Search

The malaria landscape in the Greater Mekong Subregion has experienced drastic changes with the ramp-up of the control efforts, revealing formidable challenges that slowed down the progress toward malaria elimination. Problems such as border malaria and cross-border malaria introduction, multidrug resistance in Plasmodium falciparum, the persistence of Plasmodium vivax, the asymptomatic parasite reservoirs, and insecticide resistance in primary vectors require integrated strategies tailored for individual nations in the region. In recognition of these challenges and the need for research, the Southeast Asian International Center of Excellence for Malaria Research has established a network of researchers and stakeholders and conducted basic and translational research to identify existing and emerging problems and develop new countermeasures. The installation of a comprehensive disease and vector surveillance system at sentinel sites in border areas with the implementation of passive/active case detection and cross-sectional surveys allowed timely detection and management of malaria cases, provided updated knowledge for effective vector control measures, and facilitated the efficacy studies of antimalarials. Incorporating sensitive molecular diagnosis to expose the significance of asymptomatic parasite reservoirs for sustaining transmission helped establish the necessary evidence to guide targeted control to eliminate residual transmission. In addition, this program has developed point-of-care diagnostics to monitor the quality of artemisinin combination therapies, delivering the needed information to the drug regulatory authorities to take measures against falsified and substandard antimalarials. To accelerate malaria elimination, this program has actively engaged with stakeholders of all levels, fostered vertical and horizontal collaborations, and enabled the effective dissemination of research findings.

Objectives Malaria cross-sectional surveys are rarely conducted in very low transmission settings. This study aimed to determine the prevalence and risk factors of Plasmodium infection in a near-elimination setting in southern Thailand. Methods Two cross-sectional surveys were conducted in areas of active transmission in the Surat Thani province of Thailand in January and May 2019. PCR was used to detect Plasmodium infection. Results The prevalence of Plasmodium blood infection was 0.45% and 0.61% in January and May 2019, respectively. The major parasite species was Plasmodium falciparum in January and Plasmodium vivax in May. Unexpectedly, Plasmodium knowlesi infections were also detected. Most infections, including those of Plasmodium knowlesi, were asymptomatic. Being male and staying outdoors at night-time were the only significant identified risk factors. Of people infected in January 28.0% were positive in May for the same parasite species, suggesting persistent asymptomatic infections. Conclusions Despite the very low incidence rate in Surat Thani, most malaria infections were asymptomatic. Outdoor mosquito biting at night-time is likely an important mode of malaria transmission. Unexpectedly, asymptomatic Plasmodium knowlesi infection was found, confirming previous reports of such infection in mainland Southeast Asia.

Background: Measurement of antibody titers directed against mosquito salivary antigens in blood samples has been proposed as an outcome measure to assess human exposure to vector bites. However, only a handful of antigens have been identified and the specificity and longitudinal dynamics of antibody responses are not well known. We report the protocol of a clinical trial of controlled exposure to mosquito bites that aims to identify and validate biomarkers of exposure to bites of mosquito vector species that transmit malaria and dengue in Southeast Asia and some other parts of the world. Methods: This study is an exploratory factorial randomized control trial of controlled exposure to mosquito bites with 10 arms corresponding to different species (Aedes aegypt, Ae. albopictus, Anopheles dirus, An. maculatus and An. minimus) and numbers of bites (35 or 305 bites in total over 6 weeks). Blood samples will be collected from study participants before, during and after mosquito biting challenges. Candidate peptides will be identified from published literature with antigen prediction algorithms using mosquito DNA sequence data and with immunoblotting assays carried out using protein extracts of dissected mosquito salivary glands and participants samples. Antibody titers against candidate peptides will be determined in participants samples with high-throughput cutting-edge immuno-assays. Quantification of the antibody response profile over time (including an estimate of the decay rate) and the effect of the number of bites on the antibody response will be determined using linear and logistic mixed-effects models for the continuous and the binary response, respectively. Conclusion: This research is expected to generate important knowledge for vector sero-surveillance and evaluation of vector-control interventions against malaria and dengue in the Greater Mekong Subregion. Registration: This study is registered with clinicaltrials.gov (NCT04478370) on July 20th, 2020.

