The RNA-binding KH-domain in the unique transcription factor of the malaria parasite is responsible for its transcriptional regulatory activity.

To date, only a small number of regulatory transcription factors have been predicted from the genome of Plasmodium and Apicomplexan parasites. We previously identified a nuclear factor named Prx regulatory element-binding protein (PREBP) from Plasmodium falciparum. PREBP had been suggested to bind to the cis-element in the promoter of an antioxidant pf1-cys-prx gene, thereby promoting the expression of downstream genes. PREBP has 4 putative K homology (KH) domains, which are known to bind RNA and single-stranded DNA. In this study, to understand the detailed action of PREBP in parasite cells, we first observed that in living parasite cells, PREBP was localized in the nucleus in the trophozoite and schizont stages, in which the expression of the target pf1-cys-prx was enhanced. The interaction of PREBP and the cis-element of pf1-cys-prx in the parasite cells was also confirmed. Further, the activities of PREBP deletion mutants were analyzed, and regions with repeated KH domains in PREBP seemed to be responsible for the recognition of the cis-element. These results led us to hypothesize that Plasmodium and other Apicomplexan parasites might have a transcription factor family with KH domains. Bioinformatic analysis revealed a putative ortholog group including PREBP and several Plasmodium and Apicomplexan factors with KH domains. One of the P. falciparum-derived factors, which were included in the putative ortholog group, was found to be localized at the nucleus in the trophozoite stage, indicating that it might be a novel transcription factor. The discovery of PREBP and putative transcription factors with KH domains suggested that multi-functional proteins with KH domains possibly evolved in the Apicomplexan organisms. They might play key roles in transcriptional regulatory processes that are essential for living organisms and may even represent unique drug targets for malaria therapy.

Performance of the procedure for ultra-rapid extraction and loop-mediated isothermal amplification (PURE-LAMP) method to detect malaria in Haiti.

BACKGROUND: Malaria continues to cause burden in various parts of the world. Haiti, a Caribbean country, is among those aiming to eliminate malaria within a few years. Two surveys were conducted in Haiti during which we aimed to evaluate the performance of the simple and rapid procedure for ultra-rapid extraction-loop-mediated isothermal amplification (PURE-LAMP) method with dried blood spots as an alternative diagnostic method for malaria in the context of low to very low rates of transmission. METHODS: Febrile and afebrile people were recruited from three administrative divisions within Haiti: Nippes, Sud and Grand'Anse, during the summers of 2017 (early August to early September) and 2018 (late July to late August). Their blood samples were tested by microscopy, rapid diagnostic tests (RDT), PURE-LAMP and nested PCR to detect Plasmodium infection. Sensitivity, specificity, positive and negative predictive values and kappa statistics were estimated with the nested PCR results as the gold standard. RESULTS: Among 1074 samples analyzed, a positive rate of 8.3% was calculated based on the nested PCR results. Among febrile participants, the rates in 2017 and 2018 were 14.6% and 1.4%, respectively. Three positives were detected among 172 afebrile participants in 2018 by PURE-LAMP and nested PCR, and all three were from the same locality. There was no afebrile participants recruited in 2017. The PURE-LAMP, RDT and microscopy had respective sensitivities of 100%, 85.4% and 49.4%. All of the testing methods had specificities over 99%. CONCLUSIONS: This study confirmed the high performance of the PURE-LAMP method to detect Plasmodium infection with dried blood spots and recommends its use in targeted mass screening and treatment activities in low endemic areas of malaria.

Imported severe Plasmodium falciparum infection in the first trimester of pregnancy complicated by post-artemisinin delayed hemolysis and intrauterine fetal death, a case report.

