A combined test could be useful for both diseases and would facilitate integration of the screening for gambiense HAT (gHAT) and malaria diagnosis. This study aimed to evaluate a combined prototype rapid diagnostic test (RDT) for gHAT and malaria.Malaria is endemic in all regions where Plasmodium falciparum (P.f.) Histidine Rich Protein II (HRP-II or HRP2) antigen  for malaria detection and an individual gHAT RDT based on recombinant antigens, the SD BIOLINE HAT 2.0 RDT for HAT screening. Due to the current low prevalence of gHAT in endemic regions, the set of blood samples that were collected was used to evaluate the specificity of the RDTs for gHAT, and additional archived plasma samples were used to complete the evaluation of the HAT/Malaria Combined RDT in comparison to the HAT 2.0 RDT.Blood samples were collected in the Democratic Republic of the Congo and in Uganda to evaluate the performance of a prototype HAT/Malaria Combined RDT in comparison to an individual malaria RDT based on P.f. RDT were 97.3% (95% CI: 95.5–98.6) and 97.1% (95% CI: 94.1–98.8) respectively. For gHAT, using archived plasma samples, the sensitivity and specificity were respectively 89% (95% CI: 84.4–92.6) and 93.5% (95% CI: 89.7–96.2) with the HAT/Malaria Combined RDT, and 88.2% (95% CI: 83.5–92) and 94.7% (95% CI: 91.1–97.2) with the HAT 2.0 RDT. Using the whole blood samples that were collected during the study, the specificity of the HAT/Malaria Combined RDT for gHAT was 95.8% (95% CI: 94.3–97.0).Frozen whole blood samples from a total of 486 malaria cases and 239 non-malaria controls, as well as archived plasma samples from 246 gHAT positive and 246 gHAT negative individuals were tested. For malaria, the sensitivity and specificity of the malaria band in the HAT/Malaria Combined RDT were 96.9% (95% CI: 95.0–98.3) and 97.1% (95% CI: 94.1–98.8) respectively. The sensitivity and specificity of the SD BIOLINE malaria Ag The HAT/Malaria Combined prototype RDT was as accurate as the individual malaria or gHAT RDTs. The HAT/Malaria Combined prototype RDT is therefore suitable for both malaria diagnosis and gHAT screening. However, there is a need to assess its accuracy using fresh samples in prospective clinical trials. gambiense HAT (gHAT). The main strategy to control gHAT is screening of at-risk individuals, followed by diagnosis and treatment of confirmed cases. However, this strategy and even the passive screening as currently implemented become less efficient with declining incidence, justifying innovative strategies to efficiently detect the remaining cases. All areas where gHAT occurs are also endemic for malaria, presenting an opportunity to integrate gHAT screening activities within malaria control activities. This integration is warranted by the fact that in early disease stage, gHAT patients present with signs and symptoms strikingly similar to those of malaria. In order to use malaria diagnosis as an entry point to screen for gHAT, Standard Diagnostics (SD), Republic of Korea  made a Combined prototype RDT for both malaria and gHAT, expected to be as accurate as the individual gHAT and malaria RDTs. In this study, we evaluated the accuracy of the Combined prototype RDT using whole blood samples collected in Uganda and DRC, and archived plasma samples collected in DRC, Angola and Central African Republic. We found that the Combined prototype performs just as well as individual RDTs.The annual number of reported cases of human African trypanosomiasis (HAT), also known as sleeping sickness (SS), is currently below 1,000 cases worldwide. The Democratic Republic of the Congo (DRC), the most affected country, and Uganda, which shares a border with DRC, are both endemic for  Malaria transmission is ongoing in all regions where human African trypanosomiasis , is endemic. However, the opposite is not true –4. This Glossina. HAT is caused by two subspecies of Trypanosoma brucei (T.b.) namely T.b. gambiense and T.b. rhodesiense. Trypanosoma b. gambiense is responsible for a chronic form of the disease, while T.b. rhodesiense causes a more acute disease form. Rhodesiense HAT (rHAT) is found in eastern and southern Africa. It is a zoonotic disease that only accidentally involves human beings. On the other hand, gambiense HAT (gHAT) is endemic in central and western Africa and represents >98% of all HAT cases [HAT is a vector-borne disease transmitted in sub-Saharan Africa through the bite of several species and subspecies of tsetse flies of the genus AT cases , 7, 8.gambiense HAT (referred to as HAT or gHAT), and 13 for rhodesiense HAT (referred to as rHAT). Uganda is endemic for both forms. Among the 24 countries affected by T.b. gambiense, Angola, the Democratic Republic of the Congo (DRC), South Sudan, Chad, the Central African Republic (CAR), the Republic of Congo, Guinea and Uganda represent 98% of all reported gHAT cases [Thirty-six African countries have historically been endemic for HAT, of which 24 are endemic for AT cases .Plasmodium spp. In 2018, a total of 228 million cases of malaria were reported compared to 216 million in 2016. If left untreated, malaria may result in severe complications and lead to death. In 2018, 405,000 deaths due to the disease were reported globally compared to 445,000 in 2016. The African region continues to account for more than 90% of malaria cases and deaths worldwide. DRC is second the most widely affected country in the world, after Nigeria; it accounted for 12% of reported cases and 11% of deaths in 2018 [Malaria is a vector-borne disease caused by Patients with malaria or early stage HAT often present with similar symptoms, such as headache, flu-like symptoms, malaise, joint pains, fever and chills. Diagnostic tests are therefore needed to differentiate these diseases and ensure early HAT treatment to prevent complications, such as irreversible sequelae due to invasion of the central nervous system (CNS) by parasites , 14–17. Plasmodium falciparum (P.f.) Histidine Rich Protein II (HRP-II or HRP2) antigen (Ag) [Today, in addition to microscopy, the diagnostic test that is recommended for malaria in most sub-Saharan African countries is a rapid diagnostic test (RDT) based on gen (Ag) . For HATgen (Ag) . To addrgen (Ag) –25. Thesgen (Ag) , 24.P.f. RDT. This combined test would be targeted for use in the diagnosis of malaria, with a comparative advantage that screening for HAT is done simultaneously. This would enable detection and treatment of residual cases of HAT during malaria testing, and thus contribute to efforts to prevent re-emergence of HAT and sustain its elimination.Since areas that are endemic for HAT are also endemic for malaria, both HAT and malaria RDTs could be used on the same patients. Therefore, with support from the Foundation for Innovative New Diagnostics , SD developed a prototype test that combines HAT screening and malaria diagnosis in the same cassette. Screening for HAT is based on antibody detection, and seropositive suspects have to undergo confirmatory testing by microscopy before treatment can be initiated. In contrast, malaria RDTs are based on antigen detection, and a positive result is usually sufficient for a decision to start treatment, although this depends on the national guidelines. A prototype SD BIOLINE HAT/Malaria test was made by combining the SD BIOLINE HAT 2.0 RDT  and the P.f. RDT).We report here the accuracy of the SD BIOLINE HAT/Malaria Combined prototype RDT (referred to as Combo RDT) in comparison with HAT and Malaria RDTs in individual formats .The project was carried out in conformity with the Declaration of Helsinki. The sites were appropriate health facilities with staff trained to enrol participants and collect blood samples under good clinical and laboratory practices (GCLP) conditions. The study protocol was approved by the Ethics Committee in DRC  and in Uganda . Before inclusion, written informed consent was obtained from participants or legal guardians of minors.To evaluate the accuracy of malaria diagnosis, samples of whole blood were collected from 7 study sites in the DRC and in Uganda, including 3 sites located in HAT endemic regions and 4 in regions where HAT is not endemic . The 3 sIn view of the current low prevalence of HAT in endemic regions, evaluation of the accuracy of the prototype RDT for HAT screening was done on plasma samples that had been obtained from HAT endemic countries  as part of earlier research projects, and archived at Makerere University . The whole blood samples that were collected during the current study were used to evaluate the specificity of the prototype RDT for HAT.Patients presenting