Plasmodium vivax. In the present small-scale field test, carried out in a low-transmission area of Thailand, RMOD confirmed malaria in all samples found to be infected with Plasmodium vivax by microscopy, our reference method. Moreover, the magneto-optical signal for this sample set was typically 1–3 orders of magnitude higher than the cut-off value of RMOD determined on uninfected samples. Based on the serial dilution of the original patient samples, we expect that the method can detect Plasmodium vivax malaria in blood samples with parasite densities as low as in vivo Plasmodium vivax infections.The rotating-crystal magneto-optical detection (RMOD) method has been developed for the rapid and quantitative diagnosis of malaria and tested systematically on various malaria infection models. Very recently, an extended field trial in a high-transmission region of Papua New Guinea demonstrated its great potential for detecting malaria infections, in particular  One such disease is malaria, and by virtue of increased global and local efforts significant progress has been made towards its control worldwide. Since many countries are working towards the substantial reduction of disease burden, the development of sensitive, rapid, high-throughput and low-cost malaria diagnostic methods applicable for mass-screening is a pressing issue in tropical diseases research2.Although humanity is continuously facing novel medical challenges, the management of preventable infectious diseases, which place heavy burden on tropical countries, has been a long-sought goal of the WHOPlasmodium-infected red blood cells, as it may enable the rapid and quantitative detection of the infection at low cost7. During the intraerythrocytic cycle the parasites break down the hemoglobin of their host cell and sequester its iron content into the inert, submicron-sized paramagnetic hemozoin crystals, which are distinguishing features of all blood-stage Plasmodium infections9. In the recent years, our research group has developed a new technique, the rotating-crystal magneto-optical diagnostic detection (RMOD) method, which is capable of the quantitative detection of malaria by measuring the amount of hemozoin produced during the course of the infection. The diagnostic capability of RMOD was tested in several steps using synthetic P. falciparum cell cultures and mouse infections13. Furthermore, it proved to be an efficient tool for conducting rapid, yet sensitive drug susceptibility assays14.One promising diagnostic approach is the utilisation of the magnetic properties of 15. One important—and anticipated—observation of the study was that infections with different species led to different average blood hemozoin levels, resulting in a more sensitive detection of P. vivax than P. falciparum. However, the presence of stage V P. falciparum gametocytes increased the likelihood of identifying P. falciparum infections due to their high hemozoin content. As another key finding, the mean value and the standard deviation of the magneto-optical (MO) signal for samples which tested negative with all reference methods was significantly higher than expected from previous RMOD measurements on malaria-naïve blood samples12. Accordingly, we concluded that in high-transmission settings, such as the study site in PNG, elevated hemozoin levels are likely to be maintained in the peripheral blood of a large proportion of the general population, either from concurrent low-level infections that are otherwise undetectable, or from previously resolved infections17.Recently, a detailed evaluation of RMOD has been carried out in an extended field trial in Papua New Guinea (PNG) involving almost one thousand malaria-suspected patients and multiple reference methodsP. vivax infections in low-transmission settings, here we use the technique to detect malaria in samples confirmed to be P. vivax-positive by microscopy from Thailand, a country with overall low malaria endemicity19. In addition to testing the sensitivity of the method, we evaluate the correlation between the magnitude of the MO signal and the parasite stage distributions of the samples.In order to further investigate these observations and to assess the sensitivity of RMOD for diagnosing P.vivax infections20. The main difference between RMOD and Gazelle is that the former detects the periodic modulation of light transmission induced by the hemozoin crystals co-rotating with a rotating magnetic field, while the latter measures light transmission in stationary states with and without a static magnetic field. In spite of this technical difference, the physical principle of detection is common. Thus, the magneto-optical quantification of hemozoin should support not only a qualitative, but a quantitative diagnosis in both cases. While this capability has not been investigated for Gazelle, it is an important objective of the present study.The potential of the magneto-optical approach for diagnosing malaria has also been demonstrated in a recent field test by another hemozoin-based tool, Gazelle, which showed a sensitivity of The parasite densities of the original 35 patient samples ranged from mentclass2pt{minimrings, trophozoites, schizonts, male and female gametocytes. The latter three forms contributed less than 10% to the overall parasite density in all samples with one exception, in which their proportion reached 35%. The sequestration of schizonts is a well-known phenomenon in P. falciparum infections, but the statistics of our P. vivax samples suggests that it also occurs for P. vivax21. Accordingly, the blood smears exhibited mostly rings and trophozoites, but their relative ratios varied in the sample population. A large fraction of samples (n=27) showed a relatively synchronous character and contained either predominantly P. falciparum infections, in the case of these P. vivax-positive samples more than 30% of the samples showed no ring forms, and 88% contained trophozoites in more than 10%.Besides the overall parasite density, the erythrocytic stage distribution was also determined by an expert microscopist, and the parasites were classified as As a first step, the level of the weak residual MO signal, that is present even in samples of malaria-naïve individuals, was measured on a pool of uninfected samples in order to establish a threshold that separates malaria-positive and negative RMOD incidences. Measurements on fourteen uninfected samples obtained from malaria-naïve individuals in Thailand, each measured in duplicates, yielded an average residual MO value of entclass1pt{minimaentclass1pt{minimaP. vivax-infected blood samples tested above the cut-off, yielding a 100% sensitivity on this sample set. Moreover, for more than 90% of the samples the MO values are at least one order of magnitude larger than the cut-off, implying that the method can detect lower parasite densities. To model such cases, we chose ten of the original blood samples and prepared dilution series from them using uninfected whole blood as dilution medium. The parasite densities of the original ten samples change between As displayed in Fig. 20.The MO values of the diluted samples prepared from the same infected specimen , which is a parameter calculated by assuming different hemozoin production rates of the different erythrocytic stages. When we use a subset of samples with clear dominance of rings and trophozoites, i.e., that contain schizonts and gametocytes in less than HZ parameter is a weighted parasite density, which is not equal to the actual hemozoin concentration, but is linearly proportional to it.) As shown in Fig. HZ values reaches its maximum, S and G denote the density of schizonts and gametocytes in the samples, respectively.While the MO signal shows only a moderate correlation with the parasite density, it correlates better with the estimated hemozoin content . This comparison reveals that the Thai and PNG data sets follow the same trend and the magnitude of the MO values in the two studies spans the same range. Most importantly, for 98% of the samples found P. vivax-positive in the PNG study the MO values are located above 0.94 mV/V, the cut-off level determined in the current study. This supports the assumption that a considerable fraction of false-positive MO detections in the PNG study are due to the high residual hemozoin level present in the population at high transmission settings. Whether these are ongoing asymptomatic infections or recently resolved ones is yet unclear. In a recent study Kho and co-workers found large biomass of intact asexual-stage malaria parasites in the spleen of asymptomatic and microscopy-negative malaria patients23. If some of the hemozoin produced by these otherwise undetected parasites can re-enter the peripheral circulation, it could be the source of the unexpectedly high MO signals in some of the microscopy-negative cases in our PNG study. However, to confirm this hypothesis further large-scale studies are crucial both in high- and low-transmission and/or elimination settings. Accordingly, for RMOD or any other hemozoin-based malaria detection method, the final field of application, e.g., as an in-field diagnostic device, epidemiological surveillance tool, can only be determined after elucidating the former issue. From a technological point of view, they have the benefits of requiring only minimal sample preparation, practically no expertise for operation and have the potential to be realised in a cost-efficient, field-applicable format. On the other hand, whether RMOD operation can be extended to facilitate species-specific diagnosis at least on the level of the current rapid diagnostic tests, is still the subject of our investigations. Furthermore, for its applicability in epidemiological surveillance studies, as a pre-screening tool for example, an increase in throughput of the current design is needed.To further elaborate on this, we make a direct comparison between the field samples from Thailand and PNG in Fig. in vivo P. vivax samples contain approx. 4.5 times more hemozoin than rings, while older stages (schizonts and gametocytes) contain 12–20 times larger amounts than rings. These values are comparable to the relative hemozoin production rates of rings, trophozoites and schizonts reported previously for P. falciparum cell cultures25. From this we conclude that the actual hemozoin content in peripheral blood primarily reflects the momentary hemozoin production rate, i.e., a dynamic equilibrium is maintained between hemozoin production and clearance, as also observed in rodent infections13. However, since RMOD could also confirm the infection in samples containing exclusively ring stages, whose hemozoin content is much lower than the hemozoin content of trophozoites and schizonts, a non-negligible hemozoin accumulation also has to occur in the peripheral blood during acute infections. Likely this is the reason why P. falciparum infections—which exhibit primarily very young stages in the circulation—could be diagnosed by RMOD in PNG as well, however with a lower sensitivity than P. vivax15.RMOD quantifies the concentration of hemozoin in the peripheral blood which can be present within the parasites, erythrocytes or leukocytes at the time of sampling. Since a coarse categorisation of the erythrocytic stages was carried out and the densities of the different stages were determined by expert microscopy, the hemozoin production rates of the different stages could be estimated based on the MO values of the samples. We found that trophozoites in these 28. The relation between the intraleukocytic hemozoin content and disease severity reported in former studies implies that the overall hemozoin concentration in the peripheral circulation may also serve as a useful clinical indicator26. The quantitative nature of RMOD, realising a hemozoin-based diagnosis, can also be highly advantageous in this respect.The most important requirements towards a novel method for in-field malaria diagnostics are the ease of use and high sensitivity – ideally even the detection of asymptomatic infections. However, besides the highly reliable differentiation between positive and negative cases, the quantitative assessment of disease severity might provide crucial information in certain clinical situations. In clinical practice today the parasite density is used as the primary characteristics of acute malaria infections, however, the proportion of hemozoin-containing white blood cells has also been investigated as a potential indicator of prognosisSample collection was carried out in two field-clinics in the Kanchanaburi and Ratchaburi provinces of western Thailand from May 2014 to June 2015 (n =33), while two additional infected samples were obtained in July, 2016. Patients attending to the clinics exhibiting symptoms consistent with malaria were tested for the infection using an SD Bioline Malaria Ag P.f/Pan rapid diagnosis test kit (Standard Diagnostics Inc.) and/or via light microscopic (LM) examination of thick blood smears. Once a positive diagnosis was established, a blood sample of approximately 250 Approximately 40 uninfected blood samples were collected from two consented, malaria-naïve colleagues on-site at various time points over the study period for protocol optimisation and for the establishment of an uninfected baseline signal for the RMOD. These control samples were collected, frozen and stored the same way as the infected samples. Seven samples from both individuals were utilised to determine the uninfected baseline signal and the cut-off level of the study see Fig. A while tP. vivax-infected samples on site, and analysed later by an expert microscopist at the central laboratory of MVRU, Bangkok. The morphological analysis of the parasites confirmed that all samples were infected exclusively with P. vivax. The thick blood smears were prepared using exactly one microliter of blood, thus by counting all the parasites in the total area of the smears  and an assumed leukocyte density of 30. Slides were scored as LM-positive for an individual Plasmodium species if the species was detected independently by at least two microscopists.Thick blood smears were examined independently by two microscopists, for 200 thick-film fields . The study was clearly explained to all volunteers and informed consent was obtained from all participants in the study.The study conducted in Papua New Guinea received ethical clearance by the PNG Institute of Medical Research (PNGIMR) Institutional Review Board and the PNG Medical Research Advisory Committee . All methods were carried out in accordance with the relevant guidelines and regulations of the named institutes.31. Briefly, the liquid sample containing the elongated paramagnetic hemozoin crystals is inserted into the centre of a ring-shaped assembly of permanent magnets, which creates a strong uniform magnetic field at the sample position. When the magnetic ring is rotated, the crystals follow this rotation which modulates the intensity of the light passing through the sample. The modulated intensity divided by the average intensity, which has been shown to be linearly proportional to the crystal concentration, corresponds to the measure MO values’ displayed in the corresponding figures in mV/V units10.The concept of the rotating-crystal magneto-optical setup and the underlying physical principles of hemozoin detection are described in detail in our former studiesFor the RMOD analysis in Thailand, the frozen blood samples were thawed on room temperature, mixed thoroughly and For the estimation of the RMOD sensitivity threshold two- or four-fold dilution series were prepared from ten infected samples. The infected samples were thawed and diluted with thawed aliquots of malaria-naïve blood samples frozen and stored together with the infected specimens. After the thorough mixing of the two components the final optical samples for the RMOD measurements were prepared as described above.The magneto-optical measurements in Thailand and PNG were carried out with two RMOD devices that were technical replicates using the same measurement protocols. The only differences between the two RMOD experiment series were that in PNG the samples were freshly hemolysed and measured in triplicates per blood sample, while in Thailand the whole blood samples were frozen and stored until the completion of the RMOD measurements and they were measured in duplicates per blood sample.


