Plasmodium falciparum infection is microscopic examination of Giemsa-stained peripheral blood smears. The effectiveness of this procedure for infection surveillance and malaria control may be limited by a relatively high parasitaemia detection threshold. Persons with microscopically undetectable infections may go untreated, contributing to ongoing transmission to mosquito vectors. The purpose of this study was to determine the magnitude and determinants of undiagnosed submicroscopic P. falciparum infections in a rural area of western Kenya.The gold standard for diagnosing P. falciparum infection was measured by comparing the prevalence of infection based on light microscopic inspection of thick blood smears versus real-time polymerase chain reaction (RT-PCR) targeting P. falciparum 18S rRNA gene. Long-lasting insecticidal net (LLIN) use, participation in nocturnal outdoor activities, and gender were considered as potential determinants of submicroscopic infections.A health facility-based survey was conducted, and 367 patients seeking treatment for symptoms consistent with uncomplicated malaria in Homa Bay County were enrolled. The frequency of submicroscopic P. falciparum parasites in 14.7% (54/367) of cases. All of these samples were confirmed by RT-PCR. 35.8% (112/313) of blood smear negative cases were positive by RT-PCR, i.e., submicroscopic infection, resulting in an overall prevalence by RT-PCR alone of 45.2% compared to 14.7% for blood smear alone. Females had a higher prevalence of submicroscopic infections  compared to males . The risk of submicroscopic infections in LLIN users was about half that of non-LLIN users (OR = 0.59). There was no difference in the prevalence of submicroscopic infections of study participants who were active in nocturnal outdoor activities versus those who were not active (OR = 0.91). Patients who participated in nocturnal outdoor activities and use LLINs while indoors had a slightly higher risk of submicroscopic infection than those who did not use LLINs (OR = 1.48).Microscopic inspection of blood smears was positive for asexual P. falciparum should be supplemented by more sensitive diagnostic tests in order to reduce ongoing transmission of P. falciparum parasites to local mosquito vectors.Microscopic inspection of blood smears from persons with malaria symptoms for asexual stage The online version contains supplementary material available at 10.1186/s12936-021-04012-6. Plasmodium falciparum infection may not be sufficiently sensitive to detect submicroscopic infections [P. falciparum transmission in endemic communities [Current vector control and parasite surveillance strategies have shown remarkable progress in reducing the global malaria burden . In Kenyfections . A high munities , 6.Anopheles [Submicroscopic infections have been observed not only in high transmission settings but also in malaria endemic areas with seasonal or low transmission , 8. Thesnopheles , 11, lownopheles –14, and nopheles , 15. Prenopheles –20 and cnopheles , 21–24. nopheles , 26. TheP. falciparum infection prevalence [The study was conducted at the Ngegu health facility in Homa Bay County, western Kenya. This facility had a catchment population of 6,703 persons in 2020. The sampled patient population came from the Kochia location, which is divided into smaller administrative units referred as sub-locations. Study participants were residents of Kamenya, Kanam, Kaura, Korayo, Kothidha and Kowili sublocations located near the shore of Lake Victoria at a latitude of 34.64190E and 0.38000S with an elevation of 1143–1330 m above sea level  laboratory. All the samples were tested for submicroscopic infection by RT-PCR at the ICEMR laboratory at Tom Mboya University, Homa Bay.A health facility-based survey was performed, in which 367 patients seeking malaria treatment from six sub-locations were enrolled. Patient information such as LLIN ownership and use, occupation, and participation in nocturnal outdoor activities was collected. Nocturnal outdoor activities included casting fishing nets, setting and removing fishing traps, overnight fishing, early purchase of fish by small scale traders, farming, late evening trading at open-air markets, and waiting, picking up, and dropping off clients from motorcycle taxi riders (“Bodaboda”). Patient enrolment and data collection periods occurred in July and August of 2020, which coincided with the peak of Plasmodium parasites. Slides for microscopic examination for the presence of Plasmodium parasites in patient blood samples were prepared and read by experienced hospital microscopists. Parasite density was estimated based on visualizing microscopic fields consisting of 200 leucocytes with the assumption of a standard value of 8,000 leucocytes per μL of blood. If no parasite was found after examining 200 fields at 100 × magnification, the microscopy result was declared negative. All slides were subjected to a second microscopy reading for quality control.Blood samples used in this study were obtained by antecubital venipuncture and immediately pipetted on filter paper and glass slides. Experienced hospital microscopists prepared and read Giemsa-stained slides for the presence and density of Plasmodium falciparum species-specific 18S ribosomal RNA primers and probes were used to confirm the presence of parasite DNA [Genomic DNA was extracted from dried blood spots on filter paper following a modification of the Chelex resin (Chelex-100) saponin method . Plasmodsite DNA . PCR wasP. falciparum infection and LLIN use and participation in nocturnal outdoor activities, health centre study staff interviewed and completed a questionnaire for each participant. Information on LLIN ownership and usage, engagement in nocturnal outdoor activities, and occupations , farmer, trader, fishermen, motorcycle taxi riders, teacher, security, construction, unemployed and other) were collected. Only participants who acknowledged being outdoors from 1800 h–2000 h, 2000 h–2300 h, 2300 h–0400 h, and 0400 h–0600 h due to occupational requirements were considered involved in nocturnal outdoor activities.To assess the relationship between submicroscopic Patient data were entered into Microsoft Excel v. 2016 for cleaning and analysis. Descriptive statistics such as sum, mean, standard deviation, standard error and 95% confidence interval were used to summarize the population under study. Before comparison of mean value, data normality was confirmed using the Shapiro–Wilk normality test. To determine LLIN usage across gender, age and sub-location residence, multiple mean comparisons between these variables were performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Comparisons between blood smear positive and submicroscopic  groups were performed using Pearson chi-square. Binary logistic regression models were used to determine the association between microscopic and submicroscopic infections and potential determinants such as LLIN use, engagement in nocturnal outdoor activities and gender. A multivariate analysis was used to determine the relationship between the LLIN use, nocturnal outdoor activities, microscopic and submicroscopic infections. Analyses were performed in GraphPad Prism v.8.0.1 Software and SPSS version 25 for Windows. Data were considered statistically significant at p < 0.05.2 = 6.84, df 1, p = 0.009). Study participants were predominantly > 15 years (67%), while only 5% were < 5 years. LLIN use was significantly greater among children < 5 years relative to the 5–15 and > 15-year groups  = 19.20, p < 0.0001). LLIN use was similar among participant sublocation residence as was nocturnal outdoor activity behaviour. Two study participants were pregnant. Male patients participating in nocturnal outdoor activities were 68% (57/84) whereas females were 32% (27/84). There was no significant difference in LLIN use between participants who engaged in nocturnal outdoor activities and used LLIN while indoors versus non-LLIN users who participated in nocturnal outdoor activities .Study participant gender, age, LLIN usage, sublocation residence and participation in nocturnal outdoor activities are described in Table Asexual stage malaria parasites were found in 54 (15%) of the 367 microscopically-screened blood smears. The prevalence of microscopy positive infections in males (17%) was slightly higher than that of females (13%). Children < 5 years had the lowest infection rate by microscopy (11%), while those aged 5–15 had the highest infection rates (27%), followed by adults (10%). On the basis of parasite density, children < 5 years, those age group 5–15 years, and adults recorded 219, 957 and 330 asexual parasites per microlitre of whole blood, respectively. All the slide positive results were confirmed for asexual parasite DNA by RT-PCR.P. falciparum positive by RT-PCR and labelled as submicroscopic. These infections accounted for 67% (112/166) of confirmed malaria cases. Detection of submicroscopic infections (31%) were significantly greater than that of microscopic infections (15%) . Female study participants had 36%  more submicroscopic infections than males 24% . Female patients had a significant difference between microscopic and submicroscopic infections , but males did not. Neither of two pregnant study participants had microscopy positive infection; one had a submicroscopic infection. The prevalence of submicroscopic infection was highest in children < 5 years (42%), followed by adults (30%) and children aged 5–15 years (29%). Only one of eight male patients < 5 years had submicroscopic infections, compared to seven out of 11 females of the same age.Thirty-six percent (112/313) of the slide negative samples were found to be P. falciparum (both microscopic and submicroscopic) infections (51.3%), followed by Korayo (45.7%), Kanam (44.8%), Kamenya (43.3%), Kothidha (39.1%) and Kowili (33.3%). Korayo had the highest percentage of submicroscopic infections, 38.9% , followed by Kamenya , Kaura , Kowili , Kanam  and Kothidha . The Kruskal–Wallis H test revealed a significant difference between microscopic and submicroscopic infections across all sublocations (H(11) = 41.21, p > 0.001). Moreover, Dunn’s test pairwise comparison showed a significant difference between microscopic and submicroscopic infections in Kamenya (p = 0.0320) and Korayo (p = 0.0019).There were more submicroscopic infections than microscopic infections at the sub-location level. Kaura had the highest prevalence of 2 = 0.016)  but not on submicroscopic infections  than non-users (25.4%). This implies that the chance of LLIN users harbouring submicroscopic infections was about half that of non-LLIN users (OR = 0.59). Though not significant , more patients involved in nocturnal outdoor activities had submicroscopic infections (32.1%) compared to those restricted indoors during night hours (25.3%). Participants who engaged in nocturnal outdoor activities were 0.91 times more likely to have submicroscopic infections than those who stayed indoors .The odds of females seeking malaria treatment having submicroscopic infections was 1.73 higher than males Table . Use of P. falciparum infections in patients with malaria symptoms seeking