themselves at health facilities with symptoms suggestive of malaria or HAT , aged at least 6 months (in DRC) or at least 6 years (in Uganda), and who provided informed consent, were enrolled for sample collection and characterization. Patients with severe anaemia from whom blood collection was not possible, or whose condition was such that they could not give informed consent were excluded from the study.Microscopy is currently the common test used as gold standard diagnostic for malaria and is affordable at the level of health facilities. Its shortcomings include the requirement for well-trained and experienced microscopists, rigorous maintenance of equipment, good quality supplies, and that patients with low parasite densities are often missed. Polymerase Chain Reaction (PCR) is more accurate and can detect sub-microscopic malaria infection, but it requires complex equipment, is time consuming and costly , 27. Thuet al (2012) [For microscopy, a thick and a thin blood smear were prepared from each participant. The thin smear was fixed with methanol, and all the blood smears stained with 10% Giemsa for 10 minutes. Thick blood smears were examined by microscopy for the presence of asexual parasites and gametocytes. Parasite densities were calculated as described by Adu-Gyasi l (2012)  by countPlasmodium spp., and a secondary PCR test performed on the product of the primary PCR, using primers specific for P. falciparum. The secondary PCR product was visualized on an agarose gel and a qualitative result indicating the presence or absence of a band of the expected size recorded.Nested PCR was used to detect malaria parasites and to identify the species. Amplification of the 18S rRNA gene was performed using methods and primers described previously –31. It iFor HAT, stored plasma samples collected in Angola and Central African Republic (CAR) had been pre-selected by CATT, and the ones collected in DRC had been pre-selected by CATT, the SD BIOLINE HAT RDT and/or the SD BIOLINE HAT 2.0 RDT (referred to as HAT RDT and HAT 2.0 RDT respectively) –24. ConfThe whole blood samples that were collected during the current study were preselected using the HAT RDT and HAT 2.0 RDT as screening tests in the DRC, and using the HAT RDT in Uganda. Confirmatory diagnosis of HAT was based on the CRS described above. HAT case and non-HAT control participants were considered as described above.No formal sample size calculations were performed for HAT or malaria assessments.The target was to collect fresh whole blood samples from 500 malaria case and 500 non-malaria control participants (no target numbers were set for HAT cases or non-HAT control participants). In addition, archived plasma samples from 250 HAT cases and 250 non-HAT controls were used. This was expected to provide information that would be strong enough to evaluate the prototype combo RDT, and inform decisions to start prospective performance evaluation studies in the field.Whole blood samples collected from malaria cases and non-malaria control participants were used to assess the malaria accuracy and HAT specificity of RDTs, while archived plasma samples were used to assess HAT accuracy.P.f. test , the SD BIOLINE HAT 2.0 RDT (HAT 2.0 RDT), and the prototype SD BIOLINE HAT/Malaria Combined test (Combo RDT) are all cassette-format RDTs developed by SD. They include one control band (C band) and one or two test bands for HAT and/or malaria.The SD BIOLINE Malaria Ag P.f. RDT includes one test band for the qualitative detection of the P. falciparum HRP-II antigen in human whole blood.The SD Malaria The HAT 2.0 RDT includes 2 test bands to detect antibodies against two trypanosome antigens (recombinant ISG65 and recombinant VSG LiTat 1.5) .P. falciparum HRP-II antigen  and the other to detect antibodies against two trypanosome antigens (recombinant ISG65 and recombinant VSG LiTat 1.5) (“HAT” band).The Combo RDT includes two test bands: one to detect the Plasmodium falciparum histidine rich protein (HRP-II), accession P05227.1 (UniProtKB/Swiss-Prot); T. brucei ISG65, accession XP_951587 (GenPept); and T. brucei VSG LiTat1.5, accession ABX55936 (GenPept).P.f./Pan or Ag P.f. RDT, microscopy procedures for malaria . From the second portion of blood, 4 aliquots of 500μl each were prepared and frozen in liquid nitrogen and shipped to Makerere University for malaria PCR analysis, and testing with both the SD Malaria P.f. RDT and the prototype Combo RDT.To evaluate the accuracy of the RDTs, 4 ml of venous blood were collected by trained staff of the health facility, into tubes containing heparin as anticoagulant. The blood samples were subdivided into two portions and blinded by an independent technician prior to testing. One portion of each sample was used onsite to perform the following tests and procedures: SD BIOLINE Malaria Ag Malaria RDT results obtained on site were used to manage malaria according to the country’s guidelines.The collected portions of blood described above were also used for HAT assessments as follows:one portion was tested onsite (2 ml) with HAT RDT and/or HAT 2.0 RDT and subjected to HAT parasitology testing (mHCT and mAECT).the aliquots shipped to Makerere University were also tested with the prototype Combo RDT (HAT band).The RDT results obtained onsite were used to guide confirmatory testing by microscopy, according to the HAT diagnostic algorithm.Blood samples shipped frozen on dry ice to Makerere University were thawed at room temperature and mixed to ensure homogeneity. All samples were labelled with blinding codes.P.f. RDT and to the Combo RDT , according to the manufacturer’s instructions. The procedure for performing the Combo RDT is the same as that for the SD Malaria P.f. RDT, except that the former includes two test lines .To evaluate the accuracy of malaria diagnosis, 5 μl of whole blood was applied to the SD Malaria To evaluate the accuracy of HAT diagnosis, using archived plasma samples, an aliquot of 15 μl of plasma was diluted by adding 15 μl of human blood, freshly collected from a volunteer. This was done in order to ensure that the volume to pipette for RDT testing would not be too small. The sample of whole blood from the volunteer was first checked with each of the 3 RDTs being evaluated, to ensure that it did not give any positive HAT or malaria result. Volumes of 5 μl and 20 μl were then transferred from this 30 μl aliquot and applied to the Combo RDT (HAT band) and to the HAT 2.0 RDT, respectively.Frozen blood samples were tested with the combo RDT as described above, and the result of HAT band recorded.Participant information and test results at the sample collection sites were recorded on paper case report forms (CRF). The CRFs were transferred to “Programme National de Lutte contre la Trypanosomiase Humaine Africaine” (PNLTHA) in Kinshasa (DRC) or to Makerere University (Uganda) for double data entry using a web-based clinical data management platform (Open Clinica). Data were analysed with Stata SE 15.1 software.st reader RDT results, and we assessed the inter-agreement between two independent RDT readers using Cohen’s kappa factor.Sensitivity and specificity were calculated for each test as the percentage of positive tests among cases and the percentage of negative tests among controls respectively. Accuracy was assessed by calculating Youden’s index . ConcordPositive and negative predictive values (PPV and NPV) were estimated based on the sensitivity and specificity using HAT prevalence values of 0.01%, 0.1%, 1% and 2%. The HAT prevalence in the population through active screening is currently less than 1%. However, in passive screening this proportion could be up to 1% or 2%, especially among patients that would be preselected based on signs and symptoms indicative of HAT. False discovery rate (FDR) and false omission rate (FOR), easy translation of PPV and NPV by a surveillance programme, could be estimated from PPV and NPV as described below. Based on the DRC National Malaria Control Program  annual report for 2016, about 70% of malaria suspects tested positive with malaria RDTs among people attending health facilities. We evaluated and reported the PPV and NPV considering a pre-test probability in health facility setting of 70%.PPV = sensitivity x prevalence /  + (1 –specificity) x NPV = ) / ((1 –sensitivity) x prevalence) + )FDR = 1 –PPVFOR = 1 –NPVAmong 725 participants enrolled, 486 were malaria cases and 239 non-malaria controls. Of the total participants, 459  were enrolled in HAT endemic regions, while 266  were from non-HAT regions. The median age was 24 years with an interquartile range (IQR) of 17 years (18–35 