Plasmodium falciparum exposure in areas with different malaria endemicity in north-eastern Tanzania using serological markers; PfAMA-1 and PfMSP-119.A community-based cross-sectional study was done to assess PfAMA-1 as compared to Hai 13.8% (33), χ2 = 34.66, p < 0.01. Likewise, Bondo had a higher seroprevalence 201(36.6%) for PfMSP-1 as compared to Hai 41 (17.2%), χ2 = 29.62, p < 0.01. Anti-PfAMA-1 titters were higher in malaria positive individuals (n = 47) than in malaria negative individuals (n = 741) (p = 0.07). Anti-PfMSP-1 antibody concentrations were significantly higher in malaria-positive individuals (n = 47) than in malaria-negative individuals (n = 741) (p = 0.003). Antibody response against PfAMA-1 was significantly different between the three age groups; < 5 years, 5 to 15 years and > 15 years in both sites of Bondo and Hai. Likewise, antibody response against PfMSP-119 was significantly different between the three age groups in the two sites (p < 0.001). We also found significant differences in the anti-PfAMA-1and anti-PfMSP-119 antibody concentrations among the three age groups in the two sites (p = 0.004 and 0.005) respectively. Immunological indicators of P. falciparum exposure have proven to be useful in explaining long-term changes in the transmission dynamics, especially in low transmission settings.Bondo had a higher seroprevalence 36.6% (188) for The online version contains supplementary material available at 10.1186/s13104-021-05818-y. Plasmodium falciparum is mainly responsible for 99.7% of estimated malaria cases [Africa carries the highest burden of malaria with more than 70% of all malaria cases and deaths . Each yeia cases .P. falciparum accounts for 96 percent of cases [P. falciparum malaria is poorly understood, however, evidence shows that antibody-dependent cellular mechanisms play a key role in immunity against P. falciparum malaria parasite [P. falciparum antigens [In many countries, local malaria transmission has decreased due to the extensive efforts being devoted to malaria control and elimination . P. falcparasite , 8. The antigens .P. falciparum as a type I integral membrane protein [Plasmodium species as well as the most abundant protein expressed on the surface of merozoites [. Antibodies against MSP-1 and AMA-1 antigens are potential markers of both exposure to P. falciparum and protection against the disease [Apical membrane antigen 1 (AMA-1) is expressed on merozoites and sporozoites of  protein  while Merozoites . Antibod disease , 13 and  disease .In areas with low malaria transmission, it has become extremely difficult to detect changes in transmission intensity using conventional methods such as the entomologic inoculation rate (EIR) or malaria prevalence rates. Low transmission areas (low endemicity) sometimes have low mosquito density, below the detection limits of common mosquito trapping methods , 16 and The study was conducted during April and December 2014 in two different areas of the Tanzanian mainland. The first site was Bondo in the Tanga region, inhabited by 7970 people . The secParticipants 2 years of age and above who reside in the study areas were enrolled in the study. A blood sample was obtained by finger prick, a portion of blood was used for malaria rapid test, which was performed on-site. A blood spot was prepared for each participant, then dried and stored for further analysis. A 3.0 mm diameter circle of dried blood spot  was reconstituted in 200 µl of sodium azide-phosphate buffered saline-tween (0.05%) (PBST/0.1% Azide).P. falciparum surface antigens, P. falciparum MSP 119 (PfMSP 119) and P. falciparum AMA-1(PfAMA-1) [Indirect immunosorbent Assay (ELISA)was performed using two PfAMA-1) .Plasmodium nucleic acid amplification was conducted using genus-specific reverse and forward primers (rPLU6-5′TTAAAATTGTTGCAGTTAAAACG3′ and rPLU5-5′CCTGTTGTTGCCTTAAACTCC3′) targeting small sub-unit ribosomal RNA (ssurRNA) of the parasite. A reaction mix of 20 µl per sample was used, 5 µl of template DNA extracted from participants whole blood plus 15 µl of nuclease-free water, dNTPs, Taq enzyme, buffers and salts. Amplification conditions were, 95 °C for 5 min followed by 30 cycles of 94 °C for 1 min, 58 °C for 2 min and 72 °C for 5 min then one final extension cycle at 72 °C for 10 min. Amplification products were run in Ethidium bromide agarose gel (2%) electrophoresis at 120 V, 50 watts and 120 mA. The amplified bands were visualized under ultra-violet light trans-illuminator [Parasite DNA was extracted using the simple Chelex–Saponin method, uminator , 27.2) test was used to compare two proportions.All data were analyzed using SPSS 20.0 software  and GraphPad Prism8 software . After verifying that Optical density (OD) values were not normally distributed , non-parametric tests were performed to compare the OD. The Mann–Whitney test was used for the comparison of Antibody levels of two independent groups. The non-parametric Kruskal–Wallis test was used for the comparison of more than two groups. Pearson’s Chi-squared  from Hai and 549 (69.7%) from Bondo. Males were 283 (35.9%) and females were 505 (64.1%). About 405 (51.4%) participants had more than 15 years of age, 212 (26.9%) were between 5 and 15 years and 171 (21.7%) were below 5 years. The malaria prevalence by mRDT was 8.6% (47) in Bondo and 0% in Hai (Fisher exact test *p < 0.001). By PCR, malaria prevalence was 20.4% (161), with Bondo having a higher prevalence 28.1% (n = 154) than Hai 2.9%, (n = 7), χPfAMA-1 as compared to Hai 13.8% (33), χ2 = 34.66, p < 0.01. Likewise, Bondo had a higher seroprevalence 201(36.6%) for PfMSP-1 as compared to Hai 41 (17.2%) . In Bondo, participants with more than 15 years had a significantly higher seroprevalence of PfAMA-1 61.7% (116)  and PfMSP-119 63.7 (128)  as compared to other age groups. Likewise, participants with 5–15 years and < 5 years had a higher prevalence of malaria as measured by mDRT   for 88 for PfPfAMA-1 titters were higher in malaria positive individuals (n = 47) than in malaria negative individuals (n = 741)   than in malaria-negative individuals (n = 741)   as compared in Hai (n = 239),   than those of Hai (n = 239),    , as indicated in Fig. PfMSP-119 antibody concentrations among the age groups   Fig. C, D.Fig.The purpose of this study was to use immunological markers to investigate malaria transmission patterns in areas with diverse malaria endemicities.In this study, malaria prevalence by PCR in Bondo was 28.1%. Since Bondo is a malaria-endemic area, malaria transmission occurs nearly all year long with a peak period from April to June. No significant difference was observed in malaria prevalence among age groups in the present study. This is contrary to the study conducted in 2011 which suggested a widening of the age group at risk for malaria infection to older children of 5–15 years . It is ePfAMA-1 antigens as compared to younger children and Adults. Antibodies to malaria antigens can explain long-term changes in malaria transmission dynamics [In our study, Interestingly, when the age-dependent analysis was done, older children (5–15 years) had a relatively low seroprevalence to dynamics . In 2009dynamics . In popudynamics ., which may be attributed to intense malaria interventions in Tanzania [Seroprevalence in moderate malaria transmission setting such as Bondo can play a small role in determining malaria transmission patterns although seroprevalence is almost two folds higher than Hai. A slight decline in seroprevalence was observed in the study area when compared with previous studies , 31, indTanzania , 36.PfMSP-119 was significantly higher in participants with positive malaria tests than in non-positive participants. As expected Bondo had higher antibody concentrations against both antigens as compared to Hai. Children with < 5 years present with low antibody titters suggesting a lack of recent malaria exposure and this makes the group vulnerable to the symptomatic manifestation of the disease. Earlier findings revealed that more than half of the participants reported being symptomatic and were malaria positive by mRDT [Study results showed that the overall concentration of  by mRDT . There iIn this study, immunological markers were found to be a useful indicator of ongoing malaria transmission, especially in low-endemic areas. Routine malaria surveillance can be made more effective by using these immunological markers to highlight the importance of customized and targeted control interventions.This study might not explain the recent changes in malaria transmission since it was a cross-sectional survey. A longitudinal study would have been appropriate in explaining seasonal variations in malaria infection rates across the study areas.Additional file 1: Figure S1. Map of Tanzania showing the study sites, the map was produced using ArcGIS version 10.3 software.Additional file 2: Table S1. Prevalence of Malaria by serology, mRDT, Microscopy and PCR.Additional file 3: Figure S2. A graph showing mean OD values for PfAMA-1 (A) and PfMSP-119 (B) among malaria positive and negative individuals. Presented in the Y-axis is the Log10 transformed OD values among malaria positives and negatives (X-axis).


Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene , 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.Malaria, caused by the parasite  Plasmodium protozoan and transmitted by mosquitoes, is an epidemic that predominantly occurs in tropical and subtropical regions [Plasmodium species that infect humans include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi [P. falciparum accounts for 99.7% of malaria cases in Africa and causes fatal symptoms. P. vivax induces weaker symptoms than P. falciparum but is known to occur worldwide and has a 75% infection rate in the Americas [P. vivax and P. ovale are treated using chloroquine antibiotics; P. falciparum, P. malariae, and P. knowlesi are treated with artemisinin antibiotics [P. falciparum is not detected early, it can lead to severe disease and death [Malaria, which is caused by the  regions . Malaria regions . Accordi regions . Plasmodknowlesi . P. falcAmericas . MalariaAmericas . P. vivand death . TherefoPlasmodium species under a microscope [Plasmodium, as well as distinguish between species [For malaria detection, currently available diagnostic methods include blood smears, the application of a rapid diagnostic kit (RDT), and molecular diagnostics. Blood smear, a representative diagnosis sample for malaria, is simultaneously performed with thick- and thin-layer smears, followed by Giemsa staining, in order to identify the presence of croscope . Howevercroscope  and a trcroscope . Furthercroscope . RDT rescroscope . Polymer species . However species . Thus, s species , lateral species , and qua species . Among t species ,20,21,22To date, a variety of LAMP detection methods have been developed, including colorimetric and fluorescence detection, detection using fluorescent probes, and detection using lateral flow devices . Among tPlasmodium species (Pan), P. falciparum (Pf), P. vivax (Pv), and the internal control , based on a slightly modified DARQ probe method. The limit of detection (LOD) test for the multiplex malaria Pan/Pf/Pv/IC LAMP assay was performed using four Plasmodium species, namely P. falciparum, P. vivax, P. malariae, and P. ovale. In addition, the performance of the multiplex Pan/Pf/Pv/IC LAMP assay was compared with that of the commercial RealStar® Malaria S&T PCR Kit 1.0.In the present study, we developed a multiplex malaria Pan/Pf/Pv/IC LAMP method to detect all P. falciparum, 54 P. vivax, 2 P. malariae, and 1 P. ovale) and 90 negative specimens. DNA extraction from the 208 whole blood samples was performed using the NX-48 Viral NA Kit, with a Nextractor NX-48 system , according to the manufacturer’s instructions. DNA was stored at −50 °C. All tests were performed in a blinded manner, with the operator unaware of any previous test results. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Korea University Guro Hospital (2017GR0769 (8 March 2017), 2020GR0451 (7 October 2020)).Clinical whole blood samples were collected from patients infected with malarial parasites at the Korea University Guro Hospital from June 2000 to July 2017. All clinical samples were diagnosed by microscopic examination of Giemsa-stained blood smears and in-house PCR tests at the Korea University Guro Hospital. In total, 208 whole blood specimens were used in the present study, including 118 positive specimens of four types of malaria  gene of P. falciparum and assessed for specificity via a BLAST search of sequences in GenBank . P. falciparum LAMP primers, including two outer primers (forward primer F3 and backward primer B3), two inner primers (forward inner primer FIP and backward inner primer BIP), and two loop primers (forward loop primer LF and backward loop primer LB), were designed using the Primer Explorer software . To detect all malaria species and P. vivax, we used malaria Pan and P. vivax LAMP primer sets, designed by Mohon et al. and Chen et al., respectively [P. falciparum, P. vivax, and IC, respectively. All LAMP primers and probes were synthesized by Macrogen Inc. . The LAMP primer mix was composed of 4 µM of two outer primers (F3 and B3), 32 µM of two inner primers (FIP and BIP), 10 µM of loopF primer, 6 µM of loopB primer, and 4 µM of loopB probe. The LAMP assay was run on a CFX 96 Touch Real-Time PCR Detection System  at 60 °C for 40 min. In addition, a negative control (human serum RNA and distilled water) was used as the baseline. The positive signal was determined by examining whether the signal was steep or gradual, considering the baseline, as the baseline of the LAMP assay was unstable when compared with quantitative PCR (qPCR) or RT-PCR.The multiplex malaria Pan/Pf/Pv/IC LAMP assay was performed using a Mmiso DNA amplification kit . The LAMP reaction mixture comprised of 12.5 µL of 2x reaction buffer, 0.625 µL of malaria Pan LAMP primer mix, 0.625 µL of ® Malaria Screen & Type PCR Kit 1.0  was performed with the CFX96 Touch Real-Time PCR Detection System , according to the manufacturer’s protocol. Thermocycling parameters specified in the kit were as follows: inactivation at 95 °C for 2 min, 45 cycles of denaturation at 95 °C for 15 s, annealing with fluorescence detection at 58 °C for 45 s, and extension at 72 °C for 15 s. The RealStar® Malaria Screen & Type PCR Kit 1.0 was used with two tubes, one tube to diagnose P. falciparum and P. vivax, as well as the other tube to diagnose P. knowlesi, P. malariae, and P. ovale.The RealStarPlasmodium spp., LDH partial gene sequences of P. falciparum, the 16S rRNA gene partial sequences of P. vivax, and beta-actin partial gene sequence of humans. All vectors were constructed by Macrogen, Inc. The vectors were serially diluted 10-fold, from 1.0 × 107 copies/μL to 1.0 × 101 copies/μL, to determine the LOD of the multiplex malaria Pan/Pf/Pv/IC LAMP assay. In addition, the LOD of malaria Pan/Pf/Pv/IC LAMP was tested with serially diluted clinical samples (10−1 to 10−6) infected with P. falciparum, P. vivax, P. malariae, and P. ovale. The LOD of the multiplex malaria Pan/Pf/Pv/IC LAMP for clinical malaria samples was compared with that of the RealStar® Malaria Screen & Type PCR Kit 1.0.The LOD of the multiplex malaria Pan/Pf/Pv/IC LAMP was determined using pTOP Blunt V2 vectors, including the 18S rRNA partial gene sequences of P. falciparum, P. vivax, and human actin beta vectors   were optimum for the multiplex malaria Pan/Pf/Pv/IC LAMP assay. Next, temperature gradient tests (58/60.8/65 °C) were performed to determine the optimal temperature for the multiplex malaria Pan/Pf/Pv/IC LAMP assay  under optimum conditions. The ratio of the malaria Pan, Pf, Pv, and IC LAMP primer sets was 1:1:2:1, and the LAMP assay was performed at 58 °C.To optimize the multiplex malaria Pan/Pf/Pv/IC LAMP primer set, different concentration ratios of the malaria Pan, Pf, Pv, and IC primer sets  were evaluated against the synthetic malaria Pan, 1:1:1:1) A. Three MP assay B. Among 7 to 101 DNA copies/μL  using serially diluted P. falciparum, P. vivax, P. malariae, and P. ovale clinical samples , 10−4 (Pf), and 10−2 (IC), whereas RealStar® Malaria Screen & Type PCR Kit 1.0 showed an LOD of 10−5. For the P. vivax clinical samples, the monoplex malaria Pan and Pv LAMP primer sets showed LODs of 10−4 and 10−2, respectively. The multiplex malaria Pan/Pf/Pv/IC LAMP assay presented LODs of 10−3 (pan), 10−3 (Pv), and 10−2 (IC), and the RealStar® Malaria Screen & Type PCR Kit 1.0 showed the same detection limits (10−3). For the P. malariae and P. ovale clinical samples, the monoplex malaria Pan primer set showed LODs of 10−4 and 10−1, respectively. The multiplex malaria Pan/Pf/Pv/IC LAMP assay showed detection limits of 10−3 (pan) and 10−1 (pan) for P. malariae and P. ovale. The RealStar® Malaria Screen and Type PCR Kit 1.0 showed detection limits of 10−5 and 10−1 for P. malariae and P. ovale clinical samples, respectively.The analytical sensitivity of the multiplex malaria Pan/Pf/Pv/IC LAMP assay was compared with that of the corresponding monoplex LAMP primer sets against synthetic pan, Pf, Pv, and IC vectors, ranging from 10opies/μL , Table 2® Malaria Screen & Type PCR Kit 1.0 for P. falciparum (n = 61), P. vivax (n = 54), P. malariae (n = 2), P. ovale (n = 1), and healthy patient (n = 90) whole blood specimens , the sensitivities for P. falciparum and IC were 100% and 95.08%, respectively, for the RealStar® Malaria Screen and Type PCR Kit (1.0). The sensitivity of the multiplex malaria Pan/Pf/Pv/IC LAMP assay was 100% in the malaria Pan channel (FAM), 100% in the Pf channel (HEX), and 75.41% in the IC channel (Cy5). The cross-reactivity of the Pv channel of the multiplex malaria Pan/Pf/Pv/IC LAMP assay was 100%, whereas that of the Pv channel of the RealStar® Malaria Screen & Type PCR Kit 1.0 was 98.36% against P. falciparum clinical samples (n = 61). For P. vivax clinical samples (n = 54), the sensitivities for P. vivax and IC were 100% with the RealStar® Malaria Screen and Type PCR Kit 1.0; the sensitivities of the multiplex malaria Pan/Pf/Pv/IC LAMP assay were 100% in the malaria Pan channel (FAM), 100% in the Pv channel (HEX), and 81.48% in the IC channel (Cy5). The Pf channels of the two assays showed no cross-reactivity with the P. vivax clinical samples. For the P. malariae (n = 2) and P. ovale clinical samples (n = 1), the sensitivities and specificities of both assays were 100%.To confirm the clinical performance of the multiplex malaria Pan/Pf/Pv/IC LAMP assay, the assay sensitivity and specificity were compared with those of RealStarpecimens . For P. P. falciparum and chloroquine for non-falciparum species [The accurate classification of malaria species during the early stage of diagnosis is valuable for the proper treatment of infected patients and prevention of overexposure to antimalarial drugs, as treatment policies for different species tend to vary, such as artemisinin-based combination therapy (ACT) for  species ,33. Curr species ,35.P. falciparum, and P. vivax. Developing multiplex LAMP assays lacking non-specific signals is substantially more difficult than developing multiplex qPCR, as one LAMP assay uses six or seven primer/probe mixtures, and combinations of 2–4 LAMP sets can potentially result in the non-specific amplification of several primers. In particular, in the case of LAMP with the DARQ probe method, the probe sequence corresponding to the binding region of the LAMP product needs to be designed with consideration of the low homology with sequences of other primers and LAMP products, given that it induces non-specific signals by probe-primer or probe-LAMP product interaction. Herein, the multiplex malaria Pan/Pf/Pv/IC LAMP assay was designed by combining a newly designed Pf LAMP primer set with Malaria pan and P. vivax LAMP primer sets, designed by Mohon et al. and Chen et al., respectively [P. falciparum and P. ovale, the multiplex malaria Pan/Pf/Pv/IC LAMP showed a LOD 10-fold and 100-fold higher than that of the commercial RealStar® Malaria S&T PCR Kit 1.0, respectively. For P. vivax and P. malariae, the LOD of the LAMP assay was the same as that obtained with the commercial kit. Notably, the multiplex malaria Pan/Pf/Pv/IC LAMP assay showed a slightly higher LOD than the commercial PCR kit. However, after assessing 208 clinical samples, the multiplex malaria Pan/Pf/Pv/IC LAMP assay revealed sensitivity (100%) and specificity (100%) similar to those detected with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%).In the present study, we developed multiplex differential diagnostic tools to classify the malaria Pan, ectively ,31. ThisAs the isothermal amplification method does not require an expensive temperature control device to change the temperature for target gene amplification, the isothermal amplification method has the advantage of easy application for on-site diagnosis, which is difficult to implement in general PCR ,36. UnfoP. falciparum, and P. vivax within 40 min. On evaluating 118 malaria-infected clinical samples, the multiplex malaria Pan/Pf/Pv/IC LAMP assay revealed a performance similar to the commercial RealStar® Malaria Screen & Type PCR Kit 1.0, which is based on the qPCR method. Thus, with portable multi-channel isothermal equipment, such as the Mic PCR Machine, the multiplex malaria Pan/Pf/Pv/IC LAMP assay can be applied in resource-limited settings for point-of-care diagnostics.In the present study, we developed a multiplex malaria Pan/Pf/Pv/IC LAMP assay, which can classify malaria Pan,