Background Plasmodium vivax (P. vivax) is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions. Methods We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2–4 weeks using a multiplexed Luminex® assay and identified protein-specific variation in antibody longevity. Mathematical modelling was used to generate the estimated half-life of antibodies, long-, and short-lived antibody-secreting cells. Results Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections. Conclusions Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk.

Plasmodium vivax is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions. We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2-4 weeks using a multiplexed Luminex® assay, and identified protein-specific variation in antibody longevity. Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections. Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk.

Thailand is aiming for malaria elimination by the year 2030. However, the high proportion of asymptomatic infections and the presence of the hidden hypnozoite stage of Plasmodium vivax are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to P. vivax infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of P. vivax serological exposure markers to detect residual transmission ‘hot-spots’ in Western Thailand. Total IgG levels were measured against a panel of 23 candidate P. vivax serological exposure markers using a multiplexed bead-based assay. A total of 4255 plasma samples from a cross-sectional survey conducted in 2012 of endemic areas in the Kanchanaburi and Ratchaburi provinces were assayed. We compared IgG levels with multiple epidemiological factors that are associated with an increased risk of P. vivax infection in Thailand, including age, gender and spatial location, as well as Plasmodium infection status itself. IgG levels to all proteins were significantly higher in the presence of a P. vivax infection (n=144) (t test, p18 years, pP. vivax parasites in the last 9-months, based on their IgG antibody levels to a panel of 8 previously validated P. vivax proteins. Spatial clustering was observed at the village and regional level, with a moderate correlation between PCR prevalence and sero-prevalence as predicted by the algorithm. Our data provides proof-of-concept for application of such surrogate markers as evidence of recent exposure in low transmission areas. These data can be used to better identify geographical areas with asymptomatic infection burdens that can be targeted in elimination campaigns.

Thailand is aiming for malaria elimination by the year 2030. However, the high proportion of asymptomatic infections and the presence of the hidden hypnozoite stage of Plasmodium vivax are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to P. vivax infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of P. vivax serological exposure markers to detect residual transmission “hot-spots” in Western Thailand. Total IgG levels were measured against a panel of 23 candidate P. vivax serological exposure markers using a multiplexed bead-based assay. A total of 4,255 plasma samples from a cross-sectional survey conducted in 2012 of endemic areas in the Kanchanaburi and Ratchaburi provinces were assayed. We compared IgG levels with multiple epidemiological factors that are associated with an increased risk of P. vivax infection in Thailand, including age, gender, and spatial location, as well as Plasmodium infection status itself. IgG levels to all proteins were significantly higher in the presence of a P. vivax infection (n = 144) (T-test, p < 0.0001). Overall seropositivity rates varied from 2.5% (PVX_097625, merozoite surface protein 8) to 16.8% (PVX_082670, merozoite surface protein 7), with 43% of individuals seropositive to at least 1 protein. Higher IgG levels were associated with older age (>18 years, p < 0.05) and males (17/23 proteins, p < 0.05), supporting the paradigm that men have a higher risk of infection than females in this setting. We used a Random Forests algorithm to predict which individuals had exposure to P. vivax parasites in the last 9-months, based on their IgG antibody levels to a panel of eight previously validated P. vivax proteins. Spatial clustering was observed at the village and regional level, with a moderate correlation between PCR prevalence and sero-prevalence as predicted by the algorithm. Our data provides proof-of-concept for application of such surrogate markers as evidence of recent exposure in low transmission areas. These data can be used to better identify geographical areas with asymptomatic infection burdens that can be targeted in elimination campaigns.