years old). The proportion of females was 61.9% (95% CI: 58.3–65.4).P.f. RDT, of which 473 were positive with the reference test (RT), 478 positive with the Combo RDT malaria band (471 positive to RT), 245 negative with the individual SD malaria P.f. RDT (232 negative to RT), and 247 negative with the Combo RDT malaria band (232 negative to RT) as shown in Among the 725 participants enrolled, the number that tested positive was respectively 480 for individual SD malaria P.f. RDT, 335 were enrolled from HAT endemic regions (333 positive to RT) and 145 (140 positive to RT) from non-HAT regions. Among the 478 participants positive to Combo RDT malaria band, 335 were enrolled from HAT endemic regions (333 positive to RT) and 143 (138 positive to RT) from non-HAT regions.Among the 480 participants who were positive to the individual malaria The number of participants and RDT results related to malaria assessment are detailed in supplemental P.f. RDT were 97.3% (95% CI: 95.5–98.6) and 97.1% (95% CI: 94.1–98.8) respectively. For the malaria band of the Combo RDT, the sensitivity was 96.9% (95% CI: 95.0–98.3) and the specificity was 97.1% (95% CI: 94.1–98.8).The sensitivity and specificity of the RDTs at each site are shown in P.f. RDT was of 97.9% (95% CI: 95.8–99.2) in HAT endemic regions, while it was 95.9% (95% CI: 91.3–98.5) in non-HAT endemic regions. The sensitivity of the Combo RDT malaria band was 97.9% (95% CI: 95.8–99.2) in HAT endemic regions and 94.5% (95% CI: 89.5–97.6) in non-HAT endemic regions. The specificity of the SD Malaria P.f. RDT was 98.3% (95% CI: 94.1–99.8) and 95.8% (95% CI: 90.5–98.6) in HAT endemic and non-HAT endemic regions respectively, while the specificity of the Combo RDT malaria band was 98.3% (95% CI: 94.1–99.8) and 95.8% (95% CI: 90.5–98.6), respectively.The sensitivity of the SD Malaria P.f. RDT and the Combo RDT malaria band was 99.4%. Inter-reader agreement for SD Malaria P.f. RDT and for Combo RDT malaria band was 100.0% and 98.5% respectively.Concordance between the SD malaria P.f. RDT and 0.940 (95% CI: 0.925–0.953) for the Combo RDT malaria band.Youden’s index for accuracy was 0.944 (95% CI: 0.929–0.957) for the individual SD Malaria P.f. RDT and Combo RDT malaria band. The negative predictive values (NPV) were 94% (95% CI: 90.1–96.4) and 93.1% (95% CI: 89.1–95.7) respectively for the SD individual malaria P.f. RDT and the Combo RDT malaria band.Considering the prevalence of malaria among febrile patients (prior test probability among febrile outpatients) in DRC (70% in 2016), the RDTs’ positive predictive values (PPV) were 98.7% (95% CI: 97.4–99.4) for both individual SD malaria The number of participants and RDT results per site related to HAT assessment are detailed in The frozen blood samples shipped to Makerere for testing were collected from 981 participants successfully enrolled for HAT assessment. Samples were tested with the Combo RDT (HAT band) and not with the HAT 2.0 RDT. Among the 981 participants, 7 were HAT cases and 974 non-HAT controls, 935 tested negative to the Combo RDT HAT band (933 non-HAT controls) and 46 tested positive (41 non-HAT controls) as shown in Among the 935 participants that were negative with the Combo RDT HAT band, 560 were enrolled from HAT endemic regions (558 non-HAT controls) and 375 (375 non-HAT controls) from non-HAT regions. Among the 46 participants that were positive with the Combo RDT HAT band, 39 were enrolled from HAT endemic regions (34 non-HAT controls) and 7 (7 non-HAT controls) from non-HAT regions.Using archived plasma samples, the sensitivity was 88.2% (95% CI: 83.5–92) and 89.0% (95% CI: 84.4–92.6) for the HAT 2.0 RDT and prototype Combo RDT HAT band respectively. The specificity was 94.7% (95% CI: 91.1–97.2) and 93.5 (95% CI: 89.7–96.2) for the HAT 2.0 RDT and prototype Combo RDT HAT band, respectively.Concordance between the HAT 2.0 RDT and the prototype Combo RDT HAT band was 91.4%.The inter-reader agreement was 93.9% and 94.7% for the HAT 2.0 RDT and the prototype Combo RDT HAT band, respectively.Using frozen blood samples, the overall specificity of the Combo RDT HAT band was 95.8% (95% CI: 94.3–97.0). Its specificity per site is detailed in Based on archived plasma samples, Youden’s index for accuracy was 0.829 (95% CI: 0.776–0.874) for the HAT 2.0 RDT and 0.825 (95% CI: 0.772–0.871) for the Combo RDT HAT band.The PPV and NPV at 0.01%, 0.1%, 1% and 2% HAT prevalence are detailed in P.f. RDT and HAT 2.0 RDT) in the detection of malaria and screening for HAT.The present study demonstrated that the HAT/Malaria Combined RDT prototype was as sensitive and specific as the individual SD BIOLINE malaria and HAT RDTs . On the other hand, 6 patients suffering from malaria out of one thousand people would be tested negative to the individual SD malaria P.f. RDT and 7 malaria patients out of one thousand people would be tested negative to the Combo RDT malaria band at the same prevalence.We obtained a PPV of the evaluated SD malaria s higher , 34. Theet al (2018) reported an overall HAT 2.0 prototype RDT sensitivity and specificity in both active and passive screening settings of 71.2% (95% CI: 65.7–76.6) and 98.1% (95% CI: 98.0–98.2) respectively. The HAT 2.0 prototype RDT sensitivity and specificity during passive screening, in health facility settings was 90.1% (95% CI: 84.7–95.3) and 93.7% (95% CI: 93.2–94.2) respectively, and 54.8% (95% CI: 46.8–63.5) and 99.1% (95% CI: 99.0–99.2) respectively during active screening by mobile teams [With regard to HAT screening, Lumbala le teams .et al [The sensitivity of the HAT 2.0 RDT in the present study was higher than that reported by Lumbala et al , particuet al (2018), especially in active screening setting (as well as when combining active and passive screening settings). The fact that a number of samples that were used in the current study were collected in CAR without using mAECT, the most sensitive parasitological test for HAT, could have resulted in some HAT cases being missed and wrongly considered as negative in the present study, thus resulting in an underestimate of the RDT specificity.The specificity of the HAT 2.0 RDT was significantly lower in the current study than what was reported by Lumbala Due to the relatively low specificity, the false positive rate tends to be high, especially now that HAT prevalence has decreased significantly, especially in active screening settings. With 0.1% HAT prevalence, at least 98 people out of 100 screened positive to HAT 2.0 RDT or Combo RDT HAT band would be false positive. At 1% HAT prevalence, only 14 persons out of 100 screened positive to HAT 2.0 RDT and 12 persons out of 100 screened positive to Combo RDT HAT band would be confirmed as HAT cases. Currently, a HAT prevalence of 1% can only be achieved in passive screening, and rarely in active screening. On the other hand, for every 1,000 negative screening results, 1 and 2 would be false negatives (missed cases) with Combo RDT HAT band, compared to 1 and 3 false negatives with HAT 2.0 RDT respectively at 1% and 2% HAT prevalence. As the PPV is declining rapidly with current prevalence and the progress towards HAT elimination, this will result in an increase in workload to confirm suspects, with a negative impact on health workers’ and patients’ motivation and confidence in positive test results, which would end up in missing true HAT cases. The combo RDT is intended to be used primarily for malaria testing and particularly by malaria programs in HAT endemic settings. But based on the PPV at the current HAT prevalence, it seems that while integration of HAT screening in the malaria control program is theoretically meant to increase the number of HAT tests done and therefore increase the proportion of HAT cases detected, the low PPV might quickly have a negative impact on use of the combo RDT. Indeed, introduction of the combo RDT may lead to HAT testing in sites where the HAT prevalence is even lower than 0.1% . The steady decline in HAT prevalence is leading to a decrease in the positive predictive value, while all persons identified as HAT serological suspects need parasitological confirmation by dedicated personnel. In the context of being confronted with a vast majority of false positives, logistic and financial limitations to perform parasitological HAT confirmation, there is a risk that malaria program health personnel will quickly reject the Combo RDT and return to a single malaria test. This underlines the potential shortcoming of the Combo RDT. To prevent this, it will be necessary to improve the PPV of HAT screening RDTs by for instance increasing the pre-test