treatment in Homa Bay County's six sub-locations. The use of LLINs and being female was linked to a high prevalence of submicroscopic infections. Interestingly, the effect of LLIN usage on submicroscopic infection prevalence was also observed among users who participated in nocturnal outdoor activities. Furthermore, this study was unable to establish a conclusively link between the observed microscopic P. falciparum infections and outdoor transmission. This is due to the fact that only one LLIN user participating in nocturnal outdoor activities had microscopic infections, compared to nine infected non-LLIN users engaged in nocturnal outdoor activities.In this study age, gender and use of LLINs were factors in the occurrence of submicroscopic P. falciparum slide positivity in males [P. falciparum infections and parasite density in the 5–15 age group were linked to a low number of LLIN use thus predisposed to bites from infected malaria vectors [Anopheles mosquitoes occurs at the transition point from LLINs coverage to outdoor or vice versa. With these findings, the study suggests that outdoor malaria transmission is low in the six sub-locations. This finding supports previous findings in 2018 and 2019 by Ondeto et al., (pers. commun.) on an increased population of Anopheles arabiensis collected outdoors, with blood meal results indicating a feeding preference for bovines in the study area.The study findings revealed that male patients were more likely than females to test positive for microscopic infections. This could be attributed to existing social behavioural differences, such as more males engaging in nocturnal outdoor activities, which kept them out of the intervention coverage and low LLIN use when compared to female patients. Low LLIN use and participation in activities outside of intervention coverage –34 have in males , 38. The vectors , 39, 40. vectors , 42, theP. falciparum infections [P. falciparum infections.Despite the microscopic prevalence of 14.7%, there were a large number of clinically positive cases that were missed because they were submicroscopic infections. The high levels of undetected infections may have a significant impact on existing malaria intervention strategies, potentially resulting in a plateauing of malaria cases in the future. The high number of missed cases is attributed to declining malaria prevalence, which has resulted in the area transitioning to a low malaria transmission zone . Additiofections . The redfections , host exfections , 11, andfections , 43. TheWith an odds ratio of 1:8 for detecting submicroscopic infection in males versus females, these observations were slightly higher than previously reported ratio of 1:4 in patients from low transmission zones within Belaga district of Malaysia . The higIn contrast to the previously observed link between LLIN use and microscopic infections, patients who used LLINs were more likely to have submicroscopic infections. When the study was narrowed down to establish the link between nocturnal outdoor activities, LLIN use, and submicroscopic infections, a similar observation was made. In general, patients who participated in nocturnal outdoor activities had a higher chance of having submicroscopic infections than those who stayed indoors. This was largely influenced by the high LLIN usage among those involved in nocturnal outdoor activities. These findings demonstrates that an increase in malaria vector interventions may be directly or indirectly related to an increase in submicroscopic infections in this study site. Perhaps the interventions are limiting human-vector contact, lowering both biting and sporozoite inoculation rates, implying that low biting rates are a cause of rising submicroscopic infections , 11. AddAnopheles have been successfully infected by submicroscopic infections [Despite continued IRS, LLIN use, and lower periodic malaria prevalence in the six study sublocations, submicroscopic infections persisted in this rural area of western Kenya. As a result, the accumulation of undetected and untreated infections may continue to stymie efforts to achieve long-term malaria elimination. Further, female fections , 6. Withfections , this stP. falciparum. This result suggests that combined diagnosis using microscopy in conjunction ultrasensitive Rapid Diagnostic Tests or PCR is appropriate in areas with low P. falciparum transmission.In the six study sublocations in Homa Bay County, this study found a high prevalence of submicroscopic infections, resulting in a large number of undetected and untreated patients who may serve as a reservoir for continuing transmission of Additional file 1: Table S1. Multivariate tests. Table S2. Test of between-subject effects.


Deployment of sound diagnostic tests remains a crucial component of malaria management, prevention and control in Africa. We undertook a comparative assessment of sensitivity, specificity and efficiency of three popular brands of rapid diagnostic tests (RDT) available in Nigerian market alongside with traditional microscopy.525 samples of patients that presented with acute uncomplicated malaria through clinical diagnosis were evaluated with the various tests. Total WBC count and haematocrit were also measured.Plasmodium falciparum with 280 (93.3%) cases. Co-infections of P. falciparum/vivax  and P. falciparum/malariae  were detected and confirmed with microscopy. Haematocrit values correlated inversely with parasite density .Of the 525 samples, 300 (57.1%) were found positive by Giemsa microscopy. SD Bioline had a positivity rate of 49.5% (260/525), while the positivity rate for Acon was significantly lower  and Paracheck . The sensitivity, specificity and efficiency of the three RDTs were: SD Bioline ; Paracheck  and Acon , respectively. Pre-teens aged 6-12 yrs had the highest mean malaria parasite densities with 6,631.26 at p< 0.01. The dominant malaria species was Microscopy still remains the reference standard for malaria diagnosis in limited resource settings in endemic areas. In furtherance to this, there is need for consistent monitoring of RDT product quality as part of the distribution process to end-users across Nigeria. Malaria remains one of mankind’s most dreaded diseases, with 212 million cases and more than 400 thousand deaths reported globally in 2015 [Nigeria, with a population of 134 million, records more than 100 million cases and a mortality of 300 thousand annually  and pregOne of the challenges in malaria management, particularly for children, is inaccurate diagnosis . ClinicaThe aim of this study was to undertake a comparative assessment of the efficiency of three popular brands of RDTs found in the Nigerian market alongside standard microscopy. The goal was to determine the efficiency and relative strength of each product in detecting malaria so as to enable end users to make informed choices. This in turn should aid or enhance better quality assurance and control of laboratory diagnostics for malaria and ultimately promoting better health care delivery.The study was carried out in Glory Diagnostica Laboratories Ojoo, Ibadan, the capital of Oyo State in southwestern Nigeria, dominated by the Yoruba ethnic group. The study was conducted between June and December 2012. During this period rainfall is relatively heavy in the area. Prevailing ecological and socio-cultural practices and the poor socioeconomic status of residents make malaria to be endemic throughout the year.The study adopted a non-randomised descriptive and cross-sectional study. In all, a total of 525 participants were recruited and diagnosed for acute uncomplicated malaria during medical consultation. Patient samples were obtained for RDT and Giemsa-stained microscopic slides from Mount Zion, Fajimi Memorial, Mosolape, Olufunmilayo and Olushola Hospitals, all situated in Ojoo area of Ibadan, Nigeria.Sample size was determined using the method of . BrieflyBefore sampling, temperature of each patient was measured with the aid of a mercury-type clinical thermometer. Demographic data, i.e. the age and sex of patients were recorded. Five (5) ml of venous blood from each patient was collected into a clean plastic EDTA container for haematocrit value determination and total white blood cell (WBC) count. Thin and thick blood films were Giemsa-stained for malaria microscopy. At the beginning of each test procedure, individual patient blood samples were thoroughly mixed and tested for malaria antigen reactivity separately with Acon, SD Bioline or Paracheck kits. Known positive and negative controls for malaria parasite were run along with patients’ test samples. Haematocrit or packed red cell volume (PCV) were performed by filling a plain capillary tube to ¾ levels with blood from an EDTA container by capillary flow. One end of the capillary tube was sealed with plasticine and placed in a haematocrit centrifuge and spun for 5 minutes at 5,000 revolutions per minute. The spun capillary tube was read with a haematocrit reader. Normal PCV values ranged from 40-55% and 36-45% for males and females, respectively.Plasmodium species identification, quantifying parasitaemia, and recognition of other parasite forms like schizonts and gametocytes.Thick film smears on three spots were made from fresh blood samples. The films were properly dried (without prestaining fixing) and then stained with 10% Giemsa solution and subsequently washed (after 10 min) using buffered distilled water (pH 7.2). A drop of immersion oil was applied on the dried stained slide and examined microscopically for malaria parasites using 100x oil immersion objective lens. Asexual parasite observation was regarded as a positive smear; smears were considered negative after examination of 100 high power fields did not reveal parasites. An experienced medical laboratory scientist stained the slides with Giemsa using standard methods [Two laboratory scientists independently examined the slides; a third reading of discordant results by another senior scientist was considered final. Consistent quality assurance activities were put in place to ensure that microscopy was conducted by competent and motivated medical staff supported by effective training and supervision and an adequate supply of reagents and equipment. Blood smears from patients with suspected malaria were routinely screened, blindly rechecked and validated by senior microscopists. Second and third slide external blind readers were used to ensure that discordant results were further read and final results produced. Compared to baseline assessment the first-round external quality assessment found a remarkable improvement in the quality of microscopic malaria diagnosis, from 64 to 87% agreement between slide readings.Of each blood sample 0.02 ml was added to 0.38 ml of Turk’s solution and gently mixed. After 5 min the mixture was charged into a Neubauer counting chamber and allowed to settle for 2 min before counting was done under x10 objective of the microscope.A portion of the thick film where white cells were evenly distributed and parasites well stained was selected for examination. 