailand, and Mexico. High concordances were obtained between the results from OLA-Simple and those from Sanger sequencing and even high-sensitivity HIVDR assays such as Next Generation Sequencing (NGS) with a mutation frequency cut-off at 1% ,22. The OLA-Simple is the best-developed near-POCT HIVDR thus far that has been validated for multiple key HIVDRMs from different HIV-1 subtypes. The instructional software by the assay developers also ensures the user-friendliness of performing OLA-Simple assay by inexperienced lab personnel, especially in RLS. Despite the promising approach of the OLA-Simple, it needs extra machinery to generate a DNA template, electricity to operate, and storage for lyophilized reagents. Proper training may also be necessary to avoid cross-contamination between different steps.Allele-specific PCR is one of the most widely used tests for HIVDR identification using quantitative PCR (qPCR) and is one of the many foundations of current POCTs. PANDAA is a point mutation assay developed by MacLeod et al. to tolerate the diversity of nucleotide sequences flanking the target mutation site, which showed a 96.9% sensitivity and 97.5% specificity for quantifying HIVDRMs present at ≥5% . In a trPANDAA requires no separate cDNA synthesis or PCR procedures, and the starting materials could be RNA or DNA. During the initial qPCR cycles, site-directed mutagenesis will occur to generate a population of homogenous amplicons with similar probe-binding regions complementary to the probes. This step removes any secondary polymorphisms that interfere with probe hybridization in a traditional qPCR. A target-specific probe will bind in the next stage, and qPCR results will differentiate between a mutant and a wild-type. While these primers were created by combining multiple HIV-1 subtypes to build a consensus sequence, the PANDAA assay can be curated to accommodate local HIV-1 sequence diversity . One intKouamou et al. assessed the diagnostic accuracy of PANDAA against plasma samples from patients in Zimbabwe that have acquired HIVDR . Five HIPANDAA requires a qPCR machine, but there is no need for bioinformatics support in data interpretation . One sigThe prevalence of influenza prompted McCalla et al. to develop a method to amplify RNA with engineered ssDNA probes . They reThe SMART technique uses two ssDNA probes that will bind to a specific RNA target sequence: (1) biotinylated capture oligonucleotide (BCO) attached to a streptavidin-coated magnetic bead (SMB) binds to a conserved region and (2) a SMART probe binds to the mutated region C. The twIn this assay, amplification and detection rely on the specific hybridization of the SMART probe rather than the target RNA. The SMART probe can be engineered to have favourable or unfavourable binding energies to ensure it does not bind to other oligonucleotides. Additional benefits are the use of microfluidics, which provide a close, efficient, automated system that reduces hands-on time, human error, and probable contamination. To complete this assay, a microfluidic device and a qPCR are the main components needed ,28,29. LThis assay was developed by Zhang et al. to address issues with existing POCTs only detecting one or few mutations per test. Based on a suspension array technology, they produced the MAS assay that would allow simultaneous detection of multiple major HIVDRs ,31,32,33The assay begins with adding ASPE primers to a reaction tube with a reagent mixture and the templates D. PrimerIn a follow-up study, Zhang et al. adapted the assay for subtype B by altering ASPE primers and applied it to dried blood spot specimens collected from patients on antiretroviral therapy . For botThe extreme variation among HIV-1 sequences warrants the need for specific primers to be made. In addition, as a PCR product is the starting material, raw material will have to be processed to obtain a template. There is also the requirement for a suspension array system to perform the assay and, as previously mentioned, such equipment comes with an extra burden. Considering the logistic and operational constraints as the assay is now, more development efforts are warranted to apply MAS in practice for HIVDR POCT.A preliminary study by Barany exhibited a ligase-mediated detection technique to distinguish between mismatched and complementary bases . This prThe LRA assay is a one-step, single-buffer scheme to detect point mutations from RNA. In a single tube with optimized buffer, ligase, hot-start DNA polymerase, and oligonucleotide primers are added E. The reThis method separates the ligation and qPCR reaction by exploiting hot-start polymerases. By doing so, only one reaction is active at a time, thereby achieving maximum sensitivity, which for K103N was determined as 1%. The results showed that this assay outperformed allele-specific PCR and pyrosequencing in detecting mutant specificity . To perfZhang et al. had only presented a proof of principle for this assay. The RNA template used had mutations introduced into the pol gene of the HIV-1 genome through site-directed mutagenesis. Further follow-up study and validation of this assay to examine different HIV-1 subtypes and HIVDRMs other than the K103N mutation they studied remain to be completed and are necessary before its potential for HIVDR POCT can be better assessed.Gomez et al. first developed a novel method for rapid genotyping of blood groups using a lateral flow biosensor to prevent alloimmunization, a major complication during blood transfusions . Using wThe study executed a proof-of-concept test on HIV-1 subtype B plasma specimens to rapidly detect mutations that cause resistance in NNRTIs. The assay has two major components: (1) a rapid multiplex detection system utilizing sequence-specific primer extension (SSPE) primers containing a Tag sequence and (2) a dry reagent lateral flow dipstick that generates red dots F. As theBesides the OLA-Simple, Multiplex Detection Assay is another methodology claimed to be near-point-of-care assay . It provStreptococcus pneumonia. As discussed earlier, Panpradist et al. made significant strides in modifying the OLA-Simple [This proof-of-concept assay builds on multiple techniques and previous work done by researchers. Bui et al. found success when they joined PCR with OLA to detect drug resistance in A-Simple ,44. BaseA-Simple .3 copies of pre-amplification template while mutant templates were present at 20% [The assay starts with using recombinase polymerase amplification (RPA) to amplify a portion of the HIV-1 pol region G. Afterwt at 20% .RPA not only isothermally amplifies DNA in a short time, but it also tolerates impure samples, which is favorable in places where contamination is unavoidable. Reagents of this amplification technique can also be in lyophilized form, shortening preparation time. Additionally, the assay design proves to be specific as the fork in the membrane limits the aggregation of OLA products. To complete this assay, a heat block and a tabletop centrifuge is necessary for the RPA step prior to proceeding to OLA .Although this assay can merely detect one mutation per test, it can be adapted to detect other high-impact mutations. At its current state, the assay is yet to have sample preparation as an integrated step, and more hands-on time is required when adding ELISA reagents to the membrane. Furthermore, gBlocks stocks were used as DNA template and the membrane design lacks a control region, lowering the validity of this assay. More research is needed to advance this technology towards POCT.HIV patients often elude clinics after a one-time visit because of stigma, inconvenience, travel costs, or other socio-economic factors. It is then essential for physicians to diagnose and treat the patients on the same day. POCTs for HIVDR will aid physicians when determining the best drug regimen to start with or switch to in order to achieve suppressed viral loads. As new methods arise, so do more opportunities for creating an ideal test for RLS. In this section, we explore two new technologies with the potential for simplified HIVDR typing.This melt curve analysis platform is a genotypic resistance assay that measures the hybridization, capture, and dissociation of multiple nucleic acid targets to and from surface-bound oligonucleotide probes. The probes have two parts: (1) a complementary sequence to HIV-1 strains and (2) a nucleotide triplet complementary to a codon at a drug resistance mutation. First, fluorescently labelled DNA is added to the oligonucleotide microarray at one temperature . Then, the concentration of labelled DNA captured is monitored in real-time by the probes as the solution temperature progressively increases. A time-series data is generated that defines duplex stability, which is used to identify the correct codon at the position of a drug resistance mutation. In vitro experiments were performed with HIV-1 plasma samples and culture supernatants, containing HIV-1 subtype A, B, C, D, CRF01_AE, and CRF02_AG . AlthougMyers et al. have developed the Microfluidic Biomolecular Amplification Reader (µBAR) in an attempt to combine electronics, optics, microfluidics, and molecular biology. The µBAR is a battery-powered, portable instrument capable of isothermal amplification of multiple markers with the use disposable microfluidic assay cartridges. First, the sample  is loaded onto a disposable microfluidic cartridge, where the system uses a loop-mediated isothermal amplification (LAMP) technique. The cartridge is then inserted into the µBAR, where it will control assay temperature, illuminate the chip, and monitor real-time fluorescence signals from individual reaction chambers ,48.Previously, the LAMP assay was verified to detect HIV and malaria from blood samples and TB drug resistance from sputum samples. Myers et al. have also exhibited use of the LAMP assay on the µBAR platform in detecting the HIV integrase gene. The platform also has GPS and cellphone connectivity for healthcare delivery in remote locations and epidemiological surveillance. The chip contains six inlets, meaning multiple samples can be loaded simultaneously. In its current form, the µBAR requires more work to modify for HIVDR detection .https://nanoporetech.com/, accessed on 16 June 2022). MinION is thus far the only portable device that execute NGS on DNA or RNA templates with minimal requirement for additional instrumental and technical support.The increase in using NGS technologies to detect HIVDR have been on the rise. It is noteworthy that most of available NGS platforms are not even close to the bedside POCT considering their prohibitive instrument and reagent costs, demanding technical operation and complexity in data interpretation. One exception could be the MinION platform, developed by the Oxford Nanopore Technologies , and High-Resolution Melting (HRM) have also been implemented in drug resistance detection for varying pathogens. Perhaps these methods, coupled with the rise in technological advancement, may inspire new HIVDR POCTs.The expansion of ART access comes with a growing concern for the rise in HIVDR. HIVDR monitoring is essential for effective HIV/AIDS management at individual and population levels. Conventional Sanger sequencing-based HIVDR genotyping may not be readily assessable, especially in RLS, for logistical and operational reasons. POCT offers a quick and affordable solution administered at or near patient care. The assays explored here show the progression of each test, where they stand, and adjustments that need to be made. Although the work that has been done is impressive, no such assay entirely embodies the ASSURED criteria. A fully validated POCT that satisfies the set standards and meets all the needs for HIVDR diagnostics, especially in RLS, has yet to be developed. More research still needs to be done as POCTs are indispensable in controlling the spread of drug-resistant HIV.