probability by restraining the use of the Combo RDTs to patients with clinical signs that are more specific of HAT, or using an algorithm that includes more specific molecular or serological tests. As test line positivity for malaria (antigen detection) and HAT (antibody detection) may have a different meaning, in particular for treatment decision, training, follow-up and supervision will be very important prior to introduction of the combo RDT in health facilities and / or its integration in malaria programs.The current study had two main limitations that prevent further analysis and interpretation of results. First, information related to possible malaria treatment up to 42 days prior to blood collection was not available. False positivity following a successful malaria treatment would result in an underestimated RDT specificity. The fact that this information was not collected limits comparison to other study findings, especially in regard of malaria RDT specificity.Second, due to the continued decline in HAT prevalence, it was not possible to evaluate RDT sensitivity using freshly collected blood, but only using archived plasma. The archived plasma samples had been pre-selected using various screening tests, and some of the samples were rather old (2008) albeit well stored in -80°C freezers or in liquid nitrogen. This could have had an impact on our results and would therefore make comparisons with other studies difficult. However, the fact that the current study aimed to compare the performance of RDTs using the same samples and in the same testing conditions still makes our findings relevant.Finally, the fresh blood samples that were collected and shipped frozen to Makerere University were not tested with the HAT 2.0 RDT. It was therefore not possible to compare the HAT specificity of RDTs.This study has demonstrated equivalence in sensitivity and specificity of a prototype HAT/Malaria Combined RDT for both HAT and malaria, in comparison to the individual HAT and malaria RDTs.gambiense HAT endemic areas. A similar strategy would be a good solution to the increasing problem of under-detection of rhodesiense HAT (rHAT), but a rapid diagnostic test for rHAT has so far not been developed. However, this evaluation was performed in an academic laboratory setting which differs from field conditions. Also, testing was done on frozen and/or archived samples, which may have slightly different results to those that would be obtained using fresh blood samples. Thus, there is need to conduct prospective clinical trials to confirm the performance of the HAT/Malaria Combined test under real field conditions and assess the accuracy in the local laboratory at the level of health facilities.The HAT/Malaria Combined RDT could be used to enable an integrated approach combining malaria diagnosis and HAT screening in S1 Checklist(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Data(CSV)Click here for additional data file.S2 Data(CSV)Click here for additional data file.


Plasmodium falciparum infection and the development of placental malaria.Placental malaria is associated with increased risk of adverse perinatal outcomes. While primigravidity has been reported as a risk factor for placental malaria, little is known regarding the relationship between gravidity, symptomatology and timing of The aim of this study was to investigate the relationship between the development of placental malaria and gravidity, timing of infection, and presence of symptoms. This is a secondary analysis of data from a double-blind randomized control trial of intermittent preventive therapy during pregnancy in Uganda. Women were enrolled from 12 to 20 weeks gestation and followed through delivery. Exposure to malaria parasites was defined as symptomatic (fever with positive blood smear) or asymptomatic (based on molecular detection of parasitaemia done routinely every 4 weeks). The primary outcome was placental malaria diagnosed by histopathology, placental blood smear, and/or placental blood loop-mediated isothermal amplification. Multivariate analyses were performed using logistic regression models. Subgroup analysis was performed based on the presence of symptomatic malaria, gravidity, and timing of infection.Of the 228 patients with documented maternal infection with malaria parasites during pregnancy, 101 (44.3%) had placental malaria. Primigravidity was strongly associated with placental malaria , and each episode of malaria was associated with over a twofold increase in placental malaria . Among multigravid women, the odds of placental malaria increased by 14% with each advancing week of gestation at first documented infection . When stratified by the presence of symptoms, primigravidity was only associated with placental malaria in asymptomatic women, who had a 12-fold increase in the odds of placental malaria .P. falciparum infections in pregnancy is a significant predictor of placental malaria. The importance of timing of infection on the development of placental malaria varies based on gravidity. In primigravidas, earlier asymptomatic infections were more frequently identified in those with placental malaria, whereas in multigravidas, parasitaemias detected later in gestation were associated with placental malaria. Earlier initiation of an effective intermittent preventive therapy may help to prevent placental malaria and improve birth outcomes, particularly in primigravid women.Total number of  Plasmodium falciparum is the ability of infected erythrocytes to sequester in the placenta, leading to inflammation and adverse birth outcomes [Infection with malaria parasites during pregnancy is associated with increased risk of maternal and neonatal morbidity and mortality. In sub-Saharan Africa, where an estimated 11 million pregnancies are affected by malaria infection , 3. A unoutcomes . Placentoutcomes –7.P. falciparum. A better understanding of how these factors influence placental infection in primigravid versus multigravid women may allow for more targeted public health interventions to reduce the prevalence of placental malaria and improve obstetric outcomes in both groups. Therefore, the main objective of this study was to assess whether the impact of gravidity on placental malaria varies by the presence of symptoms and/or timing of infection.Previous studies have shown that primigravidity is a risk factor for placental malaria and resulting obstetric morbidity , 9. A prThis was a secondary analysis of data collected from a double-blind, randomized trial of intermittent preventive therapy during pregnancy in Tororo, Uganda, a rural district in southeastern Uganda with an estimated entomologic inoculation rate of 310 infectious bites per person-year . ParticiAt study enrollment, participants were assessed with standardized history and physical exam including confirmation of gestational age (GA) by ultrasound. Routine visits were scheduled every 4 weeks and included collection of dried blood spots for loop-mediated isothermal amplification (LAMP). Study participant were encouraged to come the study clinic any time they were ill and if they presented with a documented fever (tympanic temperature ≥ 38.0 °C) or a reported history of fever within 24 h prior to presentation, peripheral blood was collected for a thick blood smear. If the thick smear was positive, the participant was diagnosed with symptomatic malaria and treated with artemether-lumefantrine. Because LAMP results were not available until after completion of the trial, women with asymptomatic parasitaemia did not receive treatment. For the purposes of this analysis, women who presented with fever, but later had a positive LAMP were considered to have asymptomatic parasitaemia.Plasmodium falciparum infection was defined as both symptomatic malaria (fever and positive thick smear) and/or asymptomatic parasitaemia . The primary outcome, placental malaria, was defined as any evidence of placental infection, including the presence of parasites or pigment by histopathology, parasites detected in placental blood by microscopy, or positive placental blood LAMP.Placental tissue for histopathology, placental blood for thick smears and LAMP were collected within 1 h of delivery. Women who delivered at home were visited by study staff as soon as possible after delivery for evaluation and collection of specimens. Women with evidence of any infection with malaria parasites during pregnancy, known birth outcomes, and complete placental analysis  were included in this study. P. falciparum DNA using LAMP at enrollment and every 4 weeks through pregnancy, as previously described [Dried