200 White blood cells (WBCs) were systematically counted. Asexual forms of the parasites in the field covered were estimated concurrently. The number of parasites per μl of blood was calculated using the WHO formula :200(WBCs)A slide was declared negative when there was no parasite detected/counted within 200 WBCs. Two laboratory scientists independently examined the slides; the third reading of discordant results by another scientist was taken as final.SD Bioline®: The test device was removed from the foil pouch and brought to room temperature. 20μl of whole blood was added to the sample well(s) of the test device. 3 drops of assay diluent was then added. The timer was set and a pink colour developing within 10 minutes indicated a positive result. The presence of two or three colour bands  or Pv (P. vivax) and positive control, respectively.Paracheck (Pf): Paracheck (Pf) kit components were brought to room temperature before testing. 5μl of anticoagulated blood was delivered into sample well A. Six (6) drops of the clearing buffer was delivered into well B. The mixtures are allowed to react. The timer was set and a pink colour within 15 minutes denoted a positive result.Acon: The test strip was removed from the foil pouch and used immediately. 10μl of whole blood was transferred into a specimen tube and 3 drops of buffer was added into the same specimen tube. The test strip was immersed into the contents of the test tube, taking caution not to exceed the maximum line on the test strip. The timer was set and a pink colour within 10 min indicated a positive result. Two distinct coloured lines appeared. One in the control (c) and the other in the test region. This indicated a positive result for Paracheck/Acon. A negative was indicated by no coloured line appearing in the test region but only in the control region (c).P. falciparum. The entire test kits were within the shelf life (Manufacturing date December 2012 and expiry date May 2015) and were stored in the laboratory store which was air-conditioned, albeit not on a 24 hour basis. Hence, the temperature was variable. Recommended storage temperature by the manufacturer is between 1°C and 40°C. The microscopy and RDT were done by different laboratory personnel. Each facility received training on how to appropriately collect, label and store specimens. Health workers at all participating facilities were trained to perform RDTs.The RDTs used in this evaluation were made available through the Ministry of Health and Social Welfare and included SD Bioline®, Paracheck (Pf) and Acon kits obtained through the existing national procurement and distribution system. Log books and unique ID stickers were provided to record RDT results and for specimen tracking. The test is only limited to the detection of antigens of Plasmodium species. Analysis of variance (ANOVA) was used to determine the influence of specific age groups on malaria parasite densities. This helped to determine the age group with the highest prevalence of malaria. The receiver operating characteristics (ROC) curve was used to evaluate which of the diagnostic methods yielded the best malaria diagnosis. This was plotted and interpreted using the area under the curve.Pearson’s product quotient correlation analysis was used to determine the relationship between haematocrit values and malaria parasite density in the sampled population. This determined if haematocrit levels influenced occurrence of malaria in the region. Standard error of means was generally used in a descriptive role to access distribution of data such as parasite densities by age groups and Oral informed consent was obtained from the selected patients. Ethical approval was obtained from Olabisi Onabanjo University Teaching Hospital, Ogun State, Nigeria and Kwara State University Research Committee and Ethical Review Board. Individuals were assured of voluntary participation, confidentiality of their test results and opportunity to withdraw at any time without prejudice, in line with the Helsinki Declaration .The observed difference in relative positivity between males and females  was not significant , indicatThere was a statistically significant difference across the age groups with regards to parasite density . There wPlasmodium falciparum was the most prevalent species  and negativity of 225 (42.9%) of total samples examined . This waEarly diagnosis and prompt treatment of malaria, especially in rural areas or at primary health care (PHC) level is vital to combating the disease in all endemic regions ,17. In aThis study compared the efficiency of three different but commonly used rapid diagnostic test (RDT) kits, namely Acon, Paracheck and SD Bioline along with Giemsa microscopy as the reference standard. When malaria positivity for 525 specimens were analysed using different RDTs alongside the Giemsa microscopy method, the latter was able to detect highest number of positive malaria cases. The best of the RDT kit (SD Bioline) had a malaria positivity of 260 (49.5%) of total samples diagnosed. Its positivity relative to the Giemsa method was 86.7%. Results obtained from this study confirmed that microscopy remains the reference standard and a better diagnostic tool for malaria diagnosis in the laboratory than the RDTs.The specificity (99.6%) observed with SD Bioline was almost similar to the observation made in the study of  in whoseSD Bioline is seen as the best RDT kit of the three kits analysed in this study, and is able to detect low level parasitaemia. This makes it more sensitive than the others. Besides, it has a sensitivity and specificity of 86.3% and 99.6%, respectively. Moreover, it was generally observed that the higher the parasite density, the heavier the colour intensity. In other words, the degree of parasitaemia may be estimated by the extent of colour produced on the test strip, implying that in field work, the intensity of strip colour during/after testing with this kit may be used to give a rough assessment of parasite density in the patient.In considering malaria infection according to patients’ age, the highest level of parasitaemia was observed in the age group (6-12) years. This could be as a result of the naïve nature of the immune status coupled with low blood volume as well as other comorbid factors such as malnutrition, within that age bracket, making children more susceptible to malaria infection. It is pertinent to note that  recordedP. falciparum was found to be the most predominant species among the infecting parasite species, constituting 93.3% of total malaria cases identified in this study. This is in consonance with the submission of [P. falciparum is the predominant species. P. falciparum infections are known to be highly pathogenic and since it is the most prevalent in this study, it may account for why malaria disease takes a great toll on the Nigerian population. Co-infection with other malaria parasite strains accounted for the remaining (6.7%). There was an observed 9.3% recorded for other malaria strains in past work by [p < 0.01) was in line with [Furthermore, ssion of  that in  work by . The obsine with  who had ine with . This coine with .With a temperature of >37.5°C taken as the benchmark for fever, two hundred and forty-two (242) subjects, representing 89.0% had fever symptoms out of 300 found positive for malaria parasite (p<0.05). This suggests that among people with pyrexia in the tropics, malaria parasite screening should be one of the key tests to be conducted while not excluding other probable causes. A previous study recorded 894 (55%) positive cases out of 1640 subjects having fever symptoms .Plasmodium species that are absent in blood when P. falciparum is in high concentration. In addition, the continued presence of pHRP-2 antigens in blood several weeks after treatment, even when parasites have been cleared from the blood and inability to fairly assess the degree of parasitaemia, among others, should be watched out for.It is pertinent to mention one important diagnostic advantage in microscopy which RDTs lack - the ability of the analyst to observe the morphological features of the parasite under the microscope. This makes it possible for the microscopist to identify different parasite forms and stages commonly seen under the microscope, a feature which has a lot of implications for the diagnosis of critical parasite forms like schizonts or gametocytes. The inability of RDTs to detect such parasite forms may scales down the gravity of detection of infection especially during severity, thereby contributing directly or indirectly to morbidity and mortality. Several pitfalls have been listed in using RDTs for the diagnosis of malaria . This inHowever, the establishment and maintenance of a reliable and efficient diagnostic facility for malaria at the primary health care level is dependent on many (or a combination of) factors, which can seriously degrade the utility and relevance of any malaria laboratory. These can be broadly summarised as follows: (a) Deficiencies in personnel adhering to adequate work time management, (b) substandard or inappropriate equipment: incorrect or poorly coordinated specifications, particularly of microscopes; irregular maintenance and replacement of worn-out parts; weak quality control of stains and reagents; inappropriate chain supply schedules which either cause delays in the supply of material or accumulate stocks which might exceed the shelf-life of the material, (c) organisational deficiencies: the maintenance of an efficient and relevant organisational structure calls for frequent review and analysis of the current aims and objectives of the malaria diagnostic facility as well as the necessary changes in staffing and methods to meet these requirements with available resources . From tiGiemsa staining technique has been reconfirmed in this study to be the best reference routine method for malaria diagnosis. Assessment of results from this study has shown SD Bioline as the best RDT kit among the three kits assessed, being the kit with highest sensitivity, specificity and efficiency. It may thus be used where facilities for microscopy does not exist.Plasmodium falciparum was the more prevalent malaria species among the subjects, higher parasite density was more common in co-infections. Demonstration of higher parasite density has shown that children within the age 6-12 years are at high risk of malaria infection. Results from this study indicate that the degree of malaria parasitaemia correlates inversely with the patient’s haematocrit level. Low haematocrit values found among malaria-infected subjects demonstrated that fever and anaemia are one of the major symptoms of malaria infection in Ibadan.Although Although SD Bioline has shown a proven efficiency in the diagnosis of malaria in this study, it can only be recommended as a preliminary adjunct to Giemsa staining technique in malaria diagnosis. Laboratorians in developing countries should continue to employ the Giemsa quantitative analysis for routine purposes so as to achieve effective diagnosis, disease control and efficacious treatment of malaria. It is imperative that malaria microscopy, being a reliable, affordable and accessible technique be assessed regularly against emerging technologies for malaria diagnosis.