Epidemiology of febrile illness in Uganda is shifting due to increased HIV treatment access, emerging viruses, and increased surveillance. We investigated the aetiology and outcomes of acute febrile illness in adults presenting to hospital using a standardized testing algorithm of available assays in at Arua and Mubende tertiary care hospitals in Uganda.We recruited adults with a ≥ 38.0 °C temperature or history of fever within 48 h of presentation from August 2019 to August 2020. Medical history, demographics, and vital signs were recorded. Testing performed included a complete blood count, renal and liver function, malaria smears, blood culture, and human immunodeficiency virus (HIV). When HIV positive, testing included cryptococcal antigen, CD4 count, and urine lateral flow lipoarabinomannan assay for tuberculosis. Participants were followed during hospitalization and at a 1-month visit. A Cox proportional hazard regression was performed to evaluate for baseline clinical features and risk of death.Of 132 participants, the median age was 33.5 years (IQR 24 to 46) and 58.3% (n = 77) were female. Overall, 73 (55.3%) of 132 had a positive microbiologic result. Among those living with HIV, 31 (68.9%) of 45 had at least one positive assay; 16 (35.6%) had malaria, 14 (31.1%) tuberculosis, and 4 (8.9%) cryptococcal antigenemia. The majority (65.9%) were HIV-negative; 42 (48.3%) of 87 had at least one diagnostic assay positive; 24 (27.6%) had positive malaria smears and 1 was Xpert MTB/RIF Ultra positive. Overall, 16 (12.1%) of 132 died; 9 (56.3%) of 16 were HIV-negative, 6 died after discharge. High respiratory rate (≥ 22 breaths per minute)  and low  oxygen saturation  were identified to be associated with increased risk of death.In those with hospitalized fever, malaria and tuberculosis were common causes of febrile illness, but most deaths were non-malarial, and most HIV-negative participants did not have a positive diagnostic result. Those with respiratory failure had a high risk of death.The online version contains supplementary material available at 10.1186/s12879-022-07877-3. Systemic causes of fever are notoriously challenging to differentiate clinically in tropical regions including sub-Saharan Africa (SSA), ranging from malaria to Ebola virus disease . CliniciA few decades ago during the early U.S. President’s Emergency Plan for AIDS Relief (PEPFAR) era in SSA, HIV coinfections were responsible for the majority of hospitalizations with severe febrile illness and associated with high mortality , 4–6. ThDue to high biodiversity and population density, many emerging and re-emerging zoonotic infections have been identified in Uganda particularly near the Rift Valley, further expanding the differential diagnosis of undifferentiated fever. With increasing surveillance, more cases of viral hemorrhagic fevers (VHF), including autochthonous and imported Ebola virus disease outbreaks, Crimean–Congo hemorrhagic fever (CCHF) and Rift Valley fever (RVF), have been detected in Uganda , 13–16. From August 2019 to August 2020, we recruited adults 18 years of age or older presenting to Mubende and Arua Regional Referral Hospitals (RRHs) emergency or outpatient departments designated to be hospitalized with a measured or reported temperature ≥ 38.0 °C occurring within the past 48 h or a clinical history consistent with fever within 48 h of presentation. There were 255 participants consecutively screened with 97 enrolled at Mubende RRH and 35 enrolled at Arua RRH  is located in central Uganda and serves a district population of over 688,000 people but the catchment area goes beyond the district it serves .After informed consent, participants provided information about sociodemographic and comorbid medical conditions at enrolment. History suggestive of exposure to zoonotic infections was obtained including area of residence, occupation, environmental exposures, and healthcare exposure . Also, rLaboratory tests that were conducted included complete blood count, serum creatinine, serum alanine transferase (ALT), serum aspartate transferase (AST), blood cultures , hepatitis A IgM , hepatitis B sAg , and HIV testing with consent , malaria rapid test , and thick malaria smears. A malaria smear result was stratified by any parasitaemia or ≥ 3000 parasites/μL , 21. If We estimated that a target sample size of 365 patients across sites would estimate the frequency of uncommon endemic viral infections with a 5% prevalence using a precision of 0.025 at 95% confidence. Here we report initial findings from an accrued sample size of 132 participants enrolled to date to provide clinical epidemiologic estimates, particularly in the context of the development of the Ebola virus disease outbreak in Mudende district, Uganda that started in September 2022. Summary statistics were performed for baseline demographics and prevalence of difference diagnoses. Baseline demographics, symptom prevalence, physiologic parameters, clinical laboratory results, quick sequential organ failure assessment  scores . The sam3 cells/uL with a median 129.0 × 103 platelets/uL , the median age was 33.5 years (interquartile range [IQR]: 24.0 to 46.0); 77 (58.3%) of 132 were female  were HIV-infected; median CD4 count was 198.0  in the setting of acute illness. There were 8 (17.8%) of 45 participants with a CD4 < 50. Sixteen (35.6%) of 45 were not known to have HIV; 10 were started on ART during hospitalization or within a month after hospitalization. The remaining 7 included 3 who died, 2 who had an unknown treatment history, 1 who was resistant to starting, and 1 who eloped from the hospital and was lost to follow-up. Seventeen (60.0%) of 29 known to have HIV were on ART prior to hospital presentation. Individuals with HIV were older  compared to those without HIV  p = 0.005, otherwise, they had similar baseline demographics (Table % were HIStreptococcus pneumoniae (n = 4), and an additional isolate each (n = 1) of Citrobacter freundii, Streptococcus spp., Staphylococcus aureus, Salmonella species, and Proteus vulgaris. One culture grew common contaminant coagulase negative staphylococcus, and another grew Micrococcus luteus.Overall, 73 (55.3%) of 132 had a positive microbiologic result. The most common positive test results were a malaria thick smear  or malaria rapid diagnostic test . Thirty-five of 40 (87.5%) positive malaria smears were also positive on the malaria RDT , Staphylococcus aureus, Salmonella species, or Proteus vulgaris  of 45 had at least one positive microbiologic assay. Microbiologic and rapid diagnostic test results among those with newly diagnosed or known HIV included positive results for malaria . There were 5 (11.1%) with cryptococcal antigenemia, 8 9.2%) with evidence of hepatitis A and 2 (4.4%) with evidence of hepatitis B  of 118 died; 10 in hospital and 6 after discharge or hospital transfer. Death occurred a median of 3.0 days (IQR: 1 to 7 days) from enrolment. The median age among those that died was 38.0 years , and 7 (37.5%) had HIV  compared to survivors . Fatal cases also had lower initial oxygen saturation   and causes of death after discharge were unknown. Standardized verbal autopsies were not performed. Participant attrition was in part due to hesitation to return to medical centres during the COVID-19 pandemic. Causes of death were not ascertainable with telephone follow-up. Second, the sample size is limited and associations with mortality that exist may not have been observed in our data. For example, there was no survival difference observed with HIV, but we present here our interim analysis observations and smaller effect sizes may be observed in a larger cohort. Sensitivity analyses restricted to site were not performed given limited number of fatal outcomes (n = 5) at the Arua site. Third, diagnostic test results reported here did not have extensive diagnostic testing with serology or multiplex PCR. However, testing was protocolized and in line with recommendations for rapid diagnostic testing and diagnostic assays currently available in Uganda. More expansive diagnostic testing is ongoing to understand the aetiologies of the unknown causes of febrile illness and death including emerging or neglected causes of illness including viral and rickettsial infections. Interim results are presented here to provide urgently needed updated descriptive epidemiologic information in a region with diverse causes of acute febrile illness.Taken together, these findings highlight the need to strengthen clinical and microbiology laboratory testing capacity to identify the aetiologies of acute febrile illness, particularly in settings where the epidemiology of febrile illness is shifting. These results are mostly from the pre-COVID era and aetiologies of fever may have directly or indirectly changed. Evaluation of optimization of follow-up, treatments, or diagnoses are urgent areas of study to improve outcomes among hospitalized adults in SSA.The majority of deaths occurred among those less than 50 years of age and due to non-malarial febrile illness. Rickettsial and viral aetiologies of severe febrile illness may represent an undetected and pervasive burden of disease , 32. UrgAdditional file 1: Figure S1. Enrollment and follow-up flow diagram. Figure S2. Map of Uganda with districts represented in cohorts and district population sizes. Triangle shape pins: original districts of participants that were enrolled at Arua Regional Referral Hospital. Black circle pins: districts of participants that were enrolled at Mubende Regional Referral Hospital. Map created using ArcGIS Online [GIS software]. Sources: Esri, HERE, Garmin, FAO, NOAA, USGS. Table S1. Symptoms at enrollment. Table S2. Clinical laboratory parameters. Table S3. Comparison of baseline characteristics between those with and without HIV. Table S4. Characteristics of fatal cases. Table S5. Initial laboratory values and microbiologic results stratified by fatal outcome.