Plasmodium falciparum and Salmonella typhi are major causes of fever in the tropics. Although these infections are caused by different organisms and are transmitted via different mechanisms, they have similar epidemio-logic and clinical features. This study aimed to determine the prevalence of S. typhi and P. falciparum infections and their associations with fever at 2 sites in Northern Tanzania.This was a community-based, cross-sectional study, conducted from February to June 2016, involving 128 randomly selected individuals, aged between 1 and 70 years. Sixty-three (49.2%) participants were recruited from Bondo Ward, Tanga Region, and 65 (50.8%) were recruited from Magugu Ward, Manyara Region. Blood samples were collected by venepuncture into sterile microtubes. Detection of pathogen DNA was achieved via a multiplex real-time polymerase chain reaction assay. Data analysis was done using Stata, version 14. Prevalence data were presented as numbers and percentages, and chi-square analysis was used to assess associations. P values of .05 or less were considered statistically significant.P. falciparum and S. typhi infection, respectively. Of the 63 participants from Bondo, 31 (49.2%) had P. falciparum parasitaemia. None of the participants from Magugu tested positive for Plasmodium parasitaemia. S. typhi bacteraemia was detected in 11 (17.5%) of 63 and 6 (9.2%) of 65 participants in Bondo and Magugu, respectively. P. falciparum–S. typhi coinfection was only detected in Bondo . Age was the only variable that showed a significant association with both P. falciparum and S. typhi infection; falling within the 5-to 9-year or 10-to 15-year age groups was associated with both infections . Among the 30 patients with Plasmodium parasitaemia, 7 (23.3%) had fever, whereas 2 (12.5%) of 16 patients infected by S. typhi had fever. P. falciparum infection  and P. falciparum–S. typhi coinfection  were significantly associated with fever, while S. typhi infection alone was not.Of 128 participants, 31 (24.2%) and 17 (13.3%) tested positive for S. typhi and P. falciparum were considerably prevalent in the area. One-third of the P. falciparum–S. typhi coinfected individuals in Bondo had fever. P. falciparum infection was an important contributor to febrile illness in Bondo. In the presence of coinfections with P. falciparum and S. typhi, the use of malaria rapid diagnostic tests should be emphasised to reduce irrational use of medications. In the tropics, S. typhi and Plasmodium falciparum are the most endemic pathogens,4 causing typhoid fever and malaria, respectively. The 2 diseases are associated with poverty and underdevelopment, making them more prevalent in underdeveloped countries.6 Although these diseases are caused by vastly different organisms transmitted via different routes of infection – 1 is a gram-negative bacillus transmitted via the faecal–oral route, and the other is a protozoan transmitted via the bite of an insect vector – typhoid and malaria share similar clinical and epidemiologic features.In the investigation of fever in the tropics, 2 important diagnoses to be ruled out are typhoid fever and malaria. Both cause significant morbidity, mortality, and economic loss. Despite the progress in controlling salmonellosis globally, typhoid fever remains a public health problem in several parts of the world. Despite a reported decline in incidence in many countries, the disease is still widespread, particularly in South Asia and sub-Saharan Africa, as a source of significant morbidity, mortality, and economic losses.7 The overarching aim was to produce the evidence required to support policy makers in introducing prevention efforts against invasive S. typhi infections – in particular, the introduction of safe and effective typhoid fever vaccines into routine immunisation programmes. TSAP has achieved this ambitious target, finding high incidences of typhoid fever in both rural and urban populations in several countries in sub-Saharan Africa. The results of TSAP will dictate the direction of future typhoid fever research and control endeavours in Africa, and at last provide a key piece of the disease burden jigsaw puzzle.7Typhoid fever control efforts have included the establishment of the Typhoid Fever Surveillance in Africa Program (TSAP) in 2009 – a network of 13 sentinel sites in 10 sub-Saharan African countries – created to generate high-quality, contemporary, and standardised data on the incidences of typhoid fever and invasive nontyphoidal salmonellosis in sub-Saharan Africa.8 Parallel to this are other similar programmes aimed at strategically controlling malaria, such as the National Insecticide Treated Nets (NATNETS) Tanzania “Under-five Catch-up Campaign” and “Universal Coverage Campaign”, which have contributed to an effective integrated malaria control environment through increased ITN use, subsidised tests, artemisinin-based medicines, and massive community sensitisation initiatives.9 The net result of these strategies has been a reduction in malaria incidence and child mortality rates in Tanzania.8Malaria control in Tanzania has been significantly scaled up in the past decade. The Tanzanian National Voucher Scheme (TNVS) has expanded the availability and accessibility of insecticide-treated nets (ITNs), particularly for children and pregnant women, by subsidising costs of bed nets for about a decade now.10 In most resource-limited tropical settings, definitive laboratory diagnosis of concurrent malaria and typhoid fever is based on blood smear microscopy for malaria and the Widal test or bacterial culture for salmonellosis.In malaria-endemic areas, the clinical presentation of patients in the early stages of typhoid fever is challenging because it is similar to numerous other causes of febrile illness, such as malaria. Of all symptoms, persistent fever is a prominent source of difficulty when narrowing down the differential diagnosis list.11 The WHO recommends that malaria treatment be based on diagnostic test results, but this directive has yet to gain universal acceptance. This recommendation will become particularly important as the incidence of malaria decreases, as it will become more important to distinguish cases of malaria from other febrile illnesses.12Although the introduction of malaria rapid diagnostic testing (mRDT) has reduced the diagnostic dilemma of patients presenting with fever, challenges exist that limit the use of mRDT, including the logistical challenges involved in increasing coverage to areas not accessible by road. Nonetheless, many endemic countries in sub-Saharan Africa have adopted mRDT to expand parasitological-based malaria diagnosis capacity.15 Unfortunately, there is a paucity of epidemiologic data regarding the extent of coinfection with P. falciparum and S. typhi in many parts of Africa, including Tanzania. Such data could assist clinicians to make more informed decisions when patients present with symptoms that pose a diagnostic dilemma.Despite efforts invested in the diagnosis of malaria and typhoid fever coinfections, it is concerning that diagnostic challenges continue to hinder effective malaria and typhoid control in the tropics. This is due to a combination of factors, including the nonspecific clinical presentations of the diseases, the high prevalence of asymptomatic infections in many areas, the lack of resources and insufficient access to trained health-care providers and facilities, and the widespread practice of self-treatment for clinically suspected malaria or typhoid fever.P. falciparum–S. typhi coinfections in 2 areas of Northern Tanzania for which S. typhi prevalence has not previously been determined. These data will provide useful baseline information that will clarify the magnitude of P. falciparum–S. typhi coinfection and facilitate reasonable diagnostic and prescribing decisions.We designed this study to provide baseline epidemiologic data on the prevalence of P. falciparum and S. typhi infection and coinfection and their associations with fever, was conducted from February to June 2016, in Bondo Ward, Tanga Region, and Magugu Ward, Manyara Region, in Northern Tanzania.This community-based, cross-sectional study, which aimed to determine the prevalences of S. typhi infections in these areas. Magugu is located at 4°12’ S and 35°45’ E and is about 1,392 m above sea level. Bondo is about 309 m above sea level at 5°22’60” N and 38°34’60” E. The natives of both areas are agropastoralists with moderate human–animal interaction. Both areas have 2 rainy seasons per year, with a long rainy season between February and May and short rainy season between October and December. The long rainy seasons are usually followed by high numbers of reported fever cases.17The 2 sites are located about 600 km apart and were selected based on their differing locations, climatic conditions, malaria transmission intensities, and the absence of data on prevalence of Study participants consisted of children and adults, aged 1 year and above, who were residents of the study sites, Bondo and Magugu, for at least 6 months and consented to participate in the study.2p(1-p)]/c2, where Z=1.96 for the 95% confidence level, p=the expected true proportion of (9.0%) and c=the minimal tolerable error at the 95% confidence level (0.05). This formula yielded a minimum sample size of 126. We enrolled 128 participants. Following community sensitisation activities, community members were invited to participate, and those who met the enrolment criteria were consecutively recruited into the study until the desired sample size was attained.The minimum sample size for prevalence determination was estimated using the Epi Tools online sample size calculator and the following formula: [Z19 Briefly, blood samples of 0.5 to 1 ml were collected into sterile tubes by venepuncture from all consenting participants, and about 10 ml of whole blood was used for mRDT  and microscopy. These procedures were carried out by a trained laboratory technician. For each consenting participant, 1 ml of whole blood was aseptically collected into ethylenediaminetetraacetic acid (EDTA) vacutainer tubes. The tubes were shipped to the local laboratory in dry ice and later sent to the Kilimanjaro Christian Research Institute's biotechnology laboratory and stored at –20°C for laboratory analyses. For the purposes of this study, malaria parasitaemia  was defined by detection of P. falciparum using a real-time polymerase chain reaction (RT-PCR) assay, regardless of the presence of fever or other clinical symptoms. mRDT and microscopy were done to aid clinical decision making and management.A short questionnaire was used to obtain demographic information. Fever was defined as an axillary body temperature ≥37.5°C. Thick and thin blood smears for malaria microscopy were prepared as described elsewhere.Children under 5 years of age who were found to be malaria-positive by mRDT were treated with antimalarials according to national and WHO guidelines. Adults with fever and positive mRDT results were treated with artemetherlumefantrine, the first-line antimalarial drug in Tanzania. Severe paediatric cases were referred to the nearby district hospital