Plasmodium falciparum, with a minor contribution from Plasmodium malariae and Plasmodium ovale. However, infections containing submicroscopic parasite densities can result in clinical disease. In this study, we used PCR to determine the prevalence of three human malaria parasite species harboured by suspected malaria patients attending healthcare facilities across the country.Clinical presentations of malaria in Ghana are primarily caused by infections containing microscopic densities of Plasmodium species-specific PCR was performed on DNA extracted from the dried blood spots. Demographic data and microscopy data for the subset of samples tested were available from the original study on these specimens.Archived dried blood spots on filter paper that had been prepared from whole blood collected from 5260 patients with suspected malaria attending healthcare facilities across the country in 2018 were used as experimental material. P. falciparum, P. malariae and P. ovale detected by PCR was 74.9, 1.4 and 0.9%, respectively. Of the suspected symptomatic P. falciparum malaria cases, 33.5% contained submicroscopic densities of parasites. For all regions, molecular diagnosis of P. falciparum, P. malariae and P. ovale was significantly higher than diagnosis using microscopy: up to 98.7% (75/76) of P. malariae and 97.8% (45/46) of P. ovale infections detected by PCR were missed by microscopy.The overall frequency of Plasmodium malariae and P. ovale contributed to clinical malaria infections, with children aged between 5 and 15 years harbouring a higher frequency of P. falciparum and P. ovale, whilst P. malariae was more predominant in individuals aged between 10 and 20 years. More sensitive point-of-care tools are needed to detect the presence of low-density (submicroscopic) Plasmodium infections, which may be responsible for symptomatic infections.The online version contains supplementary material available at 10.1186/s13071-022-05153-6. Plasmodium species that infect humans include Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Plasmodium knowlesi, and Plasmodium falciparum; of these  P. falciparum is responsible for most of the malaria cases in Ghana [P. malariae and P. ovale have been reported, but these species are predominantly present as mixed infections with P. falciparum [in Ghana , 2. In Glciparum , 4 and aPlasmodium parasites can result in various presentations of disease, with the uncomplicated and asymptomatic presentations being the most predominant manifestations of parasite infection. Uncomplicated malaria often presents with fever, chills, profuse sweating, headache, vomiting and diarrhoea [Infections with all human iarrhoea , whilst iarrhoea .Plasmodium parasite detection, especially for clinical cases of malaria as these usually contain high densities of parasites [Microscopy is the gold standard for arasites , 7. Micr P. falciparum is predominant and widespread, P. malariae, and P. ovale infections are usually underdiagnosed, with almost all clinical malaria cases attributed to P. falciparum infection [falciparum malaria by microscopy in P. falciparum malaria endemic countries include: (i) the very low parasitaemia of P. malariae and P. ovale; and (ii) the lack of highly skilled microscopists who can distinguish the different Plasmodium species [Accurate estimation of the density of the infecting parasites and accurate identification of the infecting parasite species are two properties of infection that are essential to the proper management of malaria, including providing information on the appropriate drug treatment , 9. In mnfection , 11. Som species –14. Mole species . The sen species .Ghana is a malaria mesoendemic country which still has some facilities to confirm the suspicion of malaria in patients suspected of having this disease  with microscopy. The aim of this study was to use PCR to determine the frequency and distribution of the three common malaria parasite species in patients with suspected malaria who were seeking treatment in 100 different healthcare facilities across Ghana in 2018.Convenience sampling was used to select between 292 and 600 archived dried blood spots on filter paper from a pool of about 2000 such samples that had been collected in the original study from patients with suspected malaria attending clinics in the 10 regions of Ghana. The adoption of convenience sampling limited the data for analysis to the selected samples, with the inherent possibility of under-/over-representation of the original study population. The selection criterion for the samples was any dried blood spot on filter paper for which there was sufficient material to punch out two 3-mm-radius spots for analysis. The samples used originated from samples taken from 5260 individuals who had been recruited as part of a larger study that enrolled 19,787 patients with suspected symptomatic malaria. Patients for this larger study were recruited from 100 healthcare facilities (10 from each of the then 10 regions of the country). Study participants ranged in age from 1 to 94 years. The demographics of the larger study population have been reported previously , 17.This cross-sectional study was conducted in the 10 regions of Ghana as of 2018: Greater Accra Region (GAR), Central Region (CR), Ashanti Region (AR), Western Region (WR), Eastern Region (ER), Brong-Ahafo Region (BAR), Volta Region (VR), Northern Region (NR), Upper East Region (UER) and Upper West Region (UWR). Ten healthare facilities were randomly selected in each of the 10 regions of Ghana , 17.The samples used in this study were collected between May and August 2018 as described earlier , 17. DemEach thick and thin blood smear obtained from the original study was read by two independent WHO-certified malaria microscopists; any disagreement on smear readings was resolved by re-examination and assessment by a third microscopist. The assessment by the third microscopists was considered to be the final decision , 17.g and the reddish supernatant decanted. The DBS punches were then washed in ice-cold PBS at 4 °C for 30 min and the supernatant again discarded. Finally, 50 μl of freshly prepared 20% Chelex-100 in distilled water and 100 μl of distilled water were added to each tube. The tubes were then heated at 95 °C for 10 min, with repeated vortexing at 2-min intervals. The tubes containing the extracted DNA were finally centrifuged at 21,000 g for 8 min, and the supernatant containing the DNA was transferred into a new sterile tube and stored at – 20 °C until use.Genomic DNA was extracted from the dry blood spots (DBS) using the Chelex extraction method , 19 with18S ribosomal RNA gene, a conserved region in P. falciparum, P. malariae and P. ovale, was amplified using nested PCR from genomic DNA using a protocol described previously [P. falciparum, P. malariae and P. ovale, respectively. The primary and secondary reactions were run at an initial denaturation temperature of 94 °C for 2 min; this was followed by a second denaturation at 94 °C for 30 s, primer annealing at 55 °C  and 58 °C (for the second reaction), both for 30 s, and extension at 68 °C for 1 min 30 s for nest 1, 30 s for nest 2  and 1 min for P. ovale (nest 2); and a final extension at 68 °C for 5 min. Positive control samples used for this study included known P. falciparum and P. ovale or P. malariae DNA samples that had been obtained as part of the Distribution 4881 of the WHO Malaria NAAT EQA programme; ultra-pure water was used as a negative control. The nest 2 PCR products were resolved in a 2% agarose gel pre-stained with ethidium bromide. The gels were subsequently visualised using the UV settings on the Vilber gel documentation system .The eviously , with miP < 0.05.All data acquired during this study were entered into Microsoft Excel , and the statistical analyses were performed with GraphPad Prism software, version 8.4.3 . The data were grouped according to regions, diagnostic tests, gender and age categories. The frequency of malaria species was determined using simple counts and proportions. The associations between frequency of species and explanatory variables were determined using chi-square statistics. Statistical significance was set at Plasmodium species in the 10 regions of the country was assessed in all dried blood spot punches from 5260 study subjects with suspected malaria. The mean age ± standard error of the mean of these subjects in the study year (2018) ranged from 14.2 ± 0.7  in the Brong Ahafo region to 27.2 ± 0.9 (95% CI: 25.5–29.0) in the Upper West region of Ghana. Female participants slightly outnumbered male ones across all regions; the Central region had the highest proportion of female participants (62.9%) , with the highest frequency identified in the Ashanti region 412/476 (86.6%) and the lowest detected in the Eastern region 41/292 (14.0%)  were missing microscopy data.The frequency of P. falciparum in 3942 of the 5260 (74.9%) individuals sampled, with the highest frequency identified in the Ashanti region  and lowest detected in the Upper West region    detected by PCR was 122/5260 (2.3%), with P. malariae accounting for 76 of the 122 (62.3%) samples PCR-positive for non-falciparum malaria. The frequency of P. malariae was highest in the Ashanti region  and lowest in the Western region , with no P. malariae infection detected in the Greater Accra region. The frequency of P. ovale ranged from 2/655 (0.3%) in the Upper West region to 13/563 (2.3%) in the Brong Ahafo region , P. falciparum–P. ovale (PfPo), P. malariae–P. ovale (PmPo) and P. falciparum–P. malariae–P. ovale (PfPmPo) mixed infections were 64/109 (58.7%), 42/109 (38.5%), 1/109 (0.9%) and 2/109 (1.8%) respectively.The majority  of the non-PmPo mixed infection was observed in the Western region of Ghana. The highest frequency of PfPo infection was observed in the Brong Ahafo region  and the lowest in the Western region . The frequency of PfPm mixed infection was high in the Volta , Ashanti , Brong Ahafo , Central (10/64 (15.6%) and Northern  regions. The mean frequency of PfPm mixed infections and PmPo mixed infections across the 10 regions of Ghana were 6.4 ± 1.6 and 4.20 ± 1.2, respectively  and Brong Ahafo  regions, where these parasites were identified , P. malariae  and  P. ovale  were significantly higher than the microscopy estimates across all the regions. A total of 33.8% (1776/5260) of the symptomatic P. falciparum malaria infections were submicroscopic. The Ashanti region contributed the least to the overall number of individuals with submicroscopic infections .The PCR estimates of falciparum infections were caused by P. malariae  and 10/11 (90.9%), respectively, of the total non-falciparum infections identified in those regions  of submicroscopic non-20, 63.3%P. falciparum detected by PCR (Pf-PCR) among the age categories 0–4, 5–9, 10–15, 16–20 and > 20 years were 719/867 (82.9%), 987/1145 (86.2%), 591/700 (84.4%), 430/547 (78.6%) and 1135/1862 (61%), respectively. The Greater Accra and Eastern regions had the lowest Pf-PCR positivity rate among individuals aged 0–4 years . The highest Pf-PCR positivity rate in individuals aged 0–4 years