Background and Objectives: Cardiovascular diseases are the main cause of death worldwide, and pulse wave velocity (PWV) is considered a predictor of major adverse cardiovascular events. The study intended to be helpful in finding methods for the preliminary assessment of PWV in primary care units. Materials and Methods: The study group consisted of 36 subjects  from the medical field (medicine students and residents) aged between 20 and 30 years: 33.3% males and 66.7% females. Two types of measurements were carried out successively: (a) measurements with the arteriograph and (b) measurements on a treadmill effort testing system, where heart rate (HR) was measured over time as a response to step function physical effort (PE). Results: The study allowed for the highlighting of some limits which, if exceeded, can be associated with high PWV values: (i) if after a moderate PE and a resting time of at least 6 min, the HR is larger than 80 b/min; (ii) if the relaxation time in a PE test of moderate intensity is larger than 1 min; (iii) if the HR measured after the subject is raised from the supine to orthostatic position is larger than 100 b/min, and (iv) if the resting HR is larger than 80 b/min. Conclusions: Steady-state HR correlates with PWV and may be used for the preliminary assessment of PWV.  The method of step function response is usually involved in electronic engineering  and in mFor instance, in characterizing the magnetic properties of materials, the stimulus is the intensity of the magnetic field, H, and the response is the magnetization, M, ; when chThe concept of step function response can be at least formally extended to any system using a stimulus and a response, for instance, in neuroscience ,5, the cCardiovascular diseases are the main cause of death worldwide, and major adverse cardiovascular events (MACEs) are a common primary outcome of interest . DefinitThere exists dedicated equipment for PWV measurement, but the equipment is only found in some hospitals. For the effective prevention of cardiovascular diseases, even in underdeveloped countries, there should be methods for evaluating PWV, which should be accessible to any general practitioner (GP).This paper presents a study on the correlation between the measured PWV and some parameters that can be assessed from the heart rate response after the sudden application of a moderate physical effort. The results of the study may be helpful in finding a method for preliminary assessing PWV in primary care units, which will help with the early recognition of susceptibility to cardiovascular diseases.The study group was selected from the medical field in a county hospital, consisting of 36 subjects, and the inclusion criterion was the statement of each subject that they did not have cardiovascular diseases and were not undergoing treatment for other diseases, with a normal ECG and heart rate response during treadmill effort recording. There were 40 volunteers at the beginning of the study, and four of them were not eligible to continue the study because of different arrhythmias during the physical effort. The study group consisted of nine medicine students (27.3%) that were in the summer practice of the Cardiology Department, with 15 residents in cardiology (42.5%), and 12 residents in diabetes mellitus, nutrition, and metabolic diseases (30.2%), all aged between 20 and 30 years. Of the total number of subjects, 12 (33.3%) were males, and 24 (66.7%) were females. The characteristics of the study group are summarized in All subjects voluntarily agreed to participate in the study, and the informed consent was approved by the Ethics Committee of the University of Medicine and Pharmacy “Victor Babeş”, Timisoara, No. 59/22.12.2021.The study was based on two types of measurements, which were carried out successively: (a) measurements of pulse wave velocity with the arteriograph and (b) measurements on a treadmill effort testing system.The stages of the measurements and the time dependence of the physical effort (in arbitrary units) are presented schematically in (I) Measurements of pulse wave velocity were performed with a TensioMed arteriograph, following the recommendations within : the sub2 and was denoted by HR-Pre.(II) After performing the measurements with the arteriograph, each subject was raised from the supine position to the orthostatic position and placed on the treadmill. In this position the heart rate was measured at the time moment tMeasurements were performed with a Labtech system from Labtech LTD Debrecen, Hungary, on the EC-12 LT Treadmill effort testing system, which was equipped with Cardiospy rest and stress software (EC-12RS PC-based Resting and Stress Test ECG System). The hemodynamic parameters were measured automatically minute by minute. Reports were automatically exported and organized using patient ID and registration time. As an emergency and automatic test stop criterion, we selected the HR value of 160 b/min. During the test, we paid special attention to the heart rate, blood pressure, changes in the ECG pattern, irregular heart rhythm, and the appearance and symptoms of the subjects.3) was measured and denoted by HR-4min;(III) The loading stage was carried out on the treadmill at a speed of 2.7 km/h and lasted for 4 min. For each subject, the heart rate (after 4 min)  The cooling period lasted one minute;4 to the time moment t5, the heart rate was measured minute by minute and denoted by HR-Relax (t), where t represents time. The heart rate value measured at the end of the recovery period  was noted by HR-6min.(V) The recovery stage, with the subject in the supine position, lasted 6 min. During the recovery period, from the time moment tp < 0.05.The schematic representations were made in Paint, whilst the plots of the results, the fit of the experimental data, and their statistical processing were performed using the OriginPro 8 software (8th version). The means, standard deviations, and Pearson’s correlation coefficients are presented. Linear regression analyses were performed between PWV and the various parameters obtained from the effort stress test, and regression equations were also presented. For correlation, a two-tailed test of significance was used. The statistical significance was set at According to the general theory of relaxation processes, if the stimulus, S, changes suddenly (following a step function), then the response, R, of the system has an exponential time dependence ,3 schem.In the case of measurements with the treadmill effort testing system, there are four moments at which the physical effort (as stimulus) changes suddenly see . The steo + A∙exp(−t/τ), and, for each subject, the parameters yo, A, and τ were determined. The significance of the fit parameters is clear from the expression of y(t) and in The typical time dependence of the heart rate measured on one subject in the recovery stage is shown in o, so yo is the resting heart rate value after physical effort. The second parameter, A, is the difference between the heart rate at the beginning of the recovery stage and the resting heart rate after physical effort (A = y(0) − yo). The time constant, τ, is the time interval after which the deviation from the resting value of the heart rate (y(t) − yo) decreases by e times, where e ≅ 2.71 is the base of the natural logarithm.When t tends to infinity, y(t) tends to y2 after the subject rose from the supine position to the orthostatic position, and HR-4min, after 4 min of moderate physical effort and HR-6min, after 6 min of relaxation in the recovery stage) are summarized in o, τ, and heart rate measured at various times during the test .The correlation between the PWV values measured with the arteriograph and each of the fit parameters of the decay function, y(t), on the recovery stage, is summarized in European studies on PWV show that, for age groups under 30 years with optimal blood pressure values, the normal values of PWV range between 4.7 m/s and 7.5 m/s, with an average of 6.1 m/s . From TaDuring physical effort, several interrelated cardiovascular changes occur, with the primary goal of increasing blood flow to the working muscles . Among to + A∙exp(−t/τ).The fact that the evolution (over time) of HR after being suddenly interrupted by physical effort is of an exponential type . The analysis of the correlation between PWV and yo showed a moderate and positive correlation . Taking into account the maximum normal value of PWV in the European healthy population aged under 30 years and the average heart rate of the healthy population (in order of 80 b/min), the dependence of PWV on yo can be divided into four quadrants  and take the maximum normal value of PWV in the healthy European population aged under 30 years  into account, the dependence of PWV on τ can be divided into four quadrants . A good correlation between the resting heart rate and PWV was also reported in references ,25,26. TOne of the parameters that can be easily evaluated for any subject  is the value of HR measured after the subject got up from the supine position to the orthostatic position, HR-Pre. We must note that among those who have an HR-Pre larger than 100 b/min, all had PWV values greater than the normal upper limit for the age group under 30 years old (which is 7.5 m/s ).o. This fact was expected because yo and HR-6min represent the steady-state HR measured after 6 min of rest in the supine position. The difference between yo and HR-6min consists in the fact that HR-6min is a measured value, whilst yo is a