BACKGROUND: Post-artemisinin delayed hemolysis (PADH) is a serious complication in patients who recover from severe malaria after receiving artemisinin-based combined therapy (ACT), including artemether-lumefantrine. In Japan, among the antimalarial drugs recommended by the World Health Organization (WHO) guideline for severe malaria, intravenous quinine gluconate is available only in 29 designated hospitals, and intravenous artesunate is unavailable. Therefore, oral artemether-lumefantrine is occasionally administered as an alternative, even though it may be a suboptimal treatment. In non-endemic settings like Japan, a lack of knowledge of malaria and the side effects, such as post-artemisinin delayed hemolysis caused by the ACT, can have critical consequences. Like our patient, being a primigravida in the early stages of pregnancy is a serious risk factor for severe malaria and must be carefully monitored. CASE PRESENTATION: This report describes a severe case of imported Plasmodium falciparum malaria complicated by fetal loss and prolonged anemia, requiring frequent blood transfusions. The patient was a previously healthy pregnant Japanese female in her 30 s. She developed a high fever 2 days after returning from Nigeria. The patient fulfilled the severe malaria criteria by WHO. On arrival, an abdominal ultrasound incidentally revealed a fetus of 5 week gestational age with a heartbeat in the uterus. Given her pregnancy and the severity of the disease, she was administered intravenous quinine 16 mg/kg as a loading dose. However, the second dose of quinine was not administered due to frequent vomiting and QTc prolongation. We initiated treatment with oral artemether-lumefantrine, and clearance of parasitemia was confirmed by microscopic observation on day 4. Miscarriage was noted on day 6 after admission. Moreover, the patient became feverish again up to 39 °C, and from days 14 to 22, the patient required multiple blood transfusions due to PADH. On day 40, follow-up was discontinued as the hemoglobin level exceeded 10 g/dL. CONCLUSIONS: In patients who recover from severe malaria after ACT treatment, monitoring the hemoglobin level for at least a month is strongly recommended for prompt identification of PADH. Travelers to malaria-endemic countries, especially primigravida women, should be provided with adequate information on the risk and prevention of infection.

False-positive fourth-generation HIV test result in a woman with Plasmodium malariae malaria.

BACKGROUND: False positive results on fourth-generation human immunodeficiency virus (HIV) diagnostic tests have previously been reported in infections with Plasmodium falciparum and Plasmodium ovale but not with Plasmodium malariae. METHODS: We report a false positive fourth-generation HIV test result in a patient with P. malariae infection. The patient's symptoms improved rapidly with antimalarial treatment and the confirmatory and repeated HIV tests were eventually negative. RESULTS: False positive results may add a variety of unnecessary burden. CONCLUSIONS: One must be aware of false positive results even with fourth-generation tests in patients with malaria, including P. malariae malaria.

Glucose-6-phosphate Dehydrogenase (G6PD) A-Variant Frequency and Novel Polymorphism in Haiti.

There are scarce data about the glucose-6-phosphate dehydrogenase (G6PD) variants in Haiti to guide public health guidelines. In this study, we investigated the prevalence of the G6PD mutations related to the A- variant. We found an allelic frequency of 35.8% for the A376G mutation and of 12.2% for the G202A mutation. We also found a novel C370T mutation concomitant with the A376G mutation in one study participant. The G680T and T968C mutations were not found. The G6PD deficient variant A202 (A376G and G202A mutations) has appreciable prevalence in Haiti (16.6%), consideration is warranted when using drugs such as primaquine, which may trigger hemolytic anemia among G6PD-deficient people.

Clinical performance testing of the automated haematology analyzer XN-31 prototype using whole blood samples from patients with imported malaria in Japan.

BACKGROUND: The automated haematology analyzer XN-31 prototype (XN-31p) is a new flow cytometry-based device developed to measure the number and the ratio of malaria-infected red blood cells (MI-RBC) with a complete blood count (CBC). The XN-31p can provide results in about one minute and also can simultaneously provide information on the malaria parasite (Plasmodium) species. In this study, clinical testing of the XN-31p was performed using blood samples from patients with imported malaria in Japan. METHODS: Blood samples were collected from 80 patients who visited the hospital of the National Center for Global Health and Medicine, Tokyo, Japan, for malaria diagnosis from January 2017 to January 2019. The test results by the XN-31p were compared with those by other standard methods, such as microscopic observation, rapid diagnostic tests and the nested PCR. RESULTS: Thirty-three patients were diagnosed by the nested PCR as being malaria positive (28 Plasmodium falciparum, 2 Plasmodium vivax, 1 Plasmodium knowlesi, 1 mixed infection of P. falciparum and Plasmodium malariae, and 1 mixed infection of P. falciparum and Plasmodium ovale), and the other 47 were negative. The XN-31p detected 32 patients as "MI-RBC positive", which almost matched the results by the nested PCR and, in fact, completely matched with the microscopic observations. The ratio of RBCs infected with malaria parasites as determined by the XN-31p showed a high correlation coefficient of more than 0.99 with the parasitaemia counted under microscopic observation. The XN-31p can analyse the size and nucleic acid contents of each cell, and the results were visualized on a two-dimensional cytogram termed the "M scattergram". Information on species and developmental stages of the parasites could also be predicted from the patterns visualized in the M scattergrams. The XN-31p showed a positive coincidence rate of 0.848 with the nested PCR in discriminating P. falciparum from the other species. CONCLUSIONS: The XN-31p could rapidly provide instructive information on the ratio of MI-RBC and the infecting Plasmodium species. It was regarded to be of great help for the clinical diagnosis of malaria.