was identified in the Brong Ahafo region . There was a significantly lower frequency of malaria in individuals aged 10–15 years and 16–20 years compared to individuals aged 0–4 years . However, individuals aged between 5 and 9 years had a higher frequency of malaria compared to individuals aged 0–4 years , with individuals from the Brong Ahafo region contributing the most in absolute numbers  within this age group. A total of 9.0% (6/67) of individuals in the age group 10–15 years from the Brong Ahafo region were infected with P. malariae. The frequency of P. malariae in individuals belonging to the age groups 16–20 years and > 20 years were highest in the Ashanti and Brong Ahafo regions  within this age group. In the Ashanti region, of the 88 individuals infected with P. ovale, three (3.4%) were aged 0–4 years.A total of nine individuals aged 0–4 years were infected with P. ovale, with individuals from the Brong Ahafo Region contributing the most  within this age group. Of the 86 individuals from the Ashanti Region aged between 10 and 15 years, four (4.7%) were infected with P. ovale  in the frequency of P. falciparum infections in male subjects  compared to female subjects . Male subjects had a relatively higher frequency of P. falciparum infections compared to their female counterparts across all the regions, with the exception of the Eastern region where PCR identified 80.7% (n = 134) of the overall female participants and 79.5% (n = 97) of the overall total male participants to be infected with P. falciparum.Overall, there was a significant difference  and 30/2100 (1.4%) of the female and male participants, respectively. The frequency of P. malariae was significantly higher  among female participants than among male participants in all regions, except for the Eastern and Volta regions  and 17/2100 (0.8%) of female and male participants, respectively. Infections containing P. ovale were detected in only four regions. The frequency of P. ovale in the Ashanti region was similar between male  and female (6/286 (2.1%) participants. In the Brong Ahafo region, P. ovale was more prevalent in female  than males  participants. In the Eastern region, P. ovale was identified in 2/166 (1%) female subjects; in the Upper West region, 4/171 (2.3%) male subjects were identified with P. ovale  in the subset of suspected malaria patients used in this study to be higher than that reported in the original larger study (16%) [Plasmodium species and on highlighting the higher sensitivity of PCR at detecting malaria parasite infections, especially the non-falciparum species, amongst patients with suspected malaria attending healthcare facilities.The use of convenience sampling to obtain the samples for this study resulted in a bias, which caused the overall frequency of dy (16%) . HoweverPlasmodium malariae and P. ovale as mono-infections resulted in symptomatic malaria cases. Plasmodium falciparum and P. ovale were more commonly detected in children aged between 5 and 15 years, whilst P. malariae was more prevalent in individuals aged between 10 and 20 years. Male subjects had a higher frequency of P. falciparum infection than female subjects, whilst P. malariae was more prevalent in female subjects. More sensitive point-of-care tools are needed to detect the presence of low-density (submicroscopic) Plasmodium infections, which can result in symptomatic infections (Additional file Additional file 1: Figure S1. Representative gel images of products from the different Plasmodium speciation reactions.


Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% . However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria.Individuals with asymptomatic infection due to  Plasmodium vivax, and provide evidence of their potential as parasitic reservoirs, even when molecular methods fail to detect the infection. Our findings reinforce the need for better diagnostic tests and open a new window of complexity to be considered in control programs.Malaria still poses as one of the most important parasitic diseases in the world. The advance of molecular diagnosis brought to light the existence of asymptomatic infections, which may represent most of the infections in some areas. Importantly, the role of asymptomatic carriers in the natural history of malaria is not completely understood. Herein we describe the general characteristics of asymptomatic individuals infected with  Plasmodium vivax is spread worldwide and although it is frequently considered to be low pathogenic, it is an important cause of morbidity and mortality in endemic areas in central and south America, and in regions of Asia and Oceania [P. vivax is challenging due to the unique biology of this species, including the early emergence of infectious gametocytes within 3 days after the detection of asexual forms, the development of hypnozoites in the liver, a potential source of later relapses, and the preferential infection of reticulocytes, which represents 0.5–2.5% of the red blood cells, which limits the parasite density [ Oceania ,2. The a Oceania . Control density ,5.P. vivax infection are mostly undetectable, neglected, and remain untreated. For this reason, asymptomatic malaria require active surveillance and poses as one of the most challenging obstacles for the control of Plasmodium infections worldwide [Patients with symptomatic malaria actively seek diagnosis and treatment. In contrast, asymptomatic (ASY) cases of orldwide ,7. Evideorldwide . Althougorldwide –11.P. vivax transmission in the Amazon basin in Brazil [P. vivax in the Brazilian Amazon and explore their characteristics, which will be relevant for future studies in this population.In the Brazilian Amazon, the incidence of malaria dropped from near 600,000 cases/year in the late 1990s to near 144,000 in 2014 , mostly n Brazil . Hence, This study was performed under protocols reviewed and approved by the Ethical Committees on Human Experimentation from Instituto René Rachou, Fiocruz, and National Ethical Council (CAAE: 59902816.7.0000.5091). All participants were informed about the objectives and procedures of the study, with voluntary participation through written informed consent.P. vivax and 287 cases of P. falciparum were notified in this town [Candeias do Jamari (8°47’41.6"S 63°42’10.9"W) is a municipality localized in the south-western Amazon region in Brazil, where malaria is endemic and the annual parasite index usually exceeds 50 cases per 1,000 inhabitants. Between 2018 and 2019, 2,394 cases of his town .P. vivax occurs during the entire year, although peaks of transmission occur during the dry season. Areas 1 and 2 had the most reported symptomatic cases in the past years, while area 3 has reported fewer cases of symptomatic malaria [From December 2018 to October 2019, four cross-sectional surveys were done in 3 areas from this municipality to identify asymptomatic infections. Candeias do Jamari has two main seasons: a wet season between October and April and a dry season between May to September. Transmission of  malaria . ScreeniP. vivax in at least one replicate were included in the Baseline study. No template controls (NTC) were included in all reactions to ensure the reliability of the results.After informed consent, 200μL of blood were collected by finger puncture and Giemsa-stained thick blood smears were performed. Blood was centrifuged, and DNA was extracted from compacted cells using a commercial kit . Extracted DNA was resuspended in 200μL of Elution Buffer to reproduce the original concentration of parasites/μL. Samples were tested at least in triplicate by qPCR and individuals with positive results for P. vivax at Screening, 47 from 108 ASY donated a new blood sample, were interviewed, and clinically examined by a physician. This date of clinical examination was considered as the Baseline in our analysis. Thirty-eight symptomatic (SY) individuals with positive thick blood smears for P. vivax performed at the Centro de Pesquisas em Medicina Tropical (CEPEM) were also enrolled in this study. Twenty healthy individuals from Porto Velho with negative parasitemia by thick blood smear and PCR, and reporting no malaria symptoms for more than two years were enrolled as controls (CTL).Four to seven days after the first positive qPCR for Blood was collected by venipuncture, and samples were used for routine blood counting and clinical biochemistry, DNA extraction, and thick smears for parasite detection. Interviews were done using a web-based tool for research data curatorship (REDCap). Heparinized blood samples from 32 ASY  and 9 SY were used in the membrane feeding assay. In this case, volunteers were included according to the availability of mosquitoes to perform the experiment. Treatment was offered to all ASY and those who chose to be treated received 3 days of chloroquine  and primaquine 7 days .Forty-nine individuals were followed weekly for six weeks to assess the development of symptoms and for parasite detection . Among those, 36 subjects were from the Baseline, and additional 13 subjects were from Screening. The study design is depicted in et al. [A qPCR addressed to a sub-telomeric non-ribosomal multicopy target (Pvr47) was done as described by Amaral et al. , with moP. falciparum was performed only in samples that were PCR positive for P. vivax to avoid the inclusion of patients co-infected with both species [P. vivax, but infected mosquitoes during membrane feeding assay (MFA), we also employed a nested PCR to confirm the result [2, 1x Taq buffer (Promega), and 0.75 units of Taq polymerase (Promega), in a 20 μL final volume. Amplification was done as follows: 92°C/2m, followed by 35 cycles of 92°C/30s and 60°C/90s and a final extension of 60°C/5m. The product was diluted 1:50 in TE buffer (Qiagen) and 2 μL were applied in 8 μL mix containing 1 μM of primer P1 and a P. vivax primer , 312.5 μM of each dNTP, 1x Taq buffer (Promega), 1.5 mM MgCl2, and 0.75 units of Taq polymerase (Promega). Amplification was as follows: 92°C/2m, followed by 18 cycles of 92°C/30s and 60°C/60s and a final extension of 60°C/5m. Products were electrophoresed and seen as ~110 bp bands in a 2% Agarose gel in a UV transilluminator.The PCR for  species . When ble result . BrieflyP. vivax blood-stage antigens PvMSP-119 [PvAMA-166 [PvDBP-IIBrz-2[Plasma from 47 ASY, 38 SY, and 20 healthy donors that reported never had malaria before was used to assess the levels of IgG against the vMSP-119 , PvAMA-1vAMA-166  and PvDBNyssorhynchus darlingi mosquitoes from an established colony (F8-F18 generations) were reared under standard laboratory conditions as described [N. darlingi is the main vector of P. vivax in the area we studied. Mosquitoes were maintained on cotton soaked with 15% honey solution ad libitum one day before and after the infection blood meal. Heparinized blood was kept at 37°C, for approximately 10 minutes, until the membrane feeding assay. Blood sample (2mL) was loaded into a glass feeder closed with parafilm membrane and maintained at 37°C by a connected heated water source. One mesh-covered 650 mL plastic cup containing 80 females 3- to 5- days old N. darlingi (F8 to F18 