value resulting from a fit. We remind the reader here that the fit can be interpreted as a form of smoothing of the experimental dependencies. In other words, yo can be seen as an average, and therefore, the Pearson’s coefficient for the correlation of yo with PWV is lower than the Pearson’s coefficient for the correlation between HR-6min and PWV.The correlation of PWV with HR-6-min is similar to that between PWV and yThe weakest correlation between PWV and the steady-state HR values during the test was recorded in the case of HR-4min, which is the HR value measured after 4 min of moderate physical effort .During any prolonged physical effort, one of the cardiovascular responses is vasodilation . For theAfter a moderate physical effort and a resting time of at least 6 min, the HR remains at values larger than 80 b/min;The relaxation time, τ, in a physical effort test of moderate intensity is larger than 1 min;The HR measured after the subject is raised from the supine position to the orthostatic position is larger than 100 b/min;Resting HR is larger than 80 b/min.The results of this study allow us to highlight some of the limits, which, if exceeded, can be associated with high PWV values:However, these must be seen as preliminary results. For their possible use in clinical practice, validation will be needed for a large number of subjects and on all age groups, for example, in a meta-study regarding the normal limits of PWV .The study shows that the use of step function physical effort as a stimulus and the recording of time-dependent HR can be used for the preliminary assessment of PWV.PWV correlates with the steady-state values of HR, both in response to a minimal effort (such as the change from the supine to orthostatic position) as well as with the steady-state values of HR during physical effort or after relaxation in the supine position. Relaxation time after moderate physical effort  is another parameter that correlates with PWV.The analyzed parameters allowed for the highlighting of some normal limits within the investigated parameters, beyond which PWV has higher values than those considered normal for the analyzed age group.


Plasmodium transmission foci, in the malaria epidemic-prone zone of Kisii highland.The vulnerable population within the malaria epidemic zone remains at risk of increased burden and fatality. This is because of unpreparedness and overstretching of healthcare capacity in the event of a full-fledged epidemic. The purpose of this study was to determine the prevalence of microscopic and submicroscopic infections, as well as map specific Plasmodium species 18S rRNA gene was used to detect the presence of submicroscopic infections on DNA extracted from dry blood spots.Patients seeking malaria treatment at Eramba health facility in the epidemic-prone zone of Kisii highland were enrolled in the study. Malaria outpatient data for the entire month of May were also included in the analysis. Patients' finger prick blood smears were examined for microscopic infections, while a real-time polymerase chain reaction targeting the 2 = 75.05, df 2, p < 0.0001). Children under the age of five had the highest positivity rate , followed by children aged 5–15 years , and individuals aged 15 years and above . Out of the 102 patients recruited, the positivity rate by microscopy was 57.8% (59/102) and 72.5% (74/102) by RT-PCR. Most of the microscopic infections  were from Morara and Nyabikondo villages in Rioma and Kiomooncha sublocations, respectively. The submicroscopic prevalence was 14.7% (15/102) and was observed only in patients from high-infection villages in Rioma  and Kiomooncha  sublocations. Across gender and age groups, females  and patients aged 15 years and above  had high levels of submicroscopic infections. There were two mixed infections of P. falciparum/P. malariae and P. falciparum/P. ovale, both from patients residing in Kiomooncha sublocation.Based on outpatient data, the malaria positivity rate was 20.7% . The positivity rate varied significantly by age group (χPlasmodium falciparum infections remained relatively high in the Marani subcounty. Infections were concentrated in two villages, which could serve as a target for future public health intervention, particularly during a malaria epidemic. However, epidemic-prone areas, particularly the highlands bordering malaria-endemic areas, remain vulnerable . BecausePlasmodium falciparum infection within epidemic zones. The purpose of this study was to determine the prevalence of microscopic and submicroscopic P. falciparum infections in clinical patients from the epidemic-prone Kisii highland in western Kenya, with the goal of mapping existing malaria transmission pockets.Increased deaths have been reported in Rift Valley epidemics-prone counties such as Baringo, Marakwet, and West Pokot as a result of a strained healthcare system during the recent malaria outbreak . For exa22.1This was a health-based survey conducted at Kisii County's Eramba health facility. The research was conducted in May 2019, when the health facility received 1115 patients seeking malaria treatment. The health facility is located on a highland adjacent to the malaria-endemic Lake Victoria basin in the Marani subcounty, which is administratively divided into locations and sublocations . Partici2.2The blood samples from finger prick were spotted on coded Whatman™ Blood Stain Cards  and smears made on glass slides. Microscopists at the hospital prepared and read Giemsa-stained slides for the presence and density of asexual parasites. Microscopy reading, estimation of parasite density and quality control were done as previously described .2.3Plasmodium DNA was stored at −20 °C. To confirm samples with parasite DNA, a species-specific real-time polymerase chain reaction (RT-PCR) targeting 18 s rRNA was performed. Amplification was carried out on a QuantStudio 3 Real-Time PCR System  with species-specific primers and probes, as previously described (Parasite DNA was extracted from DBS using the modified Chelex resin (Chelex-100) saponin method . The extescribed . The PCR2.4P. falciparum infections across gender, age groups, and sublocations. The analyses were carried out using SPSS version 25 for Windows and GraphPad Prism v.8.0.1 software. Data were considered statistically significant at p < 0.05.Patient data were entered into Microsoft Excel v.2016, and summarized using descriptive statistics. The Pearson chi-square test was used to compare microscopic and submicroscopic 2.5Maseno University's Ethical Review Committee granted ethical approval for this study (Reference No. 00456). Participants in the study provided written consent for the retrieval of patient data and the storage of their blood samples at the hospital. Parents or guardians provided consent as children's legal representatives. To avoid ethical code violations, patients' identities were not recorded.33.1P. falciparum infections than females, the difference was not significant . When compared to adults and children aged 5–15 years, children under the age of five had a higher prevalence of P. falciparum infections .Generally, the health facility attracted 1115 patients who sought malaria treatment in the month of May 2019. Females who sought malaria treatment were 78.6% (876/1115) whereas males were 21.4% (239/1115). Across age groups, 42.9% (478/1115) were adults followed by children aged 5–15 years 31.9% (356/1115) and > 5 years 25.2% (281/1115). Based on outpatient data, 935 febrile patients were examined by microscopy and 180 by the CareStart™ malaria HRP2 (Pf) rapid diagnostic kit (mRDT). The hospital's overall positivity rate was 20.7% . The positivity rate by microscopy was 20.1% (188/935), while the rate by mRDT was 23.9% (43/180). Despite males having higher proportion of fections . The var3.2Of the 935 outpatients, 102 study participants were randomly recruited. These included participants who were diagnosed by microscopy and their blood spotted on filter paper for RT-PCR assay. Most 57 (55.9%) were from Rioma sublocation whereas the rest were from Kiomooncha 37 (36.2%), Rikenye 6 (5.9%), Engoto Goti 1 (1%) and Megogo 1 (1%) sublocations. Sixty-one (59.8%) were female whereas 41 (40.2%) were males. Children between age group 5–15 years were largest group 41 (40.2%) followed by adults 38 (37.3%) and those <5 years 23 (22.5%). Female dominance was evident across two age groups as follows, 16 (69.6%) and 27 (71.1%) among <5 years and adults (≥15 years) respectively. More than half of recruited participants within the school-going age group (5–15 years) were males 23 (56.1%).3.32 = 2.98, df 1, p = 0.084). Adults had the most submicroscopic infections 21.1% (8/38) of any age group. The difference across the age groups was however not significant . These infections were only confirmed in patients from Rioma and Kiomooncha sublocations . Rioma sublocation had the highest proportion of patients with total infections (microscopic and submicroscopic) (80.7%), followed by Kiomooncha (59.5%), and Rikenye (66.7%). Females had 68.9% more total infections than males  of screened blood smears tested positive for microscopic infections. The majority of patients with positive smears 62.7% (37/59) and 27.1% (16/59) were Rioma and Kiomooncha residents, respectively. In the two sublocations, two villages, Morara in Rioma and Nyabikondo in Kiomooncha each had 12 patients with microscopic infections and contributed to 40.8% (24/59) of the slide positive rate. There were 17.6% (15/102) of submicroscopic