blood spot samples were tested for escribed , 13. Plaescribed . Any disDemographic data including maternal age, gravidity, body mass index (BMI), GA at enrollment and delivery, possession of bed net at enrollment, wealth status, and intermittent preventive therapy treatment arm were collected. Wealth status was defined as lowest, middle, and highest tertiles at study enrollment based on a composite variable of land ownership and possession of several household items . GA was Birth outcomes including GA at delivery, birthweight, and fetal and neonatal survival to hospital discharge were collected and reported in previous analysis of this data set . PretermData analyses were performed using Stata 14 . Wilcoxon Rank Sum was used to compare median values of continuous variables, all of which were non-normally distributed. Chi squared and Fisher exact tests were used to compare proportions. Multivariate binary logistic regression using placental malaria as the main outcome were performed and were adjusted for primigravidity, GA at study enrollment, and GA at the time of the first recorded infection in pregnancy. Variables were included in the multivariate model if they were significant in the univariate analysis. Multivariate analyses stratified by gravidity, symptomatology and GA at initial infection were performed to examine interactions between possible predictors. The decision to do stratified analyses was made based on the a priori hypothesis that the relationship between these predictors could not be fully explained by multivariate analysis alone. All p values were two-sided and a value of p < 0.05 was considered statistically significant.Of the 300 HIV-negative women enrolled in the parent trial, 289 (96.3%) were followed to delivery. Of these, nine 3.1%) were excluded for incomplete analyses for placental malaria including seven who did not have placental biopsies taken for histopathology and an additional two with missing results for placental blood analyses. Five women with placental malaria did not have documented evidence of parasitaemia prior to delivery and, given the inability to determine timing or number of infections, they were excluded from the analysis. Of the remaining 275 women, 228 (82.9%) had evidence of infection with malaria parasites during pregnancy and were included in the analyses. Of these, 127/228 (55.7%) did not have evidence of placental malaria and 101/228 (44.3%) had placental malaria , but this association was not seen in multigravidas. In multigravid women, each additional week of gestation at initial detected infection was associated with a 1.12-fold increase in odds of placental malaria . This trend was not seen in primigravidas. Total number of times infection was detected in pregnancy remained strongly associated with placental malaria in both primigravid and multigravid women , while there was no statistically significant association between primigravidity and placental malaria among those with any symptomatic malaria. In both groups, total number of infections in pregnancy remained highly associated with placental malaria , 14–19.9 weeks (enrollment in second trimester), and ≥ 20 weeks gestation (infection after study enrollment). Primigravidity was strongly associated with placental malaria in women whose initial documented infection occurred prior to 14 weeks , as well as in women whose first infection occurred between 14 and 19.9 weeks . Primigravidity was not a significant predictor of placental malaria in women whose initial infection occurred at or beyond 20 weeks gestation. Total number of times infection was detected in pregnancy, however, was strongly associated with placental malaria in women whose first infection occurred at or beyond 20 weeks  as well as in women whose first infection occurred between 14 and 20 weeks gestation   increased odds of placental malaria in primigravid women but not in multigravid women.When stratified by maternal gravidity, there were differences in the impact of timing of initial This is the first study to explore the relationships between gravidity, symptomatology and timing of infection and the development of placental malaria. Prior literature on how the presence of malaria symptoms during pregnancy affects development of placental malaria is sparse. However, one cross sectional study of nulliparous Nigerian women found that more than 60% of asymptomatic women had evidence of placental parasitaemia at delivery, demonstrating that even asymptomatic infection has the potential to significantly impact obstetric outcomes . This stPrevious studies exploring the impact of timing of malaria infection during pregnancy have been mixed, and few prior studies have examined the relationship between timing of infection in pregnancy and development of placental malaria despite the well-established association of placental malaria with increased adverse obstetric outcomes , 19, 20.Additionally, several studies found a negative impact on fetal growth with both early and late infection –31. MalaP. falciparum infections during pregnancy are likely to benefit all pregnant women.Taken together, the results of this study have a number of implications for public health strategies going forward. Interventions aimed at preventing, identifying, and treating asymptomatic infections are likely needed to decrease placental malaria in primigravid women, whereas the magnitude of benefit in multigravid women may be less. Particular attention should be placed on preventing and treating early infections, both symptomatic malaria episodes as well as asymptomatic parasitaemia, in primigravid women. A challenge will be engaging women early in prenatal care. Finally, interventions aimed at reducing the overall number of It is important to note that the LAMP results were not known until after delivery, and so did not impact clinical care. Future studies investigating the impact of treating based on prenatal surveillance for asymptomatic infections may improve outcomes, particularly in primigravid women who are of highest risk for adverse outcomes. Also, the effect of parasite density during pregnancy cannot be evaluated with LAMP, which is reported as positive or negative detection of parasite nucleic acid in the sample. These are important areas of future study.There were many important strengths of this study including the longitudinal, prospective design, sonographically confirmed pregnancy dating, and rigorous screening protocol for both symptomatic and asymptomatic malaria infection throughout pregnancy. Additionally, examination of the placentas for evidence of malaria was undertaken by three different methods: placental blood smear, placental blood LAMP, and histopathology. Although the aforementioned strategy for placental analyses allowed for detection of microscopic as well as submicroscopic placental infection, histopathology was the most comprehensive method for diagnosis, as it also assessed for past placental malaria. Placental blood LAMP detected one additional case, but the overall benefit is unknown at this time.There are several limitations of the study to acknowledge. Firstly, patients were enrolled between 12 and 20 weeks gestation, so details of malaria exposure in the first trimester are not known. The high proportion of patients with positive LAMP at enrollment, however, suggests widespread early exposure. Additionally, although monthly LAMP screening was performed throughout pregnancy, some malaria infections—particularly asymptomatic infections—that occurred between screening points may not have been detected. Our ability to distinguish persistent from recurrent infections as well as more definitively ascertain the timing of placental infection was also limited as parasite genotyping studies were not performed. Moreover, other obstetric comorbidities that may affect the rate of preterm birth and SGA, such as preeclampsia, bleeding or placental abruption, tobacco use, and infection were not consistently recorded.P. falciparum infections in pregnancy is a significant predictor of placental malaria regardless of gravidity, symptomatology, or timing of P. falciparum infection in pregnancy. The influence of gravidity on development of placental malaria depends on symptomatology and timing of infection. Earlier initiation of an effective intermittent preventive therapy treatment may be needed to prevent placental malaria and improve birth outcomes, particularly in primigravid women.Total number of Additional file 1: Table S1. Multivariate analyses presented without controlling for total number of times parasitemia detected in pregnancy.