generations) that had been starved for 24 h, were placed under the feeder for 30 min to allow feeding. Fully engorged females were transferred into 20cm3 cages and maintained in the same conditions of insectarium for 2 weeks.escribed . N. darl2O) for 5 min. The percentage of mosquitoes infected and the number of oocysts/mosquito were determined by examination at 40x objective on a light microscope.One-third to one-half of each group of mosquitoes were dissected on day 7 and the remaining mosquitoes on day 14 post-feeding. Mosquitoes were freeze-killed and their midguts were dissected out in phosphate-buffered saline and stained with 0.2% w/v mercurochrome (in ddHhttps://arcg.is/1H5GjP). Variables were tested for Gaussian distribution using the Shapiro-Wilk test and variables were tested according to their distribution. Mann-Whitney U test and t-student test were used for comparisons between two groups. ANOVA or Kruskal-Wallis tests were used for multiple comparisons. The Spearman’s rank correlation coefficient was used to test correlations. These analyses were performed using GraphPad Prism 8 . Longitudinal spot-matrix were done using R and ggplot2 package. The statistical significance threshold was P < 0.05, with 95% confidence intervals for all hypotheses tested.Differences between proportions were calculated by Fisher’s exact test. Odds ratios and confidence intervals (95%) were calculated by the Baptista-Pike method. Maps were edited using the software Q-GIS 3.12 and employing the SIRGAS 2000 UTM 22s coordinate system. Base-layer was from the ArcGIS  were PCR positive , while in October the Screening was conducted in an area with fewer notified cases of SY malaria (area 3). We visited all houses of area 1 in both surveys, and only 64 individuals out of 669 were sampled in more than one survey. Although all houses were visited in both surveys we could not interview or sample all the residents due to the absence of people at the time of visit or sampling refusal. There was no significant difference between the percentages of ASY from areas 1 (December) and area 2 (January), but a marginally significant difference was observed between the proportions of areas 1 and 2 when September and January were compared . In contrast, area 3 had a lower prevalence of ASY compared to the other areas , followiconf) was lower than the ASY with non-conflicting results (ASYnon-conf) whose all replicates had detected amplification  reported a previous episode of SY malaria, with 77% (34/44) and 61% (27/44) reporting respectively more than 2 and 5 or more symptomatic episodes in their lives. The time since the last SY malaria was higher than 12 months for 75% (33/44) of participants. The average age of ASY was 40 years old, and the male/female ratio was 0.8 (21/26). The average ages for SY and CTL were 39.2 and 28.9 years old, and the male/female ratios were 2.3 and 1.1, respectively. In addition, 49% (23/47) of the ASY had become PCR negative in about one week. No ASY had fever during clinical examination, while 41% (14/34) of the SY had an axillary temperature above 37.5°C. From the 47 ASY included, 34 (72%) were re-assessed 30 to 45 days after the Baseline enrollment, and none of them reported seeking or receiving malaria treatment in the previous month.No relevant routine hematological abnormality was observed in ASY. Most SY had marked lymphopenia and low counts of platelets . BiochemPvMSP-119, PvAMA166, and PvDBPIIBrz-2 were significantly higher in ASY than in CTL. Despite the levels observed in SY showed to be higher than those observed in ASY, 78% of the latter produced IgG anti-PvMSP-119 above the cutoff. Also, 63.8% and 53.2% of ASY displayed IgG levels against PvAMA166 and PvDBPII-Brz-2 above the cutoff  became PCR negative as soon as one week after the sampling and 15 were negative in all samplings throughout the study. ASY that become negative in the following week, have a higher probability of bearing parasite density below the median (14.2 copies/μL) . Also, 6 subjects had at least one positive PCR after the 3inal arm , suggestN. darlingi. From 2,823 engorged mosquitoes, 2,177 were dissected with a mean of 77.2% surviving until midgut dissection. Blood from all 9 SY were able to infect mosquitoes, with a rate of infection of 43.4% (199/458). The numbers of oocysts varied from 1–108 per midgut. Blood from 50% (16/32) of ASY was able to infect mosquitoes, with significantly lower infection rates compared to SY , blood samples from 8 ASY infected mosquitoes. The average number of infected mosquitoes was similar between both groups , most of the riparian forest is submerged due to the flooding of Jamari river. In this period, the number of SY cases is lower when compared to the dry season (May to September) ,22. AfteP. falciparum [P. vivax [The fact that ASY individuals have lower parasite densities compared to SY is well known . The conlciparum  and P. vP. vivax ,27,30.Defining ASY infection is not trivial . The fir19, as previously described [PvMSP-119 slightly decline in SY 2 months after treatment [PvMSP-119 is also short-lived in ASY [Almost 80% of the ASY had detectable levels of IgG anti-MSP-1escribed . It has reatment . Likewisd in ASY ,37. It iP. vivax monthly for longer periods [P. vivax (hypnozoites), may have an impact on the maintenance of low parasitemia in ASY, since a high proportion of recurrent infections evolve to ASY infections [P. vivax in monkeys, but evidence in humans is still absent [P. vivax, sustaining the low parasitemia and boosting the antibody levels.As reported by others ,38, our  periods . In both periods ,39. The fections ,40,41. Al absent . Thus, uN. darlingi to fed on blood of each subject. Assuming an average of 2–3μL of blood ingested by mosquito [N. darlingi in infection rates varying from 2.2–10.8%, as previously reported for N. darlingi [+/-), were able to infect mosquitoes with the same efficiency as the currently PCR positive (PCR+/+) ASY. Kiattibutr et al [P. vivax in some samples is likely due to the very low parasite density or the sensitivity of the molecular assays [In this study, we used 80–100 female mosquito  and withmosquito . Althougdarlingi  and for darlingi ,45. Impotr et al  found a r assays , but enoP. falciparum malaria, the contribution of ASY for the transmission is higher in low transmission settings, and increases with the relative proportions of subpatent infections [P. falciparum and P. vivax, to the infectious reservoir in low-endemic settings in Ethiopia. The ASY infection by P. vivax had a greater impact on the transmission compared to SY, being responsible for the majority of the infectious reservoir [P. vivax infection in our settings remains elusive. Also, it is important to notice that only adults were included in this study, and the contribution of asymptomatic malaria in children (below 18 years old) to transmission is unknown in this setting.It has been shown that in fections . Anothereservoir . Such stVery low numbers of parasites circulating may strongly impair the probability of detection by PCR, while in a real transmission scenario, high densities of mosquitoes might increase the chance of infection and maintain the endemicity. Thus, these data suggest that mass treatment might be a valuable tool for disease control, although field studies are still needed to test its efficacy compared to other control measures currently employed .P. vivax. Finally, using an artificial feeding assay, we show that blood from ASY infects colony-raised N. darlingi. Importantly, even with undetectable parasitemia by qPCR, ASY contribute with low rates of transmission to anophelines, suggesting their potential role in sustaining the P. vivax cycle in hypoendemic areas.In conclusion, we confirmed a significant proportion of asymptomatic infections in a low transmission area from the Brazilian Amazon. The distribution of asymptomatic cases in different areas is heterogeneous and is associated with a larger number of symptomatic cases in areas near the riparian forest. Low oscillating parasitemia can give false-negative results in epidemiological inquiries and the persisting silent infection fosters the conditions to turn ASY as potential reservoirs for


Malaria affects 228 million people worldwide each year, causing severe disease and worsening the conditions of already vulnerable populations. In this review, we explore how malaria has been detected in the past and how it can be detected in the future. Our primary focus is on finding new directions for low-cost diagnostic methods that unspecialized personnel can apply in situ. Through this review, we show that microfluidic devices can help pre-concentrate samples of blood infected with malaria to facilitate the diagnosis. Importantly, these devices can be made cheaply and be readily deployed in remote locations. In 2018, there were around 228 million cases of malaria worldwide, of which 405,000 were fatal, with most of them in African countries 93%) [% [3], whPlasmodium. Four different species of Plasmodium cause human infections: P. falciparum, P. malariae, P. ovale and P. vivax. Worldwide, the most prevalent species is P. falciparum (around 96%), which is the most lethal species , followed by P. falciparum (19.8%); the provinces with the most cases are Loreto, Amazonas, and Junin (95.8% of cases in the country) [Anopheles spp., and the application and design of different screening methods [Epidemiologically, Peru follows the trend of the American Region, with country) . The laccountry) ,5. To co methods . Since 2The most used diagnostic method in Peru is the Giemsa stain. Although its precision and low cost make it a powerful method, its reliance on infrastructure and specialists makes diagnostics with this technique challenging in poor and remote areas . AnotherP. falciparum from P. vivax, the two most common species of malaria, are frequent in routine microscopic diagnostics, and are therefore under-reported [Fast diagnosis and prompt treatment are necessary to prevent mild cases of Malaria from turning more serious, which could result in death. Presently, most diagnoses are performed with either optical means, which requires a microscope, or so-called rapid diagnostic tests (RDTs). The most commonly used optical method is the blood smear, which determines the presence of malaria parasites in the blood cells . Howeverreported .The same can be said for RDTs. RDTs rely on antigen–antibody interaction, resulting in a visible band on a paper strip, in the same manner as pregnancy tests. However, if there is a low concentration of malaria parasites in the blood, no band will show, leading to a negative result. The other drawback is that RDT does not screen for multiple strains of the parasite. Another promising POC method to detect malaria is loop-mediated isothermal amplification (LAMP). This method is able to detect quantities of the parasite that are below the detection threshold for other methods. LAMP has been shown to achieve specificities of up to 100% and sensitivities up to 98% .Other methods, such as DNA probes and PCR , fluoresTherefore, better, i.e., more sensitive, differential, and