in Korogwe District. Participants who were found to have salmonellosis were appropriately managed with ciprofloxacin according to national guidelines.DNA extraction and purification were done using QIAamp DNA Mini Kits  according to the manufacturer's instructions. A 200 μl aliquot of blood was lysed by QIAGEN protease and bound to a QIAamp membrane by centrifugation according to the manufacturer's instructions. The wash buffers, AW1 and AW2, were sequentially used to remove residual contaminants to improve the purity of the DNA. The purified DNA was then eluted from the QIAamp membrane using buffer AE  and stored at –20°C ready for the RT-PCR assay.TM 7 Real-Time PCR system . The Master Mix kit for Tropical Fever Core  was used to prepare the reaction mix. For a single reaction, 12.5 μl of FTD buffer, 1.5 μl of tropical fever primers and probe mix, and 1μl of enzyme mix were placed into single MicroAmp Optical 8-Tube Strips , compatible with the ViiATM 7 Real-Time PCR system, followed by 10 μl of sample. The same was done for all samples, the positive control, and the extracted negative control. The detection of pathogens was done at wavelengths of 520 nm for P. falciparum Tropical Fever 1 Primers and Probes and 620 for S. typhi Tropical Fever 2 Primers and Probes in the Master Mix of the Tropical Fever Core kit . The positive, negative, and internal controls used in this assay were commercially prepared by Fast Track Diagnostics. The positive control contained plasmids for the detection of P. falciparum and S. typhi. The negative control contained lysis buffer, and the internal control contained Streptococcus equi, which was also used as an extraction control. At the end of the run, amplification plots were reviewed in order to adjust the threshold line above all the background noise, as per the manufacturer's instructions.The RT-PCR assay was carried out in an Applied Biosystems ViiAP. falciparum and S. typhi infections) variables. The participant age groups for analysis were: <5 years, 5 to 9 years, 10 to 15 years, and above 15 years. Descriptive statistics were used to summarise the demographic and clinical characteristics of study participants. The chi-square (X2) test was used to determine associations between categorical data. Fisher's exact test was used in cases when expected counts were less than 5. A 2-tailed P value of .05 or less was considered statistically significant.Data were analysed using Stata, version 14  and categorised into demographic  and clinical . Permission was also obtained from community and local authorities. Written informed consent was obtained from each participant before enrolment, and legal guardians consented for minors. Access to the data – which were anonymised – was restricted to the research team.Demographic data are presented in P. falciparum and S. typhi infection. Sixty-three participants were screened for P. falciparum infection in Bondo, of whom 31 (49.2%) had malaria parasitaemia, in contrast with the absence of P. falciparum parasitaemia in Magugu. The prevalences of S. typhi infection in Bondo and Magugu were 17.5% and 9.2%, respectively. The prevalence of P. falciparum–S. typhi coinfection was 9.5% in Bondo, and – with no cases of coin-fection in Magugu – the overall coninfection prevalence was 4.7%.P. falciparum and S. typhi infection and participant demographic variables. When data from both sites were combined, only age was significantly associated with S. typhi infection , with 17.6% and 28.6% of the participants in the 5-to 9-year and 10-to 15-year age groups testing positive for S. typhi infection, respectively. Children younger than 5 years had the lowest prevalence of S. typhi infection. There was no association between S. typhi infection with either participant sex or study site.Plasmodium infection and age , with the 5-to 9-year and 10-to 15-year age groups having parasitaemia prevalences of 57.9% and 57.1%, respectively. The participants aged >15 years had the lowest prevalence of P. falciparum infection. We did not find any association between P. falciparum infection and sex.Similarly, there was a strong association between P. falciparum–S. typhi coinfection and sex, age, and study site were all not statistically significant.P. falciparum parasitaemia had fever, compared with only 2 (12.5%) who had fever among the 16 participants who tested positive for S. typhi infection. P. falciparum infection was associated with fever , and no association was found between S. typhi infection and fever. Among the 6 participants with P. falciparum–S. typhi coinfection, 2 (33.3%) had fever . Fever was most prevalent among 5-to 15-year-olds . There was no association between sex and fever.21 This scenario justifies the application of high-throughput and highly sensitive molecular tools to determine the causes of diseases with overlapping clinical signs and for proper management of febrile illness.The high prevalence of pathogens that cause overlapping clinical signs and symptoms, particularly fever, poses a serious challenge in diagnosing and managing febrile illness. This is particularly true in resource-poor countries, such as Tanzania, where the diagnostic infrastructure is constrained. Diagnoses are frequently provisional in the absence of adequate confirmatory tests, and it is, therefore, a common practice that antimicrobials are irrationally prescribed, leading to serious consequences, including the development of antimicrobial resistance.P. falciparum parasitaemia. This finding represents an increase in malaria parasitaemia prevalence compared with what has been observed in the past decade, during which time significant shifts have been reported. In 2009, the P. falciparum parasitaemia prevalence was 32.8% in the rainy season22; the prevalence dropped to 12% in 2011,12 with a further drop to 8.6% in 2013.23 However, a recent survey conducted in Bondo in 2016 reported a prevalence of 20.5%.18 In the absence of studies to explain the observed fluctuations in malaria prevalence in the study area, a number of explanations can be proposed regarding the outcomes of malaria control efforts implemented in the studied areas, including seasonal differences at the times when data were collected for the respective studies.About half of the participants in Bondo had 8 Other initiatives, such as the NATNETS Tanzania “Under-five Catch-up Campaign” and “Universal Coverage Campaign”, have complemented the TNVS.9 These initiatives have contributed to a substantial reduction in the incidence and prevalence of malaria in Tanzania, as reported by recent studies.9 However, a substantial impact on the disease burden has not been sustained. Challenging obstacles preventing optimal malaria control persist, including poor access to health care, poor performance of health service delivery, poor availability of proper diagnosis and treatment services, increased drug resistance, and high costs of health-care services.15 The increased malaria prevalence in Bondo suggests the possibility of a breakdown of malaria control strategies in the area. Support for malaria control at both the national and district levels has increased considerably over the past few years. The low prevalence of P. falciparum infection in Magugu has been reported for about a decade,24 likely because Magugu serves as an experimental study site for a national pesticide research institute.25In the past decade, the TNVS, implemented by the Tanzanian government, has contributed to a significant reduction in malaria through increasing the availability and accessibility of ITNs mainly targeting children and pregnant women.S. typhi infection in Bondo was high, with about one-fifth of the population testing positive for S. typhi, while in Magugu, about one-tenth of individuals were found to be infected with S. typhi. Previous studies have reported a similar uneven distribution of Salmonella infection, with the occurrence of invasive nontyphoidal salmonellosis more common in areas with high malaria transmission rates, while S. typhi is reportedly common in areas with low rates of malaria transmission.26 Wide variations in the prevalence of S. typhi infection have been reported in different parts of Tanzania and elsewhere in sub-Saharan Africa.28The prevalence of S. typhi infection prevalence in this study – an observation that was statistically significant. S. typhi infection was not associated with sex or study site. It has been reported that the most common dietary protein sources in Tanzania include milk, eggs, and meat, with urban dwellers consuming more of these products than rural inhabitants.29 These types of food are known important risk factors for human salmonellosis worldwide.30 Although the consumption of these foods is generally low in Tanzania, efforts have been made to ensure that school-aged children consume milk countrywide.31 Whether sanitary precautions are strictly observed or not, this could serve as a possible source of S. typhi infection in this age group, especially in periurban sites like Bondo. We found that malaria parasitaemia was most prevalent among participants between 5 and 15 years of age. A shift in burden of infection from being more prevalent among children under 5 to being more prevalent among school-aged children has recently been reported.18 In the absence of objective data to explain this shift, we speculate that because most malaria interventions over the past decade have targeted children under 5 and pregnant mothers,9 our findings reflect a reduction of immunity to malaria among older children, who were previously targeted by rigorous malaria control interventions when they were below 5 years of age. For both S. typhi and P. falciparum, individuals older than 15 years had the lowest infection rates, most likely explained by the build-up of specific immunity to these infections.Participants between 5 and 15 years of age had the highest P. falciparum–S. typhi coinfection was low (4.7%), and coinfection occurred only in Bondo, where the malaria parasitaemia prevalence was high. Despite the low coinfection rate, there are important implications for the coinfected individuals. There is accumulating epidemiologic and preclinical evidence supporting a causal association between malaria and nontyphoidal salmonellosis.32 However, the clinical characteristics and consequences of P. falciparum–S. typhi coinfection are not well documented, although mortality associated with coinfection has been reported to be higher than that associated with malaria alone.32The prevalence of P. falciparum infection in Bondo had fever, implying a significant contribution of P. falciparum infection to the development of fever. Besides that, 12.5% of participants with S. typhi infection had fever, reflecting an important contribution of S. typhi to the burden of febrile illness in the area. One-third of individuals coinfected with P. falciparum and S. typhi had fever.We found that nearly a quarter of individuals with P. falciparum infection were asymptomatic carriers. However, our findings do not rule out infection with other causes of fever that we did not test for. This possibility is supported by the findings of a previous study conducted in Magugu, where more than two-fifths of patients were clinically misdiagnosed as having malaria even though only less than 1% of blood films were confirmed P. falciparum-positive.11 Testing for a wider range of fever-causing pathogens would yield more specific findings to inform diagnostic and management guidelines for febrile illnesses. Our findings underscore the urgency of developing appropriate guidelines for the diagnosis and treatment of P. falciparum–S. typhi coinfection, as clinicians commonly dismiss the possibility of multiple infections during an initial patient visit. In attempts to implement an intensive malaria control programme in Tanzania, the Ministry of Health has launched a widely advertised Kiswahili slogan: “Siyo kila homa ni malaria”, literally translated as, “Not every fever is malaria”. This slogan not only reminds patients to avoid self-medication, it also reminds clinicians to carefully consider mixed infections when patients present with generic symptoms, such as fever and headache.We presume that the remaining three-quarters of afebrile participants with P. falciparum–S. typhi coinfection of 9.5% in Bondo, and we detected no coinfection in Magugu. One-third of the coinfected individuals in Bondo had fever. P. falciparum infection was an important contributor to the febrile illness burden in Bondo. Magugu was free from P. falciparum infection during the study period. Considering the presence of coinfections with P. falciparum and S. typhi, we recommend emphasising and enforcing the use of mRDT to reduce irrational use of medications. We also recommend the scale-up of typhoid fever diagnostic tools to underserved areas of Tanzania to help distinguish between malaria and typhoid fever.We report a prevalence of