Plasmodium. The most common Plasmodium species that infect humans are Plasmodium falciparum and Plasmodium vivax, which together cause the vast amount of the disease’s morbidity and mortality worldwide [Plasmodium causes a spectrum of disease ranging from asymptomatic to severe. From a biochemical perspective, malaria involves an interconnection of host and parasite through a shared resource environment, resulting in the exchange of nutrients and signaling molecules. Within the bloodstream of Plasmodium-infected hosts, perturbations in the levels of various metabolites occur, including amino acids, lipids, fatty acids, sugars, and heme metabolites [Malaria is a devastating disease caused by the protozoan parasite orldwide . From a abolites .Metabolomics is a robust tool to study host–pathogen interactions. In-depth analysis of metabolism and the associated by-products and pathways can be viewed in snapshots of time, and the biochemical fingerprints contribute greatly to our understanding of the complex interaction between hosts and pathogen . MetabolPlasmodium from a host–parasite interaction viewpoint [Within the past several years, numerous published works have emerged that employ metabolomics methodology toward the goal of better understanding malaria infection. These metabolome studies have both confirmed previous biological findings that were determined through careful molecular and cellular experimental work as well as shed light on new findings for which the biological underpinnings are still unclear. Prior reviews have provided an overview of the metabolism of iewpoint ,7 as weliewpoint . Here, tPlasmodium-infected hosts, and a number of studies have characterized these perturbations [Plasmodium arginase activity [A significant depletion of amino acids occurs in the bloodstream of rbations –22. Of trbations . While dactivity ), experiactivity ,18,21  cycle [P. falciparum [For ATP production, both A) cycle ,26,27, aA) cycle ,21,22,28lciparum  and withlciparum . GlutamiAlso implicated in severe malaria is the enzymatic conversion of tryptophan to kynurenine, which is elevated during severe malaria, resulting in the production of neurotoxic metabolites , which are thought to play a role in CM ,20 Fig . A decre2 (PLA2) is a host hydrolytic enzyme that acts on phospholipids to release lysophospholipids and free fatty acids. In humans with falciparum malaria, PLA2 activity has been associated with an enrichment of a particular downstream product, arachidonic acid (AA), which modulates inflammation [2 products, suggesting that this pathway may play a key role in the pathogenesis of CM [Acute falciparum and vivax malaria infections in humans coincide with a reduction in monounsaturated fatty acid–containing phospholipids, a reduction of lysophosphotidylcholines (LPCs), and an elevation in fatty acyl carnitines ,10,32. Tammation . Prior mis of CM .Plasmodium gametocyte stages, which are required for malaria transmission [LPCs are reduced in humans with acute malaria  9,11,32,3211,32,smission ,34. ThisMalaria is associated with a vast loss of RBCs due, in part, to parasite-mediated lysis, with hemoglobin and free heme being released in the process. Heme containing iron induces oxidative stress on RBCs  and likeP. falciparum malaria, including ethylbenzene, which has been shown to be a mosquito attractant [Although mild or asymptomatic malaria is generally tolerable compared to its severe form, low-level chronic parasite carriage is still associated with changes in host metabolism. Studies of low parasitemia in human falciparum malaria and its nonhuman primate model include altered energy metabolism pathways and reductions in multiple lipids, including sphingomyelins . Sphingotractant ,38.2 [Some alterations in host bloodstream metabolite levels during malaria are also common among hosts with other diseases. For instance, decreased arginine levels has received much attention in malaria, but this amino acid is also markedly decreased in hemolytic anemia and sepsis, pointing to similar host responses among these conditions . Additio2 , and LPC2  have alsPlasmodium derives most amino acids from hemoglobin degradation. In the process, Plasmodium produces various by-products including pipecolic acid, a catabolite of lysine , which unfortunately has failed to detect hrp2/3 gene deletion strains of Plasmodium, which may be rising in prevalence [Plasmodium infection markers  for malaria includes detection of histidine-rich protein 2 and 3  and dislic acid , glutamilic acid , and citlic acid ) could hPlasmodium. Some of these metabolic signatures overlap with other acute infectious and inflammatory responses, such as sepsis, which is characterized by catabolic distress involving a breakdown of carbohydrates, lipids, and protein stores. Overall, a notable dysregulation of amino acids and lipids occurs during Plasmodium infection, likely resulting from catabolic and anabolic processes for immune cells and parasites alike. Although there have been significant metabolic findings to elucidate host and pathogen interactions during malaria as highlighted in this review, there are still many areas to investigate to further understand metabolic roles during infection.Dynamic perturbations in host metabolites occur in individuals infected with


Plasmodium. These parasites infect not only humans but also other vertebrates, from reptiles and birds to mammals. To date, over 200 species of Plasmodium have been formally described, and each species infects a certain range of hosts. Plasmodium species that naturally infect humans and cause malaria in large areas of the world are limited to five—P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. The first four are specific for humans, while P. knowlesi is naturally maintained in macaque monkeys and causes zoonotic malaria widely in South East Asia. Transmission of Plasmodium species between vertebrate hosts depends on an insect vector, which is usually the mosquito. The vector is not just a carrier but the definitive host, where sexual reproduction of Plasmodium species occurs, and the parasite’s development in the insect is essential for transmission to the next vertebrate host. The range of insect species that can support the critical development of Plasmodium depends on the individual parasite species, but all five Plasmodium species causing malaria in humans are transmitted exclusively by anopheline mosquitoes. Plasmodium species have remarkable genetic flexibility which lets them adapt to alterations in the environment, giving them the potential to quickly develop resistance to therapeutics such as antimalarials and to change host specificity. In this article, selected topics involving the Plasmodium species that cause malaria in humans are reviewed.Malaria is one of the most devastating infectious diseases of humans. It is problematic clinically and economically as it prevails in poorer countries and regions, strongly hindering socioeconomic development. The causative agents of malaria are unicellular protozoan parasites belonging to the genus  Plasmodium. The strong negative pressure of the disease has likely forced the evolution of human populations in malaria endemic regions and the selection of some unique genetic variants. For example, thalassemia and sickle-cell disease, each of which is a genetic disorder affecting red blood cells, are commonly found in malaria endemic areas [Plasmodium species, P. vivax [P. vivax malaria is rare in these areas whereas P. falciparum malaria is prevalent [Malaria has been recognised as a serious health problem since the earliest historical times. This disease is caused by protozoan parasites belonging to the genus ic areas , and peoic areas . This coP. vivax , 4. The P. vivax , and todrevalent .P. falciparum that causes the most serious infection, remains high in economically poor countries, especially in Africa [Even in the modern world with effective antimalarials and insecticide-treated bed nets (ITNs), people in many countries remain at risk, and the number of malaria cases, particularly those resulting from n Africa . Rollingn Africa . Whilst n Africa . With thn Africa . The numn Africa , and sixn Africa .Plasmodium species infecting humans share a similar life cycle with an initial development phase in the liver and subsequent further proliferation in the blood of the host. They also show a similar susceptibility to some antimalarial drugs such as quinine, chloroquine and artemisinin, as well as the development of resistance to these drugs [P. vivax malaria can relapse after chemotherapy with drugs that kill the parasites only in the intraerythrocytic development stage [P. vivax malaria, as well as proper mosquito control, can simultaneously reduce the number of malaria incidents caused by any Plasmodium species.se drugs , 10. Trase drugs . P. vivant stage , but 8-ant stage . Thus, sPlasmodium species that naturally infect other primate hosts. But zoonotic malaria is rare, except for that caused by P. knowlesi, which naturally infects macaque monkeys such as the long-tailed and the pig-tailed macaques . Human infection by P. knowlesi has been reported since the 1960s [There have been reports of malaria in humans caused by other he 1960s , 15, butP. falciparum and P. vivax in Sarawak in Malaysian Borneo by the late 1990s–early 2000s. By contrast, the incidence of P. malariae which is rather rare in that state [Plasmodium present in patients in Sarawak was identified solely by microscopical observation of parasites in blood films. However, a molecular diagnosis combining nested PCR and DNA sequencing revealed that most of the malaria cases attributed to P. malariae by microscopy were actually caused by P. knowlesi [P. knowlesi in Sarawak, similar cases were identified in neighbouring Sabah state [P. knowlesi have also been reported in other South East Asian countries including Vietnam and Thailand [P. knowlesi malaria is now recognised as the fifth human malaria [P. knowlesi can produce gametocytes in patients who naturally acquire the infection [P. knowlesi malaria between humans has not been reported yet. The clinical importance of P. knowlesi malaria is particularly high in Malaysia, where malaria caused by the other four human-infective species has been almost completely eliminated thanks to the successful NMCP [P. knowlesi.The NMCP rolled out in Malaysia in the 1960s had achieved a dramatic reduction of the number of human malaria cases caused by at state  showed aat state . At thatknowlesi . FollowiThailand , and P.  malaria . It has nfection , althougP. knowlesi, other Plasmodium species that naturally infect non-human primates have been considered as potential threats to human health through zoonosis [Plasmodium species—P. knowlesi, P. inui, P. cynomolgi, P. coatneyi, P. fieldi and P. simiovale [P. cynomolgi has been proven to naturally cause human infection [P. inui can establish an infection when experimentally introduced into humans [P. cynomolgi) or unreported (P. inui), but they might become the next Plasmodium species to significantly affect human health in the future.Like zoonosis , 24. Macimiovale . Of thesnfection , and P. o humans . ClinicaPlasmodium implicated there are P. simium [P. brasilianum [P. simium and P. brasilianum, have been shown to be phylogenetically very close to P. vivax and P. malariae, respectively [Zoonotic malaria has been reported in South America as well. The species of . simium  and P. bsilianum , parasitectively .Plasmodium species share a similar life cycle [Plasmodium species naturally infecting humans are mosquitoes of the genus Anopheles. These mosquitoes also transmit other Plasmodium species parasitising other mammals whereas transmission of Plasmodium infecting birds and reptiles depends on mosquitoes of other genera or other blood-sucking insects [All fe cycle . It has  insects .Plasmodium life cycle begins when parasites known as sporozoites produced in the insect vector enter the blood of the vertebrate host following a bite [P. knowlesi), 48  or 72  hours. Some merozoites then differentiate into the next developmental stage called the gametocyte for sexual reproduction [P. falciparum needs to complete several cycles of intraerythrocytic propagation before it starts differentiation into gametocytes, whereas P. vivax continuously produces gametocytes even in its early intraerythrocytic propagation cycles  in around 10–18 days.The second part of the life cycle in the insect vector begins when the insect ingests the blood containing gametocytes from an infected vertebrate host. The gametocytes are activated once exposed to the specific environment of the mosquito midgut lumen, and the male and the female gametocytes differentiate to produce microgametes and macrogametes, respectively . The mic zygotes , 56. Soo zygotes . The ook zygotes . In the  zygotes , 59. Whe zygotes  when relPlasmodium cell has two distinct organelles that contain their own genomic DNA, the mitochondrion and the apicoplast (see below). It has been shown that each oocyst of P. falciparum developing in the mosquito inherits the mitochondrial genomic DNA uniparentally from the female gamete [P. gallinaceum (a species infecting chickens) but not in male gametes [Plasmodium inherits both the mitochondrion and the apicoplast only through the female gamete (macrogamete).Besides the nucleus, the e gamete . A furth gametes . This suMalaria can recur after the parasites apparently have been cleared from the patient’s blood. Recurrence is due either to a recrudescence or a relapse Table . RecrudeP. vivax is known to produce hypnozoites that cause relapses after the parasites have been cleared from the patient’s blood by treatment with antimalarial drugs such as chloroquine or quinine [P. vivax sporozoites of two or more different genotypes infecting a person, (2) sporozoites other than the one that caused the acute episode differentiating into a hypnozoite in the liver, and (3) only one of those hypnozoites causing the relapse event. External stimuli such as other infections, including P. falciparum malaria, have been suggested to activate the hypnozoite and initiate a relapse of P. vivax malaria [ quinine . There i quinine . This re malaria , althougP. ovale has long been believed to develop hypnozoites because there are reports of recurrence without a second infection by the same species. However, this view has been questioned recently because of a lack of experimental and clinical data unequivocally supporting the presence of hypnozoites in the liver [Of the other human malaria parasites, he liver , 50.P. falciparum nor P. malariae is thought to develop hypnozoites, and such cells have never been identified in either species [P. falciparum infection that persisted asymptomatically in the human body for 13 years [P. malariae case caused by blood transfusion from a donor who had probably acquired the infection over 40 years ago has also been reported [Plasmodium parasites can be maintained in the human blood over long periods of time at a very low number when their growth rate and the host’s immunity are able to maintain a subtle balance. Recurrence (recrudescence) is believed to begin when the balance is broken.Neither  species . Neverth13 years . A P. mareported . PlasmodP. vivax malaria, some recurrences of P. vivax malaria might have originated from non-hypnozoite cells, as in other human malarias [P. vivax malaria patient’s blood for malariotherapy [P. vivax does not persist as sporozoites in human blood, no hypnozoite could have developed in the recipients. Thus, these records may suggest that P. vivax malaria can recur independent of hypnozoites.Whilst there is strong evidence that hypnozoites cause relapses in malarias –69. Receotherapy –72. BecaPlasmodium cells have a haploid genome. Nevertheless, a cloned line of Plasmodium originating from a single cell generates both male and female gametocytes in the vertebrate host. This indicates that the sex of Plasmodium gametocytes is not determined chromosomally but epigenetically, and evidence explaining the mechanism is being accumulated [Throughout development in their vertebrate hosts, umulated . The gamumulated –75, and Plasmodium species that infect humans are known to be susceptible to 8-aminoquinolines such as primaquine, but not to other antimalarials such as artemisinin and chloroquine that kill the parasites in asexual intraerythrocytic development [P. falciparum gametocytes can persist for several weeks after the clearance of asexual blood stage parasites by drug treatment such as artemisinin-combination therapy [P. falciparum observed that gametocytes had a 50% survival rate of 2.6–6.5 days and that surviving cells were detectable until almost 2 months after the start of the experiment [Plasmodium carriers to prevent the parasites in them causing new malaria cases.Gametocytes of the elopment . Thus, e therapy . An in vperiment . Both thperiment , 80, as periment . People periment –84. TherPlasmodium without developing symptoms because of acquired immunity [Plasmodium in malaria patients’ blood. However, infection can be detected with more sensitive methods, e.g., molecular detection by PCR and LAMP [Plasmodium carriers can develop an episode of malaria when their immunity against the parasite is compromised, for example, when they move to a malaria-free area where they no longer have new Plasmodium infections that sustain immunity against the parasite. As a result, asymptomatic carriers may cause imported malaria [In areas endemic for malaria, many people carry immunity , 86. Oftand LAMP , or ultrand LAMP . Asymptoand LAMP . Asympto malaria , 91. Eve malaria  or organ malaria .The importance of controlling asymptomatic carriers of malaria parasites in the modern world is higher than ever. It is mainly because people’s movements have become much easier than in earlier times, thanks to economic development and transportation. In addition, the number of refugees from regional conflicts, many of which occur in malaria endemic areas, has increased. Asymptomatic carriers within migrants from malaria endemic areas ought to be identified and adequately cared for, like patients with apparent malaria symptoms, in order to prevent the spread or re-introduction of malaria into malaria-free areas , 95.Plasmodium belongs to a larger group of protozoans called the Apicomplexa, part of the superphylum Alveolata that also includes dinoflagellates and ciliates [Plasmodium, almost all apicomplexans are obligate parasites [Plasmodium species, have a vestigial, non-photosynthetic plastid called the apicoplast in the cell [a but lack chlorophyll c that is universally found in the plastids of other phototrophic alveolates. Like the apicoplast, the photosynthetic plastids of chromerids are secondary plastids [The genus ciliates . Like Plarasites , and manthe cell –99. The the cell , 101 andthe cell , 103. Rethe cell , 105. Thplastids . Featureplastids .Plasmodium carry a non-photosynthetic plastid was not clear when the organelle’s presence in the parasite was first recognised [Plasmodium apicoplast is involved in plant-type metabolic pathways including isoprenoid biosynthesis [Plasmodium to complete its development, especially in mosquitoes [Plasmodium in the intraerythrocytic cycle can keep growing in in vitro culture as long as a sufficient amount of isopentenyl pyrophosphate (IPP) is supplied in the culture medium, even when they have lost the apicoplast [Plasmodium to survive, and the plant-type methylerythritol phosphate (MEP) pathway in the apicoplast is the only source of this critical molecule in the parasite [P. falciparum is inhibited by fosmidomycin both in vitro and in vivo [Toxoplasma gondii, which also is supposed to depend on IPP supplied by the MEP pathway in the apicoplast [T. gondii to fosmidomycin has been studied, and it was suggested that poor drug uptake through the parasite’s plasma membrane is the cause [P. falciparum infects depends on a new permeability pathway induced by the parasite [Plasmodium may be able to develop resistance to the drug by mutation in unexpected genes.Unlike