faster, POC diagnostics methods are required, which can be used in remote locations when necessary. Microfluidics can play an important role in such devices. This review will give an overview of existing microfluidic-based devices that can be used for either the detection, separation, or diagnosis. At the end of the review, we will also suggest other devices envisioned to help combat malaria on all levels.One method that is suitable for use in remote locations because of its low cost and ease of operation is paper-based microfluidics, otherwise known as lateral flow devices . BecausePlasmodium falciparum histidine rich protein PfHRP2. They showed that this technique is very promising as it is easy to use and can store the reagents on the device in a dehydrated form.Implementation of improved signal amplification techniques has increased the reliability of the readout . Some ofAnother method to control the timing of reactions in a paper microfluidic device is by using microfluidic actuators (sponges), which expand at a known rate . Toley ePlasmodium falciparum L-lactate dehydrogenase (PfLDH). The sample was then loaded onto the chip, and a magnetic lens was placed underneath. By applying a current to the lens, the researchers formed virtual channels that connected the sample input to an absorbent wick. The magnetically tagged analytes were then trapped in the magnetic field while the supernatant was absorbed, leaving a large concentration of tagged PfLDH [Other paper-microfluidic devices can process pretreated blood or preconcentrate samples using magnetic markers. Using a simple 1D paper microfluidic device, researchers performed DNA extraction and loop-mediated isothermal amplification (LAMP), and detected DNA amplicons using lateral flow and antibody-based detection . This deed PfLDH . Paper med PfLDH .Although these applications are low-cost and require very little training to operate, the need for pre-processing increases the complexity of their application in situ. Because of this, other groups have researched methods that do not require tagging analytes with markers, such as magnetic beads or fluorescence tags.The rigidity of RBCs plays a significant role in the pathogenesis of malaria and can be used as a biomarker for malaria infection . MicroflThis change in cell mechanics has also been used to separate healthy RBCs from iRBCS . Using tMechanobiological methods of cell separation are extremely promising due to their ability to their high level of efficiency. Unfortunately, the most effective method requires a complex setup and a low flow rate which dramatically limits the throughput. These limitations make it a poor candidate for use in remote locations. However, other methods exist that can also leverage the difference in rigidity and have much larger throughputs.In recent years, microfluidics has been gaining momentum as a viable method to pre-process biological samples using inertial forces. This method can be used to preconcentrate iRBCs, resulting in better diagnostics of malaria. Although only a few studies have been performed with malaria-infected RBCs, many have researched similar effects in separating CTC cells from whole blood samples. This application follows the same principle as the separation of iRBCs.Re < 100. The inertial forces cause particles to migrate from one side of the channel to the other. The distance at which they focus depends on the size of the particle or their rigidity. For this to occur, the system must fulfill two criteria: (i) particle dimensions must be comparable to the characteristic length of the channel; and (ii) there needs to be a change in fluid velocity as you move from one wall of the channel to the other. These criteria create an asymmetry in the forces acting on the particle, causing it to migrate horizontally in the channel.A large amount of research has gone into understanding how the forces in microchannels change with different channel cross sectional areas and shapes ,44,45,46Studies have shown that at least two forces act on a particle in a straight channel. The shear gradient lift force orce Fwl ,48,49. T and Fwl .Secondary flows can be created by changing the channel’s curvature or by creating asymmetry in the channel. By adding curvature to the channel, a pressure gradient is created between the convex and concave walls. This gradient creates two counter rotating streams called dean vortices. Using these vortices and the interactions with the inertial lift force makes it possible to change the equilibrium position for particles in the channel. These positions depend on the physical properties of the particles and the forces acting on them.Despite the successes of these applications in CTC diagnosis, the application of inertial microfluidics to other diseases is just beginning to be explored. Given that this method allows for the separation of cells using various biomarkers, such as size, shape, and rigidity, it has been proven to be a worthwhile field of study. So far, inertial microfluidics have only been used to separate unbound aptamers from those bound to infected red blood cells . A diagrComputer simulations can give a deeper understanding of the effect of flow on particles and, thus, can help with the design of the microfluidic channel. The parameters that affect the particles in a microfluidic system include channel dimensions, flow rate, and the channel’s geometry. There is a wide range of methods for simulating microfluidics using a variety of software. One of the more popular and used software is COMSOL Multiphysics version 5.5  with the microfluidics add-on module ,53,54,55Martel et al. used COMSOL to calculate the equilibrium position of particles in straight and curved channels. It has been experimentally shown that particles suspended in a flowing liquid migrate across streamlines and reach an equilibrium position where they remain . A studySingle-cell analysis is challenging; it requires tools and systems to manipulate and characterize cells by performing tasks, such as focusing, sorting, ordering, compressing, etc. . As we hElectrical impedance represents the opposition of current flow through a given material or component and is defined as the ratio between an applied voltage and the resulting current. In the case of a MIFC, the component is a cell in a suspension medium. When an alternating voltage is applied at different frequencies, a complex impedance can be measured and calculated as follows:Already in 1956, Schwan, a pioneer in the field of bioimpedance analysis ,63, usedSince Schwan, many developments and applications have been made based on bio-impedance analysis. Ayliffe et al. made the first microfluidic device with integrated microelectrodes for single-cell impedance measurements ,69,70,71Plasmodium falciparum from a healthy RBC. The device had a measurement micro channel (30 × 200 × 5 μm) and two Ti/Au co-planar electrodes  connected to a precision LCR meter and computer. They also introduced a novel parameter to discriminate different disease states. Plasmodium vivax, i.e., the dominant species in countries such as Peru.The malaria parasite’s infection of the RBC changes the physical–chemical structure of the host cell throughout a 48-h multi-stage life cycle. These changes also affect their electrical properties. It has been observed that the impedance of a healthy red blood cell is different from a plasmodium falciparum-infected red blood cell (pf-iRBC). Ribaut et al.  were theIn this review, we have presented several microfluidic methods that have been shown to be suitable means of either pre-processing blood or detecting different pathogens present in a sample. A key advantage is that these methods are label-free; this eliminates the need for expensive reagents that may require specialized equipment and training. We presented four microfluidic methods: paper microfluidics, mechanobiological microfluidics, inertial separation, and impedance analysis. These methods can be sub-divided into methods for pre-processing  and diagnostics (paper and impedance). Mechanobiological methods are highly efficient at separating particles of different sizes and rigidities. The main disadvantage is that high enrichment rates can only be achieved at low flow rates and require an extra oscillatory flow. These flows need to be tuned correctly to achieve maximum efficiency. Although inertial separation and simpler forms of mechanobiological separation achieve much lower enrichment levels, they can be run at much higher flow rates and can speed up diagnostics. Some applications of inertial separation have been able to utilize only one fluid flow and thus simplify the operation of the final device. Using inertial separation as a means of pre-processing will increase the efficacy of the following diagnostic methods. This increase in efficacy allows POC technicians to use a wider array of tools, such as impedance analysis, which were previously unfeasible due to their low throughput or inability to identify low percentages of parasites without costly pre-processing. Recent advances in MIFC analysis have shown that it is possible to identify different stages of the parasite. These positive results, along with some of the difficulties associated with other diagnostics methods, such as PCR and RDT, make the combination of a low-cost and high-throughput pre-processing stage and a reliable and easy-to-use diagnostics stage a considerable step forward in the treatment of malaria.Although these technologies have many positive aspects, some limitations need to be addressed before using POCs in remote locations. The first limitation is the fabrication method that is typically used in making microfluidic devices. The literature shows that the majority of researchers have used photolithography as a means of fabricating molds for microfluidic chips. More recently, advances in 3D printing have also made it possible to print molds. Even with these advances in fabrication, these methods are still prohibitively expensive for lower income countries. Given that these countries are most likely to suffer from malaria, it is vital to research new and cheaper fabrication methods that can be deployed in the countries that need them. Another limitation for all microfluidic methods, except paper microfluidics, is the need for a pump to push the liquid through the system. These pumps are often large and require maintenance to perform consistently. The best pumps for microfluidics, i.e., pressure pumps, are expensive and difficult to obtain in remote areas.Even with these limitations, microfluidic separation and diagnostic methods provide an excellent way of reaching underserved communities. By utilizing microfluidic separation, it is possible to selectively remove cells of different sizes, shapes, and rigidities depending on the velocity and microfluidic effect that is being leveraged. A health care worker could then screen preconcentrated samples without any special training. A more reliable and straightforward diagnostic method would significantly reduce the mortality rates associated with infectious diseases such as malaria.