Accurate, prompt, and reliable tools for the diagnosis of malaria are crucial for tracking the successes or drawbacks of control and elimination efforts, and for future programs aimed at global malaria eradication. Although microscopy remains the gold standard method, the number of imported malaria cases and the risk of reappearance of autochthonous cases stimulated several laboratories located in European countries to evaluate methods and algorithms suited to non-endemic settings, where skilled microscopists are not always available. In this review, an overview of the field evaluation and a comparison of the methods used for the diagnosis of malaria by European laboratories is reported, showing that the development of numerous innovations is continuous. In particular, the combination of rapid diagnostic tests and molecular assays with microscopy represents a reliable system for the early diagnosis of malaria in non-endemic settings. Plasmodium consists of over 200 widely distributed species, of which at least six regularly infect humans: Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), P. ovale wallikeri (Pow), P. ovale curtisi (Poc), and P. knowlesi (Pk) [Plasmodia, such as P. cynomolgi in Southeast Asia and P. brasilianum and P. simium in South America, have been described [The genus esi (Pk) . Howeverescribed ,4.P. falciparum and P. vivax pose the greatest threat: in 2018 P. falciparum accounted for 99.7% of estimated cases in the World Health Organization (WHO) African regions; and P. vivax is the most common species in the WHO regions of Americas, accounting for 75% of infections [Among species causing malaria in humans, Plasmodium interested and the parasitic burden in the blood. Parasite count is mandatory in cases of infection with P. falciparum, because it is one of the criteria used to define severe malaria (parasitemia >4% in adults and >10% in children). Different patient management modalities are applied if the parasitemia is >2% [P. falciparum malaria [Malaria is a febrile illness and clinical symptoms of uncomplicated malaria include fatigue, headaches, muscle aches, malaise, abdominal discomfort, fever, nausea, and vomiting . Specifia is >2% . The pre malaria ,7. Accurate, prompt, and affordable diagnostic tools are also pivotal for tracking the successes or drawbacks of control and elimination efforts, and for future programs aimed at global malaria eradication. Active surveillance of the disease in each geographical area is essential for a program to succeed. The WHO Global Technical Strategy for Malaria aims, by 2030, to reduce malaria case incidence and mortality rates by 90%, compared to the 2015 baseline, to interrupt malaria transmission in at least 35 countries and to prevent its re-establishment in all malaria-free countries. The aim of surveillance is to detect all malaria infections and to investigate each individual case of infection, to differentiate imported cases, namely, infections acquired outside the areas in which they are diagnosed, from those acquired locally ,9. In fact, with 229 million cases and 409,000 deaths, especially among children (estimated in 2019), malaria is one of the most severe public health problems worldwide ,10. AlthMalaria is thought to have arrived in South Europe via the Nile Valley during the Neolithic period, from whence it has been spread to the entire continent, where it remained endemic for more than 2000 years until its elimination by 1978 . During The consistent number of imported malaria cases and the risk of reappearance of autochthonous cases stimulated several laboratories located in European countries to evaluate methods and algorithms for the diagnosis that are best suited to non-endemic settings, where skilled microscopists are not always available, especially when the diagnosis is required in emergencies outside laboratory opening hours ,14,15.In this review, an overview of the studies performed in the period 1999–2021 by European laboratories, concerning the evaluation and the comparison of methods for the diagnosis of malaria, was reported.Plasmodia in blood film, alternative methods can be used in areas where training and expensive equipment can be introduced, such as staining with fluorescent dyes having affinity for the nucleic acid    directlyPlasmodia at the species and stage levels, and allows their quantification in peripheral blood in order to monitor patients with malaria, including follow-up during specific therapy. It is noteworthy that microscopy requires specific skills rarely available in non-endemic settings, especially when cases of mixed or sub-microscopic infection occur [Overall, microscopic examination provides rapid and inexpensive detection and identification of on occur . AlthougRapid diagnostic tests (RDT) are immunochromatographic assays for quickly (15–20 min) establishing the diagnosis of malaria infection by detecting specific malaria antigens in blood ,18. The The availability of commercial kits  providinPlasmodium falciparum-specific histidine-rich protein 2 (HRP2), Plasmodium pan-specific lactate dehydrogenase (pLDH), and pan-malarial aldolase for Plasmodia infecting humans [P. falciparum: it is produced by asexual stages and gametocytes of such Plasmodium, and it is expressed on red blood cells’ (RBCs) surface. pLDH is expressed at high level in asexual stages of P. falciparum, P. ovale, P. vivax, and P. malariae human malaria parasites. Aldolase is a pan-specific enzyme involved in the glycolytic pathway of the malaria parasites [The antigens currently used in RDTs available are g humans . HRP2 waarasites . The limarasites .Several European laboratories evaluated the performance and/or the usefulness of commercial RDTs for their inclusion in the malaria diagnosis workflow, as reported in P. falciparum malaria, the false-negative results observed can be attributed to a low level of parasitemia that appears to be critical for this assay [Different performances among the commercial assays were observed; however, overall, the authors concluded that RDTs are useful supporting tools for the diagnosis of malaria in non-endemic settings. Though they cannot be considered as unique diagnostic methods, these tests help the operator to achieve a rapid and easy to perform interpretation, especially if a trained microscopist is not always available; this can avoid delay in the management of life-threatening malaria cases ,28,31,32is assay . Furtheris assay ,26,31. Iis assay ,31. FalsTogether with the risk of false negative and false positive results, RDTs could miss double infections and are not able to quantify the parasitemia and distinguish among the parasitic stages .P. falciparum detection and pLDH antigens for P. vivax detection might be the best way to realize a reliable RDT for malaria diagnosis. In this light, the sensitivity in detecting species other than P. falciparum and P. vivax is very low [However, based on their results and the scientific literature in the topic, Grobush and colleagues  concludevery low ,33.P. falciparum assessed by microscopy, particularly when an inexperienced observer is involved in the diagnosis. However, for the reasons already exposed, they cannot substitute microscopy and their use is not recommended for following the response to antimalarial treatment. Furthermore, the currently available RDTs are not able to detect P. knowlesi [The observations reported by these authors meet with the Guideline for the laboratory diagnosis of malaria by Bailey et al. in 2013 , intendeknowlesi ,35.RDTs have been proposed to be used as self-diagnosis technique for high-risk groups, such as travellers in endemic areas after appropriate instructions and training to allow prompt treatment and avoid over-diagnosis of malaria on-site; although some recent results are encouraging, this application is still controversial ,36.Although microscopy is still the reference method because of the reasons described above, and RDTs provide valid support for diagnosing malaria, molecular assays have been proposed as a confirmatory method. In particular, they are crucial in cases of sub-microscopic parasitemia and when morphologic characteristics overlap, and/or when parasite morphology has been altered by drug treatment or improper storage of the sample . CDC sugIn Europe, molecular methods have been largely evaluated ,54,55,56Plasmodia involved [Overall, nucleic acid amplification tests (NAATs) are at least 10-fold more sensitive than microscopy . The liminvolved . Plasmodium genome. In particular, this reference target includes a genus-specific sequence of approximately 1.2 kb containing all the Plasmodium human-infecting species-specific sequences, which have been characterized and sequenced [The first target considered—and it is still used as a reference target—is the 18S-rRNA gene, present in 5–8 copies per equenced ,53,58,59P. falciparum stevor multigene family, telomere-associated repetitive element, and P. vivax Pvr64 sequence [P. falciparum parasite has one mitochondrion, whereas mature gametocytes have 4–8 mitochondrial organelles [varATS) gene, located on the chromosomal genome, and cytochrome b (cytb) on the mitochondrial genome. The authors assumed that, as previously demonstrated, most individuals with asexual parasites also have sub-microscopic gametocyte carriage. Interestingly, cytb PCR detection in follow-up samples later than varATS PCR may be due to the detection of gametocytes, as hypothesized by the authors. However, based on their observations, the authors concluded that it is unclear whether the DNA detected after treatment originated from residuals of destroyed parasites or live gametocytes [Newly developed NAATs include additional target genes, such as mitochondrial DNA (mtDNA), which allows the detection of all human malaria species together with 18S-rRNA, and other targets focusing on single species detection, such as sequence . The 18Sganelles . In a stganelles . The difetocytes .Plasmodia not only at the genus level, but at the species level too [P. falciparum infections with low parasite density and it is important to P. malariae and P. ovale malaria because the sensitivity of RDTs can be very low [In all the studies cited in evel too ,54,55,56evel too ,56. Thisvery low .Plasmodia species [Microscopy is the gold-standard method, and that cannot be avoided; however, different laboratories include in their workflows the molecular assays in ways that best suit their needs. In some laboratories, for example, the molecular assay is performed when species identification is problematic or in cases of strong suspicion of malaria with negative results by conventional methods ,55. Roug species ,40,49,50 species . This prPlasmodia detection, the correlation between real-time PCR’s cycle threshold and parasitemia was also assessed, as previously performed [Real-time PCR assays are highly sensitive and specific, and far less labor-intensive. They are performed in a closed system where post-PCR handling is not required and limit the possibility of contamination together with a good rapidity, although they cannot be strictly considered a rapid technique for the initial diagnosis of malaria requiring more than 1 h ,53. Furterformed . Unsatiserformed .Plasmodia 18S-rRNA for diagnostic purposes and epidemiological and drug studies [Among the different available molecular techniques, a faster and simpler method than real-time PCR for the diagnosis of malaria is a real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) assay evaluated in Amsterdam ,54 that  studies ,54.