Imported Malaria at a Referral Hospital in Tokyo from 2005 to 2016: Clinical Experience and Challenges in a Non-Endemic Setting.

In this study, we reviewed imported malaria cases observed at the National Center for Global Health and Medicine, Tokyo, between 2005 and 2016, to comprehend their demographic and clinical characteristics. Data on 169 cases were used to analyze demographic information; data on 146 cases were used for the analysis of clinical information. The median patients' age was 34 years, and 79.3% of them were male. The proportion of non-Japanese patients increased and surpassed that of Japanese patients after 2015. In 82.2% of the cases, the region of acquisition was Africa, and Plasmodium falciparum was the dominant species (74.0%) followed by Plasmodium vivax (15.4%). We observed 19 (18.4%, 19/103) severe falciparum malaria cases. Mefloquine was the most commonly used drug for treatment until the early 2010s; atovaquone/proguanil was the most commonly used after its licensure in 2013. Although none of the patients died, four recrudescence episodes after artemether/lumefantrine (A/L) treatment and one relapse episode were observed. Overall, malaria was diagnosed on median day 4 of illness, and, thereon, treatment was initiated without delay. Diagnosis on day 5 or later was significantly associated with severe disease in Japanese cases (odds ratio = 4.1; 95% CI = 1.2-14.3). We observed a dominance of falciparum malaria, an increase in the number of non-Japanese cases, late treatment failure after A/L treatment, a low relapse rate, and an association between delayed malaria diagnosis and higher disease severity. Pretravel care and early diagnosis are necessary to reduce malaria-related mortality and morbidity in settings such as ours.

No Plasmodium falciparum Chloroquine Resistance Transporter and Artemisinin Resistance Mutations, Haiti.

We obtained 78 human blood samples from areas in Haiti with high transmission of malaria and found no drug resistance-associated mutations in Plasmodium falciparum chloroquine resistance transporter and Kelch 13 genes. We recommend maintaining chloroquine as the first-line drug for malaria in Haiti. Artemisinin-based therapy can be used as alternative therapy.

Combination of PURE-DNA extraction and LAMP-DNA amplification methods for accurate malaria diagnosis on dried blood spots.

BACKGROUND: Malaria is one of the most important parasitic infectious diseases for which almost half of the world's population is at risk. Although several diagnostic methods are now available to detect the infection, more sensitive and applicable tests are still required in the field. The loop-mediated isothermal amplification (LAMP) method is a DNA amplification tool in which the DNA amplification can be achieved by incubation at a stable temperature. A malaria detection kit based on this methodology has already been commercialized and is being used in some countries. The kit includes two reaction tubes: one targeting the common Plasmodium genus (Pan tube) and the other specifically targeting Plasmodium falciparum (Pf tube). In parallel, a simple DNA extraction method, the procedure for ultra rapid extraction (PURE), which can produce a DNA solution suitable for the LAMP reaction without the use of a centrifuge, has also become available. In this study, the sensitivity of the combination of the PURE and LAMP methods (PURE-LAMP) was evaluated with archived dried clinical blood samples of imported malaria cases, including P. falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. RESULTS: Using a nested PCR as the reference, 117 samples including 46 P. falciparum, 7 P. vivax, 9 P. ovale, 4 P. malariae, and 51 negative cases were tested. The PURE-LAMP Pan correctly identified 64 of the 66 positives and the 51 negatives. Among the Pan-positive samples 45 P. falciparum were also detected with the PURE-LAMP Pf. The PURE-LAMP Pan and PURE-LAMP Pf had respective sensitivities of 96.96% (95% CI 89.47-99.63) and 97.82% (95% CI 88.47-99.94) and common specificity of 1. CONCLUSION: The PURE-LAMP system is accurate when used with dried blood spots and extendable to the field.

Case Report: Plasmodium knowlesi Infection with Rhabdomyolysis in a Japanese Traveler to Palawan, the Philippines.

Skeletal muscle is known to be damaged by falciparum malaria via sequestration of infected erythrocytes. We present a case of rhabdomyolysis caused by Plasmodium knowlesi infection. The patient had fever, myalgia, and muscle weakness 5 days after returning to Japan from Palawan, the Philippines. Blood test revealed thrombocytopenia and an elevated creatine kinase level. Although rhabdomyolysis resolved with fluid therapy, fever of 24-hour cycle continued and thrombocytopenia intensified. On day 7 of illness, Giemsa-stained thin blood smear revealed malaria parasites, with a parasite count of 2,380/µL, which were morphologically indistinguishable between P. knowlesi and Plasmodium malariae. Rapid diagnostic test showed a negative result. The pathogen was later confirmed to be P. knowlesi by nested polymerase chain reaction (PCR). The patient was successfully treated with artemether/lumefantrine. This case suggests that knowlesi malaria might be able to cause skeletal muscle damage.