Andrographis paniculata is an annual herbaceous plant widely used in many East Asian countries to help relieve symptoms of the common cold, fever, and non-infectious diarrhea. It is often co-administered with prescribed drugs. The present work aimed to study the interactive effects of A. paniculata extracts and the cancer chemotherapeutic drug, 5-Flurouracil (5-FU), on the expression of four common drug metabolizing cytochrome P450 s (CYP450), including CYP1A2, CYP3A4, CYP2C9, and CYP2D6, in human hepatocellular carcinoma HepG2 cells. Two different types of A. paniculata extracts were tested, the ethanolic extract at the first true leaf stage (FTLEE) and the water extract at the mature leaf stage (MLWE), along with their major phytochemical compound, andrographolide (AP1). The results showed that FTLEE, MLWE, and AP1 sensitized HepG2 cells to the cytotoxicity of 5-FU. Western Blotting assay demonstrated that both extracts and AP1 single treatments decreased CYP2C9 and CYP2D6 but increased CYP3A4 levels. 5-FU single treatment reduced the level of CYP1A2 and CYP2D6. Combination of 5-FU only with FTLEE produced an increase in CYP1A2 level compared to 5-FU treatment alone. Combination of either extracts or AP1 with 5-FU produced a further reduction of CYP2D6 levels. The data suggest the influence of harvesting stage of A. paniculata on CYP1A2 induction, and the synergistic effect of A. paniculata and 5-FU on CYP2D6 reduction. The combinations of A. paniculata with anti-cancer drugs should be critically considered. However, the interactive effects of A. paniculata extracts or AP1 with 5-FU on CYP450 s need more preclinical studies.

Cholinergic receptors, such as α7-nicotinic acetylcholine receptor (α7-nAChR) and M3-muscarinic acetylcholine receptor (M3-mAChR), have been demonstrated to serve a significant role in the proliferation, differentiation and apoptosis of leukemic cells. However, the expression of these receptors in samples from patients with leukemia remains unclear. The present study aimed to determine the expression of M3-mAChR and α7-nAChR in the bone marrow or peripheral blood of 51 patients with leukemia, including acute myeloid leukemia (AML; n=33), acute lymphoblastic leukemia (ALL; n=13), and chronic myeloid leukemia (CML; n=5). Peripheral blood mononuclear cells (PBMCs) were also isolated from healthy subjects (n=5) for comparison. Western blot analysis was performed to determine the protein expression profiles, and a pattern of decreased α7-nAChR levels in patients with leukemia was observed. Among the leukemia types, the lowest expression of α7-nAChR and M3-mAChR were identified in patients with T-cell ALL/lymphoma (T-ALL). CML exhibited the highest level of M3-mAChR, which was significantly different from APL and AML-M4, yet not from healthy subjects (P

Mutations of isocitrate dehydrogenase isoform 1 and 2 (IDH1 and IDH2) genes have been identified in glioblastoma and acute myeloid leukemia (AML). However, little is known about the molecular alterations of IDH genes in preleukemic disorders with a propensity to transform to AML. We performed polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) followed by direct sequencing to detect IDH mutations in 237 patients with myeloproliferative neoplasms (MPNs; n=108), myelodysplastic syndrome (MDS; n=22), paroxysmal nocturnal hemoglobinuria (PNH; n=41), and aplastic anemia (AA; n=66). No IDH1 R132 and IDH2 R172 mutations were identified in the entire cohort, whereas IDH1 G105G allele was detected in 4/108 MPN (3.70%), 2/22 MDS (9.09%), and 2/41 PNH (4.88%) patients. Three IDH2 R140Q mutations were found in 2/108 MPN (1.85%) and 1/22 MDS (4.54%) patients, while one IDH2 G145G allele was found in 0.92% (1/108) of MPN patients. Overall, our data suggest that IDH mutations are rare in the preleukemic disorders and may not be the major initial step in AML leukemogenesis.