Plasmodium vivax presents control challenges due to several reasons, among them the increasing possibility of failure of P. vivax treatment due to chloroquine-resistance (CQR). Despite limited reports of CQR, more extensive studies on the actual magnitude of resistance are still needed. Short-time recurrences of malaria cases were analyzed in different transmission scenarios over three years , selected according to malaria incidence. Multilevel models  were used to evaluate association of short-time recurrences with variables such as age. The zero-inflated Poisson scan model (scanZIP) was used to detect spatial clusters of recurrences up to 28 days. Recurrences compose less than 5% of overall infection, being more frequent in the age group under four years. Recurrences slightly increased incidence. No fixed clusters were detected throughout the period, although there are clustering sites, spatially varying over the years. This is the most extensive analysis of short-time recurrences worldwide which addresses the occurrence of P. vivax CQR. As an important step forward in malaria elimination, policymakers should focus their efforts on young children, with an eventual shift in the first line of malaria treatment to P. vivax.In Brazil, malaria caused by  Plasmodium vivax presents control challenges for several reasons, among them the possibility of increased failure of P. vivax treatment. Chloroquine is an antimalarial used as a first-line drug in the treatment of the blood phase of the parasite, but chloroquine resistance (CQR) remains a hurdle. Treatment failure appears as a problem in Brazil and various countries of South America [P. vivax malaria in the Amazon [Malaria caused by  America ,2, Centr America , and oth America ,5. Even  America . Brazil e Amazon ,8,9.P. vivax differs from P. falciparum by the presence of hypnozoites, a latent form present in the liver [P. vivax malaria consist of three categories: recrudescence, relapse, and reinfection. Recrudescence is the failure to clear asexual blood stages from the circulation, related to ineffective blood schizonticidal treatment. Relapse involves the re-emergence of the disease resulting from the activation of hypnozoites from the liver. Reinfection, in turn, consists of a new infection acquired by the individual through the bite of an infected vector [Malaria caused by he liver , which md vector .However, relapse episodes may be mistakenly classified as recrudescence or, in regions of high incidence, as reinfections . ResistaThe National Malaria Control Program (NMCP) of the Brazilian Ministry of Health, classifies recurrences depending on the following times after a first infection: (1) recrudescence, from the 3rd day to the 28th; (2) relapse probable, from 29 to 60 days, and (3) likely reinfection, above 60 days [P. vivax, clinical trials must be performed [In order to evaluate the resistance of antimalarial drugs caused by erformed . Howevererformed , includierformed .P. vivax malaria literature, there are many reports of CQR, but further large-scale studies on the extent of this resistance are still needed [In the l needed . The anaThe study area covers all the Brazilian Amazon from where all malaria notifications are mandatory in the National Malaria Surveillance System  database of the Health Surveillance Department of the Ministry of Health, active since 2003. The data provided allow surveillance by municipal, state, and federal agencies .P. vivax malaria in the Amazon from 2003 to 2016, classified as High (2005), Intermediate (2010), and Low (2015)  . In thesAlthough SIVEP-malaria is a very robust and comprehensive information system, no information on patient follow-up is available. Thus, linking multiple notifications for the same potential patients has some hurdles: the absence of a unique identifier, typos when writing patient names, duplicated entries, and homonymous patients. In the absence of a unique identifier, we use the names of patients along with other personal variables, via linkage tools ,11. The We obtained a selection of notification pairs identified as likely to be from the same patients by automatic verification, applying a probability threshold (probability > 0.7). The next step of notification inspection includes visual confirmation of homonyms, possible siblings (twins), and duplicity. A new identification (ID) for notifications from patients deemed as recurrent were provided.P. vivax malaria in a given year had its percentage of recurrence estimated up to 28 days.The number of days between each individual event of infection was considered the recurrence time within 28 days of infection. Total number of recurrences in the municipality was obtained by counting the number of individuals who were positive from the 5th day to the 28th day, after the first entry in SIVEP-malaria. Recurrence ratio was calculated by dividing the number of recurrences by the number of infections in the municipality. Thus, each municipality that presented records of For analysis of the risk factors, we used the individual information on those who recurred up to 28 days, and the comparison group was composed of individuals who had only one entry in SIVEP-malaria. The predictor variables were age (by age group) and the annual parasitemia index (API).A binomial regression model with random effects in the municipality of likely infection provides the effects of risk factors for recurrence up to 28 days, as odds ratios or predicted probabilities. A multilevel model was applied considering the first level as the individual, and the second level as the municipality.We used a spatial cluster detection tool with counting data. Because of the study design, some municipalities presented zero recurrences and, in order to deal with that, scan statistics for zero-inflated Poisson (ZIP) models were applied accordingly, which uses a priori information of the expected value of ZIP . The decThe SIVEP-malaria databases were imported, and data curated and analyzed in the R software (version 3.5.0), Rstudio Server (version 1.2.1) installed on a server with Linux CentOS operating system version 7, 64 bits, with 90 GB of RAM and 12 physical processing cores. R software packages for larger databases  were used, in addition to GAMLSS, lme4, hnp, soundexBR, scanstatistic, sf, spedp, SpatialEpi and tmap. The significance level for the tests was 0.05.Access to SIVEP-malaria was duly authorized by the NMPC, respecting the non-disclosure of the identity of patients. The project was submitted to and approved by the CEP-INI/FIOCRUZ  .Results were organized to describe the percentages of recurrences up to 28 days at the level of the Brazilian Amazon as a whole, and then its spatio-temporal distribution in the municipalities.The data in The three maps represented in P. vivax malaria, recurrence rates in 2015 are not far from those from the peak incidence of P. vivax malaria in 2005. Even in the scenario with many P. vivax cases, the general pattern of recurrences up to 28 days remained.A total of 351/566 (2005) municipalities in the region had zero recurrences up to 28 days, mostly those outside the states of Amazonas and Pará. Over time, the municipalities in these two states, once with more recurrences, experienced a recurrence prevalence decrease. Despite the decline in the number of municipalities with recorded cases of Using information at the individual level, we list age group (age) as the main risk factor for the probability of recurrence up to 28 days. First, for comparison purposes, we performed the analyzes only with the age group as a risk factor . We founThe predicted probability of recurrence up to 28 days in ages under four years old is above 3% and below 5%. For this age group, the confidence intervals present intersections between them across the studied years. For the range of 4 to 5-year-olds, and subsequent ages, the probabilities are smaller, and confidence intervals are non-overlapping with that for below four years old. This difference becomes more evident in the year 2010 .Time to the first recurrence, on average, was close to 28 days in the children under 4 years old group. For the other groups, this average time was apparently higher and this is consistent for the three scenarios evaluated .In order to assess the effects of the annual parasitemia index (API) on recurrences, we estimated probabilities of recurrence among municipalities in the three incidence scenarios, demonstrating that the probabilities, regardless of incidence, tend to increase as the API increases . The incClusters of recurrence up to 28 days were detected in the higher transmission scenario in Roraima , Pará (Oeiras do Pará and surrounding), and Amazonas  A, the fiWhen comparing the age group related risk of recurrence up to 28 days to recurrences up to 42 days, no significant differences were found . HoweverP. vivax treatment in Brazil, except in pregnant women [In the three transmission scenarios assessed, recurrence ratios up to 28 days were always estimated below 10% in most of the municipalities. This finding is in agreement with those from clinical trials using the same treatment schemes as in this study ,14. Recunt women ,14. Prevnt women . FurtherP. vivax was already associated with likely recrudescence, defined as episodes that occur before the 16th day from the first episode, and with likely relapse or recrudescence, when the second episode occurs between the 17th and 28th day after the first [Resistance to he first . Althoughe first . There ahe first ,23. Casehe first ,25.Malaria elimination relies on a pack of specific components and measures. A comprehensive Information System is one of them. Since 2003, the MoH of Brazil defined SIVEP-malaria as the main information system for malaria surveillance in the Amazon and a huge database has been constructed since ,19. HoweThe findings presented here demonstrate that the probability of recurrence up to 28 days is associated with the age group. In addition, high incidences of vivax malaria in the municipalities increase the variations in recurrence. Recurrences at low API are mostly composed of resistance. The probability of recurrences in municipalities with a high incidence of vivax malaria possibly increases; however, great uncertainty still remains, since municipalities with extreme incidence make inference harder . The anaRecurrence rates for children under 4 years showed strong association with the locality of infection in the Amazon. Earlier studies have demonstrated similar findings ,9, incluThe time to first recurrence was lower in the children under 4 years old group as already described . We alsoFurthermore, a more frequent recurrence in children may be linked to developing more symptoms, for example, fever, or even another unrelated infection, mistakenly notified as malaria due to selection bias . By contP. vivax malaria currently relies on chloroquine, identifying chloroquine-resistance is essential in efforts towards malaria eradication. Maintaining a surveillance of short-time recurrences as a proxy for CQR is a very important approach toward such a goal.The findings presented here show that children aged less than 3 years old are an important group regarding recurrences related to CQ resistance potentially associated with drug dosing. Decision makers should consider this population carefully in order to achieve malaria elimination. Since the first-line treatment of