A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi.

A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient's blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a "fast PCR enzyme". In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the "fast PCR enzyme", with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.

Identification of a novel and unique transcription factor in the intraerythrocytic stage of Plasmodium falciparum.

The mechanisms of stage-specific gene regulation in the malaria parasite Plasmodium falciparum are largely unclear, with only a small number of specific regulatory transcription factors (AP2 family) having been identified. In particular, the transcription factors that function in the intraerythrocytic stage remain to be elucidated. Previously, as a model case for stage-specific transcription in the P. falciparum intraerythrocytic stage, we analyzed the transcriptional regulation of pf1-cys-prx, a trophozoite/schizont-specific gene, and suggested that some nuclear factors bind specifically to the cis-element of pf1-cys-prx and enhance transcription. In the present study, we purified nuclear factors from parasite nuclear extract by 5 steps of chromatography, and identified a factor termed PREBP. PREBP is not included in the AP2 family, and is a novel protein with four K-homology (KH) domains. The KH domain is known to be found in RNA-binding or single-stranded DNA-binding proteins. PREBP is well conserved in Plasmodium species and partially conserved in phylum Apicomplexa. To evaluate the effects of PREBP overexpression, we used a transient overexpression and luciferase assay combined approach. Overexpression of PREBP markedly enhanced luciferase expression under the control of the pf1-cys-prx cis-element. These results provide the first evidence of a novel transcription factor that activates the gene expression in the malaria parasite intraerythrocytic stage. These findings enhance our understanding of the evolution of specific transcription machinery in Plasmodium and other eukaryotes.

2-Cys peroxiredoxin of Plasmodium falciparum is involved in resistance to heat stress of the parasite.

In the cytoplasm of Plasmodium falciparum, two peroxiredoxins: PfTPx-1 and Pf1-Cys-Prx, are expressed at different time-points of the parasite cell cycle during the intraerythrocytic stage. In the present study, to gain insight into the functions of Prxs in the cytoplasm of P. falciparum, we investigated the heat stress sensitivity of the previously established PfTPx-1 KO line and found that PfTPx-1 disruption renders the parasite hypersensitive to heat stress. In addition, we established Pf1-Cys-Prx knockout (KO) parasite lines. The phenotypes of Pf1-Cys-Prx KO lines were different to those of the PfTPx-1 KO line and did not show hypersensitivity to reactive oxygen species, reactive nitrogen species, chloroquine or heat stress. These results suggest that the function of Pf1-Cys-Prx in the parasite cytoplasm is independent from that of PfTPx-1. The hyperthermal protective function of the PfTPx-1 is obviously important for the parasite physiology in the human patient body, in which it must survive repeated incidences of fever.

Evaluation of the NOW Malaria Immunochromatographic Test for Quantitative Diagnosis of Falciparum and Vivax Malaria Parasite Density.

The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density.

Target proteins of the cytosolic thioredoxin in Plasmodium falciparum.

The target proteins of a cytosolic Trx (PfTrx-1) in Plasmodium falciparum with Trx-affinity chromatography were examined. Based on the Trx protein reduction pathway, we generated a cysteine mutant of PfTrx-1, which captures the target protein as a mixed disulfide intermediate. A number of proteins were captured with PfTrx-1(C33S) immobilized on resin and were eluted by DTT treatment. The PfTrx-1(C33S) immobilized resin-captured proteins were trypsin-digested and analyzed on a liquid chromatography-mass spectrometry system. Analysis of the sequence data against databases assigned 20 proteins, four of which had been found previously in P. falciparum, with the remaining 16 being new targets. The potential Trx-target proteins included those in pathways such as the redox cycle, protein biosynthesis, energy metabolism and signal transduction. We captured 4 enzymes in the glycolysis pathway (hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase and L-lactate dehydrogenase (LDH)) as Trx-targets, and we found that PfTrx-1 enhanced the activity of PfGAPDH and PfLDH.

Genetic evidence for Plasmodium falciparum resistance to chloroquine and pyrimethamine in Indochina and the Western Pacific between 1984 and 1998.