infections, with female patients infected more than males . These iocations . Two paran males . Both in4In May, the malaria positivity rate among clinically ill patients was high in the epidemic-prone Marani subcounty. Males and children under the age of five were the most affected groups in this epidemic-prone zone, according to the total number of reported malaria cases in the facility. The infections were zoned to two major sublocations, Rioma and Kiomooncha, with transmission foci traced to Morara and Nyabikondo villages, respectively. The levels of submicroscopic infections, on the other hand, were low and were only confirmed on patients from the two sublocations. Females bore a disproportionate share of the submicroscopic infections compared to males, with high prevalence of total infections and parasite densities still being recorded among children aged 5–15 years.As observed in endemic zones, more females in Kisii highland sought malaria treatment at health facility as compared to males . FurtherP. falciparum. Morara and Nyabikondo villages in Rioma and Kiomooncha, which have a high number of microscopic infected individuals, serve as a guide for possible focal parasite or vectorial intervention targets in the event of a future outbreak of P. falciparum infections in the Marani subcounty.The majority of the microscopic infections observed were focal and could be traced back to Rioma or Kiomooncha sublocations. The two sublocations also had the highest number of patients seeking malaria treatment and are adjacent to each other while bordering an endemic region, Homa Bay County. Their proximity to endemic areas may predispose residents to malaria infections , as wellSubmicroscopic infections were relatively low, with females and adult patients having the highest levels. The slightly higher prevalence of this type of infection in females and adults supports previous findings and has been linked partly to immunity . However5This study established high malaria positivity rates and low levels of submicroscopic infections in five sublocations in the epidemic-prone region of Kisii county. The transmission was focalized to two main sublocation of which two key villages were observed as sentinel sites with the potential targeted parasite or vectorial intervention in case of malaria epidemic.10.13039/100000002National Institutes of Health (U19 AI129326 and D43 TW001505).The work was supported by Grants from the Authors have no conflict of interest to disclose.


Plasmodium falciparum histidine-rich protein 2 rapid diagnostic test (PfHRP2 RDT) in the detection of falciparum malaria in Akure, Nigeria. A cross-sectional and hospital-based study involving 601 febrile volunteer participants was conducted in Akure, Nigeria. Approximately 2–3 mL venous blood samples were obtained from each study participant for parasitological confirmation by microscopy and PfHRP2-based malaria RDT. Thick and thin films were prepared and viewed under the light microscope for parasite detection, parasite density quantification, and species identification, respectively. Dry blood spot samples were prepared on 3MM Whatman filter paper for nested PCR. The overall prevalence of microscopy, PfHRP2 RDT, and nested PCR were 64.89% (390/601), 65.7% (395/601), and 67.39% (405/601), respectively. The estimates of sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and Youden’s j index of microscopy and RDT were 96.30, 100.00, 100.00, 92.89, 97.50, 0.963, and 95.06, 94.90, 97.47, 90.29, 95.01, and 0.899, respectively. Malaria RDT recorded higher false negativity, compared microscopy (4.94% vs. 3.70%). A near perfect agreement was reported between microscopy and nested PCR, and between PfHRP2 RDT and nested PCR with Cohen’s kappa (k) values of 0.94 and 0.88, respectively. This study revealed that PfHRP2 RDT and microscopy continues to remain sensitive and specific for falciparum malaria diagnosis in the study area.Malaria remains a major public health challenge worldwide. In order to ensure a prompt and accurate malaria diagnosis, the World Health Organization recommended the confirmatory parasitological diagnosis of malaria by microscopy and malaria rapid diagnostic test (RDT) prior to antimalarial administration and treatment. This study was designed to evaluate the performance of nested polymerase chain reaction (nested PCR), light microscopy, and  Malaria is one of the deadliest infectious diseases worldwide, particularly in tropical and sub-tropical regions of the world, including Nigeria. Meanwhile, the prompt treatment control, management, and elimination of malaria disease is basically predicated on early and accurate detection of malaria parasites in infected individuals ,2. HowevPlasmodium lactate dehydrogenase (pLDH), and the aldolase [P. falciparum. However, Plasmodium lactate dehydrogenase (pLDH) and aldolase can be employed to detect all the four species of Plasmodium that infect humans, though they are inadequate for differentiating between P. vivax, P. malariae, and P. ovale, hence the name pan-pLDH and pan-aldolase [P. falciparum histidine-rich protein 2 (pfhrp2) gene deletions [Apparently, microscopy is advantageous in detecting and quantifying malaria parasite species, and it is quite cheap and excellent when used by an adequately trained microscopist . Howeveraldolase . One majaldolase ,8. Rapidaldolase ,10. Addieletions . Therefoeletions .Regarding nested PCR, it is most suitable for field research, and this technique has been confirmed by several studies to be highly sensitive and specific, such that it can detect a submicroscopic malaria parasite with a low density of 10 parasites/μL of blood . The morObviously, based on plethora of reports from previous studies conducted in malaria-endemic settings, it is well-established that malaria RDT and, to an extent, microscopy have low performance for the detection of submicroscopic malaria infections ,14. For P. falciparum as the dominant species [The study was carried out in some selected health facilities in Akure South local government area (LGA) of Ondo State, Southwestern Nigeria. The selected health facilities include Orita Obele Basic Health Centre, FUTA University Health Centre, State Specialist General Hospital, and Don Bosco Medical Centre. Akure South LGA is the most populous LGA in Ondo State, with an estimated population projection of 471,100 people in 2015 . It is l species .This research was a cross-sectional and hospital-based study conducted between October 2019 and February 2020. There were four randomly selected health facilities, consisting of Orita Obele Basic Health Centre, FUTA University Health Centre, State Specialist General Hospital, and Don Bosco Medical Centre. A face-to-face interview was employed to collect important information, such as age, sex, and occupation, from each of the volunteered individuals visiting the health facilities. Recruitment of participants was carried out in the outpatient section of the health facilities, with the assistance of health personnel. The inclusion criteria were fever with temperature ≥37.5 °C, resident within the community for a minimum of 6 months, completing the questionnaire, submission of the blood sample, and readiness to provide informed consent. Generally, all study participants recruited were individuals presenting with symptoms suggestive of malaria infection, as determined by the assisting health officer. Out of the 615 potential participants initially selected, only 601 individuals eventually satisfied the inclusion criteria and participated in the study, thereby giving a response rate of 97.72%. The participants’ recruitment flow chart is shown in 2 × P (1 − P)/M2, where N is the sample size, t is the normal standard deviation, which is 1.96, P is the previous malaria prevalence around the study area, and M is the degree of precision, which is 5% (or 0.05) [Regarding the study sample size, it was computed by using the single population proportion estimate formula of Araoye: N = tor 0.05) . By emplor 0.05)  and a co® filter paper , air-dried in a dust-free area, wrapped in plastic sample bags, placed in sealed zip-lock plastic bags with silica gel to prevent DNA degradation, and stored at room temperature until use.The blood samples were collected intravenously with the assistance of a well-trained laboratory technician. Approximately 2–3 mL venous blood was collected from each volunteer participant. Thereafter, the blood samples were transferred into ethylenediaminetetraacetic acid (EDTA) tube to prevent the coagulation of the blood samples. Additionally, 2–3 drops of blood were aliquoted on 3 MM WhatmanPlasmodium parasites prevalence, parasite density, and their exact species. The thin film was used in the identification of Plasmodium species, while parasite density was determined using the thick smear. Each slide was assumed to be negative when no parasite was observed after approximately 200 microscopic oil immersion fields have been observed. Additionally, all the slides were stored in secured slide-boxes, and quality assurance was carried out on 10% of all the positive slides by an expert laboratory microscopist for species identification and confirmation, and there was no difference after the quality assurance was performed. Afterwards, all the blood samples were confirmed by nested PCR. Moreover, all participants positive for falciparum malaria were treated according to national standards and guidelines with artemether-lumefantrine (AL).Thick and thin blood films were prepared from the blood