Several tools exists to evaluate copy-number variation (CNV) from sequencing read data. Here, the aim is to investigate whether larger CNVs in leukemia can be extracted directly from already called variants - without specialized algorithms. Methods: 12 cases of leukemia, with 6 paired and 6 unpaired, were included (Table 1). Following exome sequencing and reference alignment variants were called with GATK to produce recalibrated sets in variant call format (VCF). Passed single nucleotide polymorphisms (SNPs) were limited to depth of coverage > 29x. Each chromosome were visualized as frequency scatter plot and smoothened histograms of frequency distribution in order to inspect frequency bands and number of peaks in histograms. Kolmogorov-Smirnov test was employed to compare distributions between paired samples. Results: None of the AML cases with normal karyotype displayed CNVs and no significant changes in frequency distributions were detected. In the relapse of paired T-ALL a Chr 4 trisomy and a 33×10−6 bp copy-number neutral loss of heterozygosity (CN-LOH) on Chr 9 were detected. Collectively, 7 aberrations confirmed standard cytogenetics, 8 new aberrations were added to the cytogenetical profile and 5 could not be detected as shown in Table 1. The aberrations not detected were, as expected, balanced translocations (3) and microdeletions (2). Discussion: No previously found large aberrations escaped detection, when disregarding translocations. In addition, other CNVs not detected by conventional cytogenetics were found by SNP analysis. Moreover, the added information is in agreement with the literature: 5% of T-ALL patients display del(11)(p12p13) and +12 is one of the most common cytogenetic changes of CLL, e.g. Thus, we suggest that CNV profiling can be initiated by directly evaluating frequencies from existing variant calls. This may ease implementation of “closed toolboxes”, enable analysis of single unpaired samples and function as a direct complement to array-CGH or FISH to detect CN-LOH. Table 1SamplesPairedCytogeneticsCNV detectedNK-AML5YesNormal karyotypeNoneT-ALL relapse1Yes4+, 3p26.2 and Xp22.2 micro-deletions+4, partial del(9p)AML M01YesNormal karyotypePartial del(11p)AML M31Not(15;17)NoneCML1Not(9;22)NoneT-ALLNodel(5) t(7;14)Partial del(5q), +4q, +9p, del(11p), del(12q)CLL1NoNot analyzed+12AML M01NoNormal karyotypePartial +11 Citation Format: Marcus C. Hansen, Laura L. Herborg, Anne S. Roug, Sadudee Chotirat, Chirayu U. Auewarakul, Eigil Kjeldsen, Peter Hokland. Resolving copy number variations from existing exome SNP data without the need for sophisticated software. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3648.

Introduction Exome sequencing can detect somatic mutations at an unprecedented scale. However, high false-positive rates due to multiple technical contributors affecting signal-to-noise ratio is an unsolved inherent problem. Furthermore, the current literature does not consistently assess or report the allelic burden of detected and scrutinized mutations, potentially leading to focus on irrelevant mutations. We hypothesized that this issue can be addressed by a structured usage of allele frequencies in combination with detected mutations. Employing improved techniques to assess the molecular signature shortly after diagnosis could also result in improved risk stratification. Here, we present proof-of-principle that identification of relevant mutations, and extent of clonality, can be accomplished by pairing hypothetical allelic burden deduced from sequencing reads to leukemic burden in individual samples. Methods 7 samples were used for whole exome sequencing derived from bone marrow aspirated from two different patients. Of these, 4 were leukemia samples (diagnostic and two relapse samples of one T-cell/myeloid mixed-phenotype leukemia and a diagnostic from AML, M0), reflecting different clinical situations, and 3 samples served as controls (keratinocytes, and fibroblasts in the first, remission in the second) to include different clinical situations. Raw data processing, mutations analysis and downstream analysis of sequencing data was performed as described earlier (Hansen & al., MethodsX 2015, Hansen & al., Br J Haematol. 2015), following GATK Best Practice workflow and MuTect default parameters. Cluster analysis was utilized to mathematically divide somatic SNVs into read frequency clusters on the basis of squared Euclidean distance, thus enabling the retrieval of mean allelic burden and expected allele frequencies juxtaposed with flow cytometry-derived leukemic blast percent. The initial condition of the classification model was default set to accommodate separation of background (cluster 1: noise or low read), heterozygous mutations (cluster 2), and an outlier bin for possible homozygous mutations (cluster 3). Results and discussion In order to reference the leukemic burden of a given sample we employed data from immunophenotyping as a surrogate marker for malignancy. Without