Plasmodium falciparum resistance to chloroquine and pyrimethamine is widely distributed in malaria-endemic areas. The origin and geographic spread of this drug resistance have been inferred mainly from records of clinical resistance (treatment failure). Identification of the Plasmodium falciparum chloroqunie resistance transporter (pfcrt) gene and the dihydrofolate reductase (dhfr) gene as target genes of chloroquine and pyrimethamine, respectively, has made it possible to trace the history of genetic resistance to these two drugs. However, evidence for genetic resistance has been limited because of scarcity of archival specimens. We examined genotypes of pfcrt and dhfr in Indochina (Thailand, Myanmar, and Laos) and the Western Pacific (the Philippines, Indonesia, and Papua New Guinea) between 1984 and 1998 by testing samples obtained from malaria cases imported to Japan. Results show that 96% (28 of 29) and 77% (20 of 26) of samples had resistant genotypes of pfcrt and dhfr, respectively, substantiating the inferred history of clinical resistance in these geographic areas during this period.

5' sequence- and chromatin modification-dependent gene expression in Plasmodium falciparum erythrocytic stage.

Plasmodium falciparum, the human malaria parasite, is evolutionarily distant from other eukaryotes. Genome-wide analyses of transcription-associated proteins have revealed a relative paucity of putative regulatory transcription factors and an abundance of putative chromatin remodeling machinery, suggesting that this parasite has a transcription regulatory system that is distinct from those of other eukaryotes. Here, we have analyzed transcriptional regulation of the peroxiredoxin genes, pf1-cys-prx and pftpx-1, which show different expression patterns in P. falciparum. The reporter assays revealed the presence of putative enhancers in the 5' regions of these genes. Although pf1-cys-prx shows trophozoite/schizont stage-specific transcription, a putative cis-acting enhancer sequence in pf1-cys-prx was constitutively active when inserted into the 5' region of pftpx-1. Electrophoretic mobility shift and DNase I footprinting assays showed that this enhancer region is the target of trophozoite/schizont stage-specific DNA binding proteins. In addition, chromatin immunoprecipitation assays showed that the increased levels of histone acetylation in the 5' region of pf1-cys-prx and pftpx-1 correlate with the transcriptional activity of these genes. Recruitment of PfGCN5 histone acetyltransferase to the pf1-cys-prx enhancer in trophozoite/schizont stage was observed. These results suggest that P. falciparum possesses a sophisticated system of transcriptional regulation during intraerythrocytic stages that is managed by coordinated interactions of unique cis-elements and trans-acting factors and chromatin modifications.

Disruption of the Plasmodium berghei 2-Cys peroxiredoxin TPx-1 gene hinders the sporozoite development in the vector mosquito.

To investigate the physiologic role of cytosolic 2-Cys peroxiredoxin of Plasmodium berghei (PbTPx-1), we infected the vector mosquito Anopheles stephensi with a parasite carrying a targeted knockout of pbtpx-1 (Prx-KO). The number of Prx-KO midgut oocysts at 14-15 days post-feeding (pf) was comparable to that of the parent strain (WT); however, the numbers of sporozoites that formed in midgut oocysts and accumulated in the salivary gland of Prx-KO-infected mosquitoes by 21 days pf were decreased to 10-20% and 3-10%, respectively, of those values in WT-infected mosquitoes. A higher frequency of DNA strand breaks was detected in Prx-KO oocysts than in WT oocysts. Sporozoites carrying the targeted disruption had reduced infectivity in mice; however, the knockout did not affect the ability of the sporozoite to reach the liver parenchyma and initiate exo-erythrocytic form (EEF) development. TPx-1 may be involved in development during exponentially multiplying stages, such as sporozoites and EEF.

Peroxiredoxins in malaria parasites: parasitologic aspects.

Malaria is one of the most debilitating and life threatening diseases in tropical regions of the world. Over 500 million clinical cases occur, and 2-3 million people die of the disease each year. Because Plasmodium lacks genuine glutathione peroxidase and catalase, the two major antioxidant enzymes in the eukaryotic cell, malaria parasites are likely to utilize members of the peroxiredoxin (Prx) family as the principal enzymes to reduce peroxides, which increase in the parasite cell due to metabolism and parasitism during parasite development. In addition to its function of protecting macromolecules from H(2)O(2), Prx has also been reported to regulate H(2)O(2) as second messenger in transmission of redox signals, which mediate cell proliferation, differentiation, and apoptosis. In the malaria parasite, several lines of experimental data have suggested that the parasite uses Prxs as multifunctional molecules to adapt themselves to asexual and sexual development. In this review, we summarize the accumulated knowledge on the Prx family with respect to their functions in mammalian cells and their possible function(s) in malaria parasites.