in EDTA for the detection and quantification of malaria parasite species . The thiParasite density was calculated as the number of parasite/µL of blood, assuming an average leucocyte count of 8000/µL of blood for an average individual . TherefoP. falciparum histidine-rich protein 2 antigen in blood samples by following the manufacturer’s instructions. The test kits were kept at an optimal temperature between 2–30 °C, and the results were read after 20–25 min. Briefly, approximately 5 µL from each blood sample was aliquoted on the sample window of the PfHRP2 RDT cassette. Thereafter, three drops of diluent were added. The PfHRP2 RDT was left intact at room temperature for a period of 20 min, and the results were recorded as positive or negative as appropriate after checking the cassette for the presence of distinct lines.The CareStart™ PfHRP2 RDT  was employed for the rapid detection of the ® Blood and Tissue Kit, cat. No. 69506  by following the manufacturer’s instructions. Moreover, genomic DNA was eluted in 50 mL and was kept at −20 °C until nested PCR analysis. Regarding the nested PCR, Plasmodium genus-specific and species-specific primers targeting the 18 small subunits ribosomal RNA (18S rRNA) genes were designed according to Snounou and Singh [Plasmodium culture were used as the positive control, while ultra-pure water was employed as the negative control. The primers used are shown in Genomic DNA was extracted from the 3 MM Whatman filter paper dried blood spot (DBS) using the DNeasynd Singh , with miPlasmodium nested PCR product was measured using a 50 base pairs ladder. The nested PCR products of 205 base pairs, as observed on the gel, were thereafter purified and sequenced.The electrophoresis of the secondary nested PCR amplification products was carried out in a 2% agarose and 0.5X Tris-acetate EDTA (0.5X TAE buffer). Thereafter, the gel was stained with ethidium bromide and subsequently visualized using the ultraviolet–violet (UV) gel documentation system . The size of the p-values ≤ 0.05. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic accuracy, and Youden index (J) were calculated by using the GraphPad Prism . Computations were carried out using the contingency table. The total accuracy was computed through the formula: Accuracy = (TP + TN)/(TP + FP + FN + TN), where TP = True Positive, TN = True Negative, FN = False Negative, and FP = False-Positive.Data entry and analyses were conducted through Statistical Package for Social Sciences (SPSS), version 22.0 . The presence or absence of malaria disease was computed, and the variance in the prevalence between the age groups and sex was calculated using a chi-square test at 95% confidence level. Statistical significance was set at Prior to the commencement of the study, the ethical protocol was reviewed, and approval was obtained from the Ondo State Ministry of Health, with reference number OSHREC/10/01/2019/082, and the Ethical Review Committee of the Federal University of Technology, Akure, Nigeria. Additionally, permission was sought from the hospital management board of all the health facilities in the study areas. Written informed consent and accent were sought from each of the participants and caregivers or guardians after a detailed explanation of the study protocol and procedures, coupled with the risk and benefits of the study. All confirmed malaria-positive individuals were treated according to the national guidelines. This study was conducted in accordance with the Helsinki Declaration of 1975 and as amended in the year 2000.Out of the 615 potential participants enrolled on the study, only 601 (97.72%) met the criteria for inclusion in the study. Therefore, a total of 601 individuals were screened for malaria parasite using PfHRP2 RDT, microscopy, and nested PCR, of which males were 341 (56.7%), while females were 260 (43.3%). Additionally, with respect to the age group, the highest age group examined was ≥20 years, with 310 (51.6%), followed by the age group 13–19 years, with 230 (38.3%), while the least age group examined in this study was ≤12 years, with 61 (10.1%) participants. Similarly, the study sites and the number of participants included Orita Obele Health Centre, 100 (16.64%); FUTA Health Centre, 203 (33.78); State Specialist Hospital, 197 (32.78%); and Don Bosco Health Centre, 101 (16.81%) .P. falciparum by microscopy, PfHRP2 RDT, and nested PCR techniques is shown in The malaria prevalence by microscopy, PfHRP2 RDT, and nested PCR were 390 (64.89%), 395 (65.72%), and 405 (67.38%), respectively. A total of 385 samples were observed to be positive by all three diagnostic techniques. However, 15 samples were positive only by nested PCR and 10 samples only by PfHRP2 RDT. No sample was detected by microscopy only. The Venn diagram comparing the proportion of prevalence of Moreover, p < 0.0001) decreased as age increased. The age group ≤12 years had the highest sensitivity and specificity (100.00 (92.44–100.00) vs. 100.00 (78.47–100.00)). However, the age group ≥20 years had the least sensitivity of 94.39% (C.I: 90.23–96.84) and a high specificity of 100% (C.I: 96.74–100.00). Similarly, the sensitivity and specificity of PfHRP2 RDT significantly (p < 0.0001) decreased with an increase in age. The age group ≤12 years significantly (p < 0.0001) had the highest sensitivity and specificity compared to the age group ≥20 years, which had the least sensitivity and specificity.The findings from this study highlight the high performance of microscopy and histidine-rich protein 2 RDT, with respect to nested PCR, which was employed as the standard reference. The World Health Organization (WHO) recommended that all suspected cases of malaria should be promptly and accurately confirmed by RDT and microscopy, which is considered the gold standard technique ,24. SimiApparently, this study revealed that nested PCR significantly detected a higher malaria prevalence (67.39%), compared to microscopy (64.89%) and RDT (65.72%). Studies have shown that nested PCR can detect a low parasite density of 10 parasites/μL of blood . The obsP. falciparum [P. falciparum and P. vivax to be 95.3% and 96.4%, respectively. Similarly, other studies have demonstrated high sensitivity of PfHRP2 RDT and light microscopy in Nigeria [Currently, malaria RDT is the major method of malaria diagnosis in Africa, where it represented approximately 75% of all diagnostic testing for suspected malaria infection in public facilities in 2017 . Though lciparum . Similarlciparum  on PfHRPlciparum , who rep Nigeria ,29,42 an Nigeria ,43,44. On the other hand, the sensitivity obtained in this study was higher than that obtained in a systematic review and meta-analysis conducted by Yimam , who repApparently, the health officers understood how to use PfHRP2 RDTs correctly without any difficulty, which therefore confirms that PfHRP2 RDTs are simple tests to perform and interpret . The PfHPlasmodium parasites. Additionally, it could result in a significant wastage of drug resources on false-positive individuals. Thus, it will be appropriate to use a better sensitive diagnostic tool for malaria infection confirmation before treatment.While microscopy revealed a zero false-positive result in this study, malaria RDT showed a false-positive rate of ≃5%. Apparently, the false-positive rate of PfHRP2 RDT observed in this study was lower than the false-positive rate of PfHRP2 RDT reported from some other high malaria-endemic regions, such as Zanzibar , Ethiopipfhrp 2 and pfhrp 3 genes from the parasite. However, available information revealed that the deletion of pfhrp 2 and pfhrp 3 genes from the parasite is very unlikely in this study. [P. falciparum parasite density during antigen-antibody immunological reactions [Furthermore, in this study, microscopy showed a false-negative rate of ≃4% among submicroscopic individuals, while PfHRP2 RDT showed a false negativity of ≃5%. Our findings in this study were lower than the false-negative results of microscopy and PfHRP2 RDT obtained in other studies conducted in malaria-endemic settings ,56. The s study. ,61. Ratheactions ,63. Obvieactions ,63. ThusFurthermore, our findings revealed that the sensitivity and specificity of microscopy and PfHRP2 RDT decreased with increases in age. This could likely be due to the low parasite density or submicroscopic malarial infection, which may likely be undetected by microscopy and PfHRP2 RDT techniques ,46,59. In view of the findings, which revealed that PfHRP2 RDT had a high sensitivity detection level in this study, it could be recommended that PfHRP2 RDT could be employed, along with the gold standard method of microscopy, for better detection and treatment of malaria-infected individuals . MeanwhiConclusively, although the sensitivity and specificity observed in this study were quite high, false-negative rates of ≃4% and ≃5% were obtained for microscopy and PfHRP2 RDT, respectively. Therefore, besides the routine malaria RDT and microscopy used as the major diagnostic tools in Nigeria, nested PCR and other sensitive diagnostic tools, such as loop-mediated isothermal amplification (LAMP), should be included as additional options where it is feasible. Even though nested PCR tools are relatively costly, with respect to the standard diagnostic tool, their subsequent advantage in malaria control and elimination cannot be underemphasized. Therefore, in order to ensure proper malaria control and elimination in the study area, the use of a more sensitive malaria diagnostic tool is highly imperative.