®-Eiken Chemical Co., Tokyo, Japan) for the detection of P. ovale malaria. The LAMP results were discordant in 2.6% of samples as compared to the nested-PCR used as a reference method: it remains to be determined whether there were false positives by LAMP, or false negatives with very low parasitemia by nested-PCR, as already reported [P. ovale endemic areas [One of the most recent evolutions of DNA amplification for malaria diagnosis is the development of commercial assays based on the DNA loop-mediated isothermal amplification (LAMP) that reduce the analysis time within the 2-h delay recommended for the diagnosis and ensure a simple technical process and a high sensitivity ,44,47. Areported . The autPlasmodia other than P. falciparum. Although the improved sensitivity is evident, their adoption and inclusion in the workflow should be deeply evaluated.Dakić and colleagues  encouragPlasmodium DNA detected in blood is probably a sign of active infection, even if no parasites are detected by microscopy [Plasmodium falciparum DNA for (NAT)-based assays that can be used for quality control and in the determination of the analytical sensitivity of different assays [It cannot be ignored that they detected the parasitic DNA while not distinguishing among DNA belonging to live parasites, residual DNA of destroyed asexual blood stage parasites, and circulating gametocytes which can remain in sub-microscopic quantities after successful therapy, thereby risking false positives due to the persistence of DNA after a malaria episode’s resolution, and as a consequence, unnecessary malaria treatment interventions ,45,46. Hcroscopy . Furthert assays .These considerations strengthen the need to carefully apply molecular techniques to the diagnosis of malaria.P. knowlesi in travellers with suspected malaria returning from Southeast Asia. The detection of P. knowlesi is mandatory, since the infection can be fatal if not treated promptly; however, its identification by microscopy is particularly difficult because of the morphological resemblance of early trophozoites to P. falciparum and later erythrocytic stages to P. malariae [One of the main current challenges is the detection of malariae . In thismalariae ,63.micrA summary of the key features of, and the desired improvements for, microscopic examination, RDTs, and NAATs for the diagnosis of malaria, are reported in Together with molecular assays, other novel techniques have been developed for the diagnosis of malaria, particularly those detecting hemozoin ,79,80,81In 2010, Mens et al.  evaluateThe magnetic susceptibility of hemozoin has led to the development of innovative detection methods based on nuclear magnetic resonance (NMR) and on magnetic resonance relaxometry (MRR)  excited surface acoustic wave (SAW) sensor . The autIn conclusion, therefore, the described novel techniques that search for hemozoin are not yet tools applicable to the diagnosis of malaria, but they could be promising solutions, after improvements, for future diagnostic systems.In Malaria is a rare diagnosis in Europe, but it is a medical emergency. A travel history is the key when malaria is suspected, and it is mandatory in patients with fever. There are no specific clinical signs or symptoms of malaria, although fever is seen in almost all non-immune patients. Migrants from malaria-endemic areas may have few symptoms.Plasmodium species are being distinguished. Even under ideal conditions, reliable distinction of the infecting Plasmodium species can be very difficult, if not impossible. Particularly, P. vivax and P. ovale cannot always be easily differentiated based on morphology; distinguishing P. knowlesi from P. malariae can be very challenging; P. ovale wallikeri and P. ovale curtisi are morphologically identical; P. cynomolgi is morphologically indistinguishable from P. vivax; and P. simium and P. brasilianum cannot be distinguished by microscopy from P. vivax and P. malariae, respectively. The limit of detection is also not ideal, because sub-microscopic asymptomatic individuals with low parasitemia remain undiagnosed and untreated, and also enable the transmission cycle to continue in the community. Malaria diagnostics should be performed immediately on suspicion of malaria, and the gold-standard is microscopy of Giemsa-stained thick and thin blood films. The quantification of malaria parasites can be used to make clinical management decisions and to monitor responses to treatment. Microscopy diagnosis is prone to human error, owing to its subjective nature. An inherent weakness of microscopy is the dependence on morphological features when A RDT may be used in parallel, but should not replace microscopy . It is aMolecular methods have demonstrated to be more sensitive and specific than microscopy, allowing the detection of missed cases and correctly identifying the species of Plasmodia of medical interest, with the final result of improving the early diagnosis of all cases of imported malaria ,20. HoweThe proposed algorithm takes into account these observations and the essential contribution of the genus- and species-specific DNA amplification assays for accurate diagnosis of malaria.According to WHO Global Technical Strategy for Malaria 2016–2030 , the futThis review showed that diagnostic laboratories in malaria non-endemic settings provide excellent diagnosis of malaria, especially regarding the detection of P. falciparum. Despite the limitations of current diagnostic methods, they continue to play important roles in dealing with the current global malaria situation, including decreasing its incidence. Diagnostic tools are critical for ensuring the appropriate care for each patient, and in this light, the development of numerous innovations continues.


Understanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission.Plasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44–94% for P. falciparum and from 23–72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings , parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings , >65% of gametocyte carriers were adults. Per survey, 37–100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes .Interventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling. Plasmodium vivax and Plasmodium falciparum cause the vast majority of all human malaria cases. Across all transmission settings, a large proportion of infections of the two species remain asymptomatic. These infections are not diagnosed and treated by control programs focusing on clinical cases. They can carry gametocytes, the sexual stage of the parasite that establishes infections in mosquitos, thus asymptomatic infections contribute to transmission. In order to determine who is likely to contribute to transmission, gametocyte densities were measured by sensitive molecular methods in afebrile individuals in four countries. The proportion of infections with gametocytes varied greatly among surveys, and was higher in regions that had experienced low transmission for extended periods of time. In moderate-high transmission settings, gametocyte densities were particularly high in children below six years, highlighting the importance that interventions to reduce transmission include this age group. The majority of gametocyte carriers was positive by light microscopy. The comprehensive data on gametocyte carriage presented here lays the foundation for the development of more effective screen and treat activities to reduce malaria transmission. A variety of malaria control interventions aim to reduce the transmission of parasites from the human to the mosquito host. Vector control tools such as bed nets and indoor residual spraying  lower thInterventions that reduce human-to-mosquito transmission are most effective when they target individuals within a population who contribute most to transmission. Not all individuals with blood-stage parasitemia are equally infectious to mosquitos. Only a small fraction of all parasites in the human host develop into sexual stages termed ‘gametocytes’; parasite development in the mosquito relies on uptake and subsequent mating of male and female gametocytes .P. falciparum gametocytes are sequestered in extravascular sites such as bone marrow for 1–2 weeks [P. falciparum endemic countries. As a result of the continued release of sequestered gametocytes after treatment, and their circulation for days to weeks, individuals can carry gametocytes for several weeks after treatment [P. falciparum gametocytes. Low-dose primaquine has been shown to reduce duration of gametocytemia after treatment [Developing –2 weeks , 8. Game–2 weeks . Mature –2 weeks –12. Mostreatment . Primaqureatment , 16.P. vivax gametocytes infective for mosquitoes appear within 2–3 days after blood-stage asexual parasitemia commences and might circulate for only 2–3 days [P. vivax gametocytes are sensitive to common drugs such as chloroquine or artemether-lumefantrine. In drug trials, up to 90% of patients were gametocyte-free one day after treatment, and virtually none carried gametocytes at day 7 [2–3 days , 18. Gam2–3 days –21. P. vat day 7 –22.P. vivax [In most malaria endemic regions the vast majority of infections are asymptomatic, i.e. not associated with fever. 