plastids of photosynthetic organisms, the apicoplast is non-photosynthetic, and almost all gene products encoded in the tiny organellar genome are predicted to be involved in either transcription or translation . Therefocognised . Howevercognised , it gradynthesis , type IIynthesis  and haemynthesis . These psquitoes , 115 andsquitoes , 117. Hoicoplast . This suparasite . One of parasite , and gro in vivo , 122. Byicoplast . The molhe cause . Anotherparasite . Althougparasite , PlasmodCryptosporidium and Gregarina do not have the apicoplast [P. falciparum and T. gondii cannot survive if they lose the organelle [Cryptosporidium species encodes a remarkably large number of putative amino acid transporters compared to apicomplexans with an apicoplast [Some apicomplexans such as icoplast , while Prganelle , 128. Thrganelle . By conticoplast . These sCinchona spp.) contains an agent with antimalarial activity. This substance, quinine, has been used in the treatment of malaria since then [From ancient times, various plant products have been used in folk medicine to treat malaria. In the seventeenth century, it was shown that the bark of South American quina-quina trees ; it is especially high in the genome of P. falciparum—nearly 80% in coding regions and approaching 90% in non-coding regions. The genome of P. falciparum has been shown to be prone to small genetic changes such as indel mutations [Plasmodium developing drug resistance.It has been reported that clinical isolates of h repair , 140. Geutations . The higPlasmodium depends on the species. It can be extremely narrow as in P. falciparum, which infects humans but not African apes that are phylogenetically very close to humans [P. relictum, which is known to infect more than 100 different species of birds around the world, classified into different families and orders [Plasmodium species are sub-categorised into distinct subgenera based on their morphology and ranges of vertebrate hosts and vectors [Plasmodium as a whole is polyphyletic and that the taxonomy of the order Haemosporidia, which consists of Plasmodium and other related genera, has many conflicts [Plasmodium seems to be monophyletic. Plasmodium species that infect mammals generally belong to either one of three subgenera, Laverania, Plasmodium or Vinckeia, apart from some species recently identified from ungulate hosts [Laverania, Plasmodium and Vinckeia consist of parasite species that infect apes, monkeys and rodents, respectively.The natural host range of o humans . The rand orders . Traditi vectors . It has onflicts . Neverthte hosts , 145. ThPlasmodium species that infect birds, which are transmitted by a wide variety of mosquitoes including Culex and Aedes, mammalian malaria parasites belonging to subgenera Laverania, Plasmodium and Vinckeia are transmitted only by anopheline mosquitoes [Plasmodium species can complete their development from gametocytes to infectious sporozoites only in anopheline mosquitoes. However, this does not mean that these parasites cannot developmentally differentiate from gametocytes in non-anopheline mosquitoes [Culex bitaeniorhynchus was fed with human blood containing gametocytes of P. falciparum or other human malaria parasites [P. falciparum fed to C. quinquefasciatus developed ookinetes that soon lysed and never formed oocysts [P. falciparum is killed by the mosquito’s immune system when the ookinetes are exposed to the haemolymph of non-anopheline mosquitoes [Unlike squitoes . This issquitoes . There warasites . However oocysts . This susquitoes .Plasmodium between humans or animals; different Anopheles species have different habitats, feeding behaviour and preference for the animal species from which they suck blood [It is not true that all anopheline mosquitoes are equally important in transmitting ck blood . These dck blood .Plasmodium species, P. falciparum belongs to subgenus Laverania, whereas all the others belong to subgenus Plasmodium. P. knowlesi that causes zoonotic malaria in humans also belongs to the subgenus Plasmodium. Generally, P. falciparum only infects humans in natural conditions [P. falciparum from bonobos (Pan paniscus) kept at the Lola ya Bonobo Sanctuary in the Democratic Republic of the Congo, though the mitochondrial haplotype map they drew indicated that the isolates from bonobos were genetically distant from P. falciparum parasites infecting humans [P. falciparum Laverania species that is phylogenetically closest to P. falciparum is P. praefalciparum, which infects gorillas but not humans or chimpanzees [Plasmodium species of subgenus Laverania so far described show a strong host specificity in their natural transmission [Laverania species coexist revealed that the three sylvan Anopheles species collected in the survey, An. vinckei, An. moucheti and An. marshallii, carried multiple Laverania species whose vertebrate host specificity varied [Laverania species is not solely caused by specific association between anopheline vectors and vertebrate hosts. A recent comparative genomics study between Laverania species revealed that these parasites have striking copy number differences and structural variations in multiple gene families in their genomes [stevor family, which has been shown to be involved in host-parasite interactions in P. falciparum [Laverania species to determine their strong host specificity. Rh5, a member of another gene family, has been shown to be important for Plasmodium species of subgenus Laverania to bind to erythrocytes of specific hosts [EBA165, a member of the erythrocyte binding-like (EBL) gene family, is pseudogenised in the genome of P. falciparum but not in other Laverania species’ genomes. A recent study showed that P. falciparum becomes capable of binding to ape erythrocytes but loses the ability to bind human erythrocytes when a functional EBA165 product is expressed [EBA165 product was a key step in the emergence of the human-infecting P. falciparum from its P. praefalciparum-like ancestor that probably did not infect humans.Of the four human nditions , though nditions . Krief eg humans . The nonmpanzees . As withsmission , 155. A y varied . This in genomes . Variatilciparum , showed ic hosts . EBA165,xpressed . This suPlasmodium species which were extremely close to P. vivax or P. malariae [P. vivax-like isolates from mosquitoes collected in the survey in Gabon made a cluster with P. simium, one of the two South American zoonotic Plasmodium species [P. brasilianum, the other South American zoonotic Plasmodium, is probably an amphixenotic variant of P. malariae that acquired infectivity to the simian hosts while also keeping its original human infectivity [P. vivax and P. malariae can switch their host more easily than the species of subgenus Laverania [Plasmodium including P. knowlesi and other simian species causing zoonotic malaria are not well understood.In the mosquito survey in Gabon described above, it was also found that the three sylvan species of Anopheles carried malariae . In a ph species . Anotherectivity . All theaverania . HoweverPlasmodium. Thanks to the discovery of natural and chemically synthesised antimalarials, malaria has become a disease curable by chemotherapy. Together with mosquito vector control using insecticides, treatment of malaria patients with synthetic antimalarials such as chloroquine and artemisinin has dramatically reduced the burden of malaria in the modern world compared to the past. Nevertheless, malaria is still prevalent, killing hundreds of thousands of people globally every year.Humans have long suffered from malaria, the disease caused by One of the problems that hinder control of malaria is the emergence and spread of chemotherapy-resistant parasites –162. To Plasmodium can be easily carried from endemic areas to non-endemic areas in the modern world because of increased movement of people. Malaria-free countries and areas are steadily increasing, but imported malaria is a commonly shared threat against health in many countries and areas. Plasmodium species can be retained in the human body for a long time without causing any overt symptoms. Asymptomatic Plasmodium carriers can start developing malaria spontaneously and spread imported malaria in malaria non-endemic countries and areas. These people may also spread the parasite when they are involved in blood transfusions and organ transplantation as donors.Another problem is that P. falciparum and other human-infecting Plasmodium species share a characteristic that lets them infect humans, which is the outcome of convergent evolution. Currently, Plasmodium species that naturally cause malaria in humans are limited, but other species may also acquire natural infectivity to humans and start causing new zoonotic malaria at any time. The physical distance between humans and non-human animals such as other primates can depend on the degree of local development, and this could affect the chance of non-human Plasmodium species becoming the cause of zoonotic malaria in humans.Plasmodium species may change their insect hosts as well. Unlike the Plasmodium species that infect mammals, avian malaria parasites develop and produce infectious sporozoites in non-anopheline mosquitoes. This implies that mammalian Plasmodium species also could acquire resistance to the immune system of non-anopheline mosquitoes such as Culex and Aedes and use them as transmission vectors. Some non-anopheline mosquito species can breed even in harsh environments such as in tunnels, on the coast or in urbanised areas, and can be cosmopolitan [Plasmodium species that can cause malaria in humans acquired the capability of completing its mosquito stage development in non-anopheline mosquitoes, its impact on human society could be substantial.opolitan , 164. IfPlasmodium changes dynamically due to both the parasites’ nature and the activities of humans. Understanding the basic biology of the parasites that leads to these changes, and applying the knowledge to malaria control, should help to achieve a healthier global society.The relationship between humans and