Plasmodium knowlesi, P. cynomolgi, P. inui and P. cynomolgi are the emerging cause of malaria in humans. The epidemiological study of simian malaria in primates provides useful information in identifying the risk of human-macaques Plasmodium infection.The aim of Malaysia to eliminate malaria nationwide by 2020 seems need to be prolonged. Whilst Malaysia has successfully eliminated human malaria transmission, simian malaria parasites such as Plasmodium parasites in macaques. The most prevalent Plasmodium species in macaques was P. inui (49.27%) and the least prevalent was P. fieldi (4.76%). The prevalence of simian malaria was higher in East Malaysia compared to Peninsular Malaysia. The mono, dual and triple infection types were the most common among macaques.This study was performed to gather all available data in terms of simian malaria epidemiology study among macaques in Malaysia over the last two decades. This systematic review was conducted according to the PRISMA guidelines to select appropriate articles as references. Data searches were performed through international databases such as Google Scholar, PubMed, CrossRef, Scopus, Web of Science and Science Direct for original articles published from 2000 until 2021. The review identified seven simian malaria epidemiology studies in Malaysia over the 20-year study period. Most studies were conducted in Peninsular Malaysia  followed by East Malaysia . All studies showed positive detection of The non-human primates like macaques are the reservoir of simian plasmodium in Malaysia. Hence, the study of host epidemiology is an important insight to public health management as there is a high occurrence of simian malaria in Malaysia. The right measurement can be taken as well to prevent the transmission of simian malaria from macaques to humans. Plasmodium species that can cause zoonotic malaria in humans. Close contact of human and macaques cause zoonotic transmission of simian malaria. The simian plasmodium such as P. knowlesi, P. inui and P. cynomolgi have been found infecting humans in Malaysia; mainly in East Malaysia (Borneo). Zoonotic malaria poses great risk to public health as prolonged in treatment often lead to fatal outcomes. Hence the knowledge of prevalence and diversity is important to access, this can therefore enlighten the authorities to plan a control strategy that will minimize the zoonotic transmission between non-human primate host to human. This systematic review has summarised all publish data of macaques-plasmodium infection from the year 2000–2021 by using PRISMA guidelines. Our result showed that P. inui (49.27%) is the most prevalent Plasmodium species found in macaques, followed by P. cynomolgi (33.05%) and P. knowlesi (26.86%). Simian plasmodium prevalent was also found higher in East Malaysia (97.0%) compared to Peninsular Malaysia (45.18%). The significant increase of simian malaria incidences in human have jeopardized the national malaria elimination programme. Hence, this study provides a compact insight into the plasmodium epidemiology of macaques in Malaysia.Macaques are the most abundant primates in south east Asia including Malaysia. Due to deforestation, macaques came closer to human settlements searching for food. Macaques like the long-tailed and pig-tailed harbouring several  Plasmodium are the causative agents of the malaria disease. Over the recent years, the burden of clinical malaria in Malaysia was predominantly due to infection by P. knowlesi, a malaria parasite of macaques. Many studies have shown that parasites are frequently transmitted from wild non-human primates (NHP) like long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) or those in captivity to humans in a shared habitat  of combination to obtain relevant articles. This systematic review was conducted according to the guidelines of PRISMA  [This systematic review was conducted using published studies on the detection and identification of nalysis) .Plasmodium diagnosis method, as well as the Plasmodium species and the number or percentage of infection.The inclusion criteria for the article selection were: (a) the primary simian malaria research conducted in Malaysia; (b) the study subject were primates, specifically macaques; (c) the articles were published from the year 2000–2021; (d) full-text articles in English; (e) the articles must also provide a description of sample size, study site, Plasmodium in vector; (d) Plasmodium genomic research; (e) diagnostic and antimalaria; (f) geographical and landscape.For exclusion criteria, this study excludes review articles and articles that focused on: (a) human malaria; (b) simian malaria infection in human; (c) Plasmodium detection, the number/ percentage of Plasmodium positive infection as well as the type of simian malaria infection were extracted.All searched articles were imported and combined into the Microsoft Excel 2019 software and duplicated files were removed. Based on the predetermined inclusion criteria, two independent review authors (SJ and NAS) determined qualified studies based on titles, language and abstracts. From the selected articles, relevant information such as name of the first author, year of publication, study region , study state, period of sampling, study subject, sample size, analysis method for Plasmodium species infection in macaques, type of simian malaria infection and simian malaria in different regions were calculated and tabulated.Based on the extracted data, descriptive analysis was conducted using Microsoft Excel 2019 software. The total number of samples collected in each state of Malaysia was represented in map. The prevalence of Plasmodium analysis, human infection articles and geographical and landscape articles. As a result, seven articles were selected in this study for data extraction [The literature search from six databases has generated 1347 results, which composed of 660 articles from Google Scholar, 89 from PubMed, 141 from CrossRef, 46 from Science Direct (Elsevier), 332 from Scopus and 79 from Web of Science. After duplicate articles have been removed, 967 articles were left for screening. After the screening of foreign languages, titles, and abstracts, 536 articles were left for further detailed evaluation . Most artraction ,18,26–30Among all the included studies, one study (14%) was published from year 2000–2010 , and sixMacaca fascicularis (long-tailed macaque) and M. nemestrina (pig-tailed Macaque) were known to be the subjects in all seven studies. One of the studies has included Pongo pygmaeus (Bornean Orang-Utan) in their study, but the data were excluded due to insufficient details [Plasmodium parasite. details . Meanwhi details . The samSix out of seven (86%) studies had utilised nested PCR ,18,26–29Plasmodium parasites in their subjects. The highest percentage of Plasmodium positive subjects is 100% [Plasmodium positive percentage from macaques’ blood samples is 59.98%.All studies showed positive detection of  is 100% , meanwhi is 100% . OverallPlasmodium species in macaques. The prevalence of P. knowlesi infection was shown in all studies (7/7) [P. cynomolgi, P. coatneyi, P. fieldi and P. inui prevalence were shown in 4/7 studies [Plasmodium species, P. inui has the highest average prevalence (49.27%) and P. fieldi has the lowest prevalence average (4.76%). Plasmodium knowlesi have almost the same prevalence average (26.86%) as P. coatneyi (26.82%) with only 0.04% indifference. Meanwhile, the average prevalence of P. cynomolgi (33.05%) is the second highest among the five Plasmodium species. One study had observed quite number of human plasmodia like P. vivax, P. ovale and P. malariae in their macaques’ blood samples but the data was not included [es (7/7) , 26–30 w studies ,26,27,29Plasmodium species respectively. Overall, dual infection (17.59%) was discovered mainly in an individual macaque and quintuple infection (0.92%) is less likely to occur. However, there were not many differences between prevalence of mono, dual and triple infection with only 0.03%-0.18% differences. All in all, macaques in Malaysia were likely to be infected by either one, two or three of Plasmodium in an individual.The prevalence of simian malaria infection type was shown in P. cynomolgi (9.00%) and P. fieldi (6.50%) compared to the rest of the simian plasmodium. For dual infection, macaques were often identified with the mix of P. cynomolgi with P. inui (10.00%) and the mix of P. knowlesi with P. inui (6.0%). The triple infection in macaques were predominantly comprise with the mix of P. knowlesi, P. cynomolgi and P. coatneyi (10.50%). Lastly, for quadruple infection, macaques were found most with the combination of P. knowlesi, P. cynomolgi, P. coatneyi and P. inui (23.00%).Subsequently, the details of the infection type were breakdown in The prevalence of infection in different regions of Malaysia ware shown in Plasmodium infection compared to Peninsular Malaysia, and Pahang state in Peninsular Malaysia has a higher prevalence compared to Klang Valley.Next, two of the state in Peninsular Malaysia; Pahang and Klang Valley which made up of Selangor and KL were compared for the prevalence of infection. These two states were selected among all other states because the number of studies has been carried out was more than one; three studies have been conducted in Pahang ,28,29 anP. knowlesi [Plasmodium in simian host species have been carried out [Plasmodium among simian host species in Malaysia that is based on seven full-text English publications