50–80% of infections are not detectable by microscopic inspection of blood smears , 24. SinP. vivax –33. It iP. falciparum and P. vivax gametocyte carriage across a range of transmission settings, five cross-sectional surveys involving a total of 16,493 individuals were conducted in Brazil, Thailand, Papua New Guinea (PNG) and Solomon Islands [P. vivax being the predominant parasite . Blood samples were collected from the general population irrespective of symptoms of malaria illness. Total blood-stage parasites and gametocytes were quantified by LM and sensitive qPCR and RT-qPCR assays using the same methodology across all studies, allowing for the first direct comparison across transmission intensities.To advance our understanding of  Islands –31. The The study was approved by the PNG Institute of Medical Research IRB (1116/1204), the PNG Medical Research Advisory Committee , the WEHI Human Research Ethics Committee , the Case Western Reserve University Hospitals of Cleveland Medical Center , the Solomon Islands National Health Research Ethics Committee (HRC12/022), the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand , the Brazilian National Committee of Ethics (CONEP) (349.211/2013), and by the Committee of Ethics for Clinical Investigation from Barcelona Hospital Clinic (7306/2012). Prior to sample collection, the aims of the study were explained to all individuals and informed written consent was obtained from participants or, in case of minors, from their guardians.Details on study sites are given in The study sites experience little or moderate seasonality in transmission. In PNG, the rainy season is from December to April. Both surveys were conducted in May to July, i.e. after the rainy season. There is minimal seasonal variation in Solomon Islands; samples were collected in May to June. In Thailand, the peak transmission season is from April to July, and samples were collected in September and October. In Manaus, Brazil, highest incidence occurs from May to September. Half of the samples were collected in September to early January, and the other half in August to September.Form each participant, 250 μL blood was collected by finger prick into 2 mL EDTA microtainers (BD). Hemoglobin levels were determined using the HemoCue handheld meter. For RNA extraction and gametocyte detection, 50 μL blood was transferred into tubes containing 250 μL of RNAprotect (Qiagen) in the field. Samples in RNAprotect were kept on ice packs in the field and transferred to -80°C storage every evening and kept there until RNA extraction. The remaining 200 μL blood were kept in the EDTA microtainer, also kept on ice packs, and transferred to -20°C storage until DNA extraction.pfs25 and pvs25 [DNA was extracted from 200 μL whole blood kept in EDTA microtainers and eluted in 200 μL elution buffer. Parasites were quantified by qPCR using the 18S rRNA gene as target . The assnd pvs25 .pvs25 and pfs25 copy numbers, were included in the analysis. Expert microscopy was conducted in PNG, Solomon Islands, and for parts of the survey in Brazil. To determine multiplicity of infection (MOI), P. falciparum infections were genotyped by msp2 [P. vivax infections were genotyped by msp1F3 and MS2 [Procedures for sample collection, qPCR, and RT-qPCR were standardized between all sites. Results of individual cross-sectional surveys have been published previously –31, 35.  by msp2 , and P.  and MS2 .The following definitions are used: ‘proportion gametocyte-positive infections’ describes the number of gametocyte carriers divided by the number infected with asexual parasites and/or gametocytes; ‘population gametocyte prevalence’ is the prevalence of gametocytes among all individuals, infected and non-infected .10 transformed for all calculations. To correct for imperfect detection of gametocytes and include low-density infections without gametocytes detected in multivariable models, +0.1 was added to all gametocyte density values prior to log10-tranformation. For multivariable analyses, individuals were grouped into age classes ≤6, >6–12, >12–20, and >20 years. Survey and age group were included as fixed effects. The ratio of pfs25 or pvs25 transcripts per parasite genome was assessed, representing the proportion of gametocytes among all parasites. Infections  with densities at the technical limit of detection of 0.25 copies/μL blood (i.e. 1 DNA/RNA template per PCR) were excluded from correlation analysis as quantification is imprecise at very low densities, and including them at a set density of 0.25 copies/μL would artificially increase correlation. All data is available in supplementary file Multivariable regression models were used to predict factors associated with the proportion gametocyte-positive infections and gametocyte density. Parasite densities were logP. falciparum by qPCR ranged from 0.14%  to 18.5% , and P. vivax prevalence from 3.3%  to 19.7%  , with no clear trend between population parasite prevalence and mean parasite densities  were asymptomatic, i.e., not accompanied by measured or reported febrile illness in the preceding 48 hours.Prevalence of 96/2517) . Light m by qPCR . Mean paensities . In eachpfs25 RT-qPCR, the proportion of gametocyte-positive infections differed significantly between surveys .By PNG 2010 . In univpvs25 RT-qPCR, P. vivax gametocytes were detected in 22.6% and 23.5% of infected individuals in PNG 2014 and Solomon Islands, and in 48.9%, 53.7%, and 71.5% of infections in PNG 2010, Brazil, and Thailand .By P<0.001, . This repfs25 or pvs25 transcripts per P. falciparum or P. vivax genome among gametocyte-positive samples was plotted , while no significant change was observed for P. vivax . A febrile episode and/or antimalarial treatment in the preceding 2 weeks increased the proportion gametocytes significantly for P. falciparum , with 9.0-fold more gametocytes per 10-fold increase in genome density.In multivariate analysis across all surveys, parasite density was a strong predictor for the probability that a sample was gametocyte-positive . Each 10ensities . The cor density . ConcordP. falciparum positive individuals, the odds to detect gametocytes was 54% lower (P<0.0001) and gametocyte densities were 69% lower (P<0.0001) in individuals co-infected with P. vivax. Reported malaria or anti-malarial treatment in the past 2 weeks was associated with higher P. falciparum gametocyte prevalence and densities . In Thailand and Brazil, for both species, and for P. vivax in PNG 2014, no other factors than parasite density were significantly associated with gametocyte density.Factors affecting gametocyte density were assessed independently in PNG 2010 (moderate-high transmission), PNG 2014 (recently decreased transmission), and Thailand and Brazil pooled (long-time low transmission) . For PNGAs a result of the correlation of parasite and gametocyte density and the decreasing parasite densities with age in both PNG surveys, for both species the majority of individuals with detectable gametocytes were children. 48–78% of gametocyte carriers were <12 years . In contP<0.0001, Pf: n = 676, P = 0.471; Pv: n = 1501, P = 0.512). Likewise, P. falciparum and P. vivax gametocyte densities were not affected by an interaction between DNA copies and survey .When including parasite density in multivariable analysis to correct for differences in mean density among surveys, the proportion gametocyte positive infections, and gametocyte densities of both species differed significantly between surveys  of LM-positive individuals. Among LM-negative individuals, gametocytes were detected in 40.2% (170/422) of individuals. Pfs25 transcript densities were nearly 10-fold higher in LM-positive individuals . The ability of LM to detect P. falciparum gametocyte carriers differed significantly between surveys , P<0.001, , and appectively .P. vivax gametocytes were detected by RT-qPCR in 64.9% (235/362) of LM-positive individuals, but in only 16.7% (163/976) of LM-negative individuals. Mean gametocyte densities in LM-positive individuals were almost twice as high compared to LM-negative individuals . Thus, approximately 50% of P. falciparum and 59% of P. vivax gametocyte carriers were positive by light microscopy.P. falciparum and P. vivax infections carrying detectable gametocytes in 5 cross-sectional surveys representing distinct malaria-epidemiological contexts. The proportion of gametocyte-positive infections in community surveys is heavily impacted by the sensitivity of the assays used for parasite and gametocyte detection [We observed substantial differences in the proportion of etection . The usePlasmodium species (as determined by RT-qPCR) were positive by LM for asexual parasites. A lower proportion of gametocyte carriers was LM-positive in Madang 2014 (both species) and Solomon Islands (P. vivax), where transmission had declined in the years prior to the surveys [P. falciparum in Solomon Islands), expert LM or other tools such as RDT remain sufficiently sensitive to identify the majority of gametocyte carriers.Blood-stage parasite densities were a strong predictor for gametocyte positivity and could largely explain differences between surveys. In most surveys, the majority of gametocyte carriers of both  surveys , 30, andP. vivax. This contrasts findings from P. falciparum in Africa, where school-age children were proposed to contribute most to transmission densities [Pronounced age trends in gametocyte carriage were evident in PNG and Solomon Islands. Prevalence of infection peaked in children or adolescents, and parasite densities decreased rapidly with increasing age, most likely due to the acquisition of immunity. As a result, the vast majority of gametocytes were detected in children below 6 years, especially for ensities , 41. In P. vivax, a constant proportion of gametocytes among all parasites was observed irrespective of parasite density. In the case of P. falciparum, high proportions of gametocytes were observed in a subset of infections with low-to-moderate densities The 2-week sequestration of developing P. falciparum gametocytes results in a temporal lag of peak gametocytemia following peak parasitemia [P. falciparum gametocyte density reflects parasite density in the preceding 2 weeks.Apart from parasite density, limited effects of transient factors on gametocyte densities were observed. Multiple clone infection or hemoglobin levels did not affect gametocyte carriage of either species. For asitemia . Thus, iasitemia . This isasitemia . LongituP. falciparum gametocyte positive infections and lower gametocyte densities were observed in individuals co-infected with P. vivax. It is not known whether co-infection results in an adjustment of the gametocyte conversion rate, or whether multi-species infection is a surrogate marker for higher exposure, and thus, higher levels of gametocyte-specific immunity.As an exception to the limited impact of transient factors, a lower proportion P. vivax infectivity in Brazil [P. vivax, in Thailand a steep increase in infectivity was found at densities (by LM) of 10–100 parasites/uL, with little effect if densities increased further [Mosquito feeding assays have repeatedly shown a correlation between parasite density and infectivity. Few studies have included asymptomatic individuals and individuals negative by LM for gametocytes and asexual stages –50. Withn Brazil , individ further . This depfs25 and pvs25 RT-qPCR, is expected to allow for better predictions of infectivity [Studies assessing gametocyte densities and infectivity over time will be required to determine what proportion of low-density infections will rise in density and become highly infective. The importance of male gametocyte densities to predict infectivity is increasingly recognized. In low density infections, male gametocytes might be the limiting factor for onward transmission . Measuriectivity . Due to ectivity , it is uP. falciparum in Brazil and Solomon Islands compared to PNG. Recent malaria control activities have resulted in significant changes in vector composition and biting behavior in the study sites [P. falciparum populations in East Africa [gametocyte development gene 1 (gdv1), which is essential for early gametocyte development, was found to be under strong selection in P. falciparum populations in regions of different endemicity in West Africa [P. vivax [Even after correcting for different mean parasite densities between surveys, a higher proportion gametocyte positive samples were found for both species in Thailand and for dy sites , 53. A rt Africa , and thet Africa . In CambP. vivax . The preThe probability to detect gametocytes was closely correlated to blood-stage parasitemia in different transmission settings. The vast majority of all infections with high gametocyte densities (as determined by RT-qPCR) could be diagnosed by microscopy. Pronounced age trends of gametocyte carriage in areas of moderate to high transmission was observed. Even though the contribution to transmission is influenced by vector exposure and transmission blocking immunity in addition to gametocyte prevalence and density, the age trends suggest that interventions to reduce transmission will have the greatest effect when targeted towards children. In contrast, in order to achieve elimination in low transmission settings individuals of all ages need to be protected from vector contact, which is often not the case .S1 Data(TXT)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.