dated from 2008–2021.The aim of Malaysia to eliminate malaria nationwide by 2020 seems to need to be prolonged. Whilst Malaysia has successfully eliminated the transmission of human malaria , a largeknowlesi –34. In oried out ,18,26–30M. fascicularis) and pig-tailed macaques (M. nemestrina). These macaques are known as the reservoir of five common simian Plasmodium species namely P. knowlesi, P. cynomolgi, P. coatneyi, P. fieldi and P. inui. This study has found that the most prevalent Plasmodium in macaques was P. inui, followed by P. knowlesi and P. cynomolgi and the least prevalent was P. fieldi. Studies in the Philippines [P. inui was highest among all, but it was lowest in the study conducted in Singapore [P. fieldi was high in Thailand (30%) [Plasmodium parasites at each sampling site were also affected by few variables, which include differences in host genetics, susceptibility of host toward parasite, differences in the vectors between sampling sites and the frequency of sampling on one individual host [P. vivax, P. ovale and P. malariae [P. knowlesi with human plasmodium also observed in the sampled subject [The main simian malaria host in Malaysia is the long-tailed macaques  , Singapond (30%)  and the nd (30%)  but it wnd (30%) ,26,27,29nd (30%) . The reaual host . There w% , SingaPlasmodium species infection in macaques were detected in Malaysia by the PCR diagnosis method. Though the microscopic examination of Giemsa-stained thick and thin film is the most common malaria diagnostic method. However, it was time-consuming and misdiagnosis of the Plasmodium species is common particularly when the parasitaemia is low and inexperience staff [P. knowlesi infection may resemble P. malariae infection and other malaria infections such as P. falciparum and P. vivax [P. cynomolgi would have been misidentified as P. vivax under the microscope [Plasmodium species are indistinguishable on regular microscopy, hence upgrading microscopy training will be ineffective. Therefore, Plasmodium species detection by using molecular method; PCR-based was adapted. Nested and real-time PCRs are relatively simple, highly specific and have greater sensitivity of detection even when the parasitaemia is low (as low as 2–5 parasites/μl) [Plasmodium 18S subunit rRNA gene [P. knowlesi and P. malariae for human infection cases in Sabah have been subjected to a PCR test. In 2011, molecular detection was expanded to Sarawak and Peninsular Malaysia [The presence of multiple ce staff . The P. P. vivax  and P. ccroscope . As thesites/μl) ,42. FurtRNA gene –47. WhenRNA gene . Since 2Malaysia .Plasmodium species at once. In Malaysia, the commonly found Plasmodium species in mono infection is P. cynomolgi, and mix of P. cynomolgi with P. inui for dual infection; this have found similar in Singapore [Plasmodium species mixture for triple and quadruple infection in Malaysia macaques are P. knowlesi + P. cynomolgi + P. coatneyi and P. knowlesi + P. cynomolgi + P. coatneyi + P. inui respectively. The plasmodium mix of triple and quadruple infection were also found in the studies at Singapore and Cambodia but their infection percentage were not as high as Malaysia. The multiple infections in a host are complex, it has been observed that the parasitaemia of Plasmodium parasite fluctuates over time within a host [Plasmodium species from one another [Plasmodium present in each individual host [Plasmodium species in a primate host as the light microscopy detection on Giemsa-stained blood films method are imperfect and often misdiagnosed the actual Plasmodium species.This study found that the mono, dual and triple infections were the most common infections detected in primate hosts in Malaysia and similar observations were reported in Thailand , Singapoingapore  and Cambingapore . Meanwhin a host ,38. Due  another , 38. Eveual host . NonethePlasmodium compared to macaques that live in urban/city areas [P. falciparum or P. vivax transmission, people infected by P. knowlesi are usually exposed to agricultural settings or forests due to the vectors’ exophagic nature [Anopheles mosquitoes as well as macaque populations. To add, P. knowlesi transmission was also found to be higher in large intact forest patches, pulpwood and oil palm plantations with fragmented landscapes in Sabah [P. knowlesi, as confirmed by specific antibody responses and data on asymptomatic infections indicates that women have a higher number of non-clinical infections [In comparison between the prevalence of simian malaria between regions, this study found the simian malaria prevalence in East Malaysia (97.00%) was higher compared to Peninsular Malaysia (45.18%). The forest cover has played an important role in the prevalence of the disease as studies have found that macaques in rural/forested areas were more infected by ty areas ,37. The ty areas , meanwhity areas . In genety areas . These cty areas . Unlike c nature . Naturalc nature . Furtherc nature  showed Lin Sabah . Based ofections ,55.Anopheles mosquitoes of the Leucosphyrus group mainly feed on macaques in the forested areas at higher elevation [On the other hand, the urban area such as Klang Valley has lower prevalence in simian malaria due to the lack of competent vectors. This is because vectors for simian malaria in Malaysia which is the levation ,56,57. Tlevation . Neverthlevation . Not onllevation –60. The levation . TherefoP. knowlesi in humans. The first case of a naturally acquired human infection with P. cynomolgi in Malaysia was a 39-year-old Malay woman from Hulu Terengganu located at the East Coast of Peninsular Malaysia. She lives in a malaria-free modern housing area and had no previous history of malaria. However, there was a small, forested area with infrequent sightings of long-tailed macaques behind her house [P. cynomolgi and P. knowlesi co-infections [P. cynomolgi infections among indigenous communities in four states  in Peninsular Malaysia [Plasmodium inui and P. coatneyi infections were also detected among the indigenous communities in Melaka and Perak state Peninsular Malaysia respectively [When macaques move from the forested area to forest fringe and human settlement, the vectors may have followed their host and adapt to human settlement as well . On thater house . Next, sfections . A recenMalaysia . Plasmodectively . In Peniectively .P. knowlesi cases rosed from 376 to 4131 cases [knowlesi malaria cases in Malaysia from year 2010–2018 were the highest (18687 cases) compared to the other South East Asia country like Indonesia (481 cases), Brunei (73 cases), Myanmar (49 cases), Thailand (44 cases), Vietnam (38 cases), Laos (10 cases) and Philippines, Singapore, together with Cambodia for less than 10 cases [A recent systematic review also revealed the malaria cases from predominantly causes by human malaria parasite were shifting to simian malaria parasites . Human m31 cases . Subsequ10 cases . Hence, knowlesi malaria but zoonotic malaria as whole and ensuring that procedures are in place to allow for timely accurate diagnosis and treatment, particularly in high-risk areas. The use of mosquito’s repellent, patch, and net need to encourage mainly for people that live or entering high-risk area.While The National Malaria Elimination Strategic Plan that targeted to achieve “malaria-free” status by 2020 were challenged due to the rising case of zoonotic malaria in human and there was no new strategic plan for elimination of zoonotic malaria to date. To reduce simian plasmodium transmission to humans, present malaria prevention initiatives may need to be revised or new approaches created. By the finding of this study shown, the authorities like The Wildlife Department might screen macaques for malaria parasites and restrict their population together with managing the human-macaques conflict. Parallel to this, public health policies not only should focus on raising awareness of Plasmodium can be detected through saliva, urine and a faecal sample of an infected individual. Thus, simian malaria detections from urine and a faecal sample of macaques should be considered for the epidemiology study in the future. Additionally, the future surveys of macaque hosts should be combined with the studies of the Anopheline vectors, because zoonotic malaria does not exist where there are no macaque-mosquito-human interactions.In the period of 20 years, the number of local simian malaria epidemiology studies were relatively low and among them, only seven articles fulfilled our criteria. The lack of local simian malaria studies might be due to the invasive specimen collection of the blood samples from macaques as this required a proper trapping system and highly trained experts to handle the animals. Therefore, a non-invasive specimen collection method that can substitute for blood sample collection such as urine and faecal sample collection has been introduced by several studies ,67, revePlasmodium species that infected primates such as P. knowlesi, P. cynomolgi, P. inui and P. cynomolgi are emerging causes of malaria in humans. In addition, this study confirmed that these four species together with P. fieldi were detected among macaques in Malaysia. The prevalence of simian malaria was higher in East Malaysia and the mono, dual and triple infection type is the most common among macaques. More simian malaria epidemiology study among macaques together with Anopheles vectors should be carried out in Malaysia to assess the risk of macaques-human infection.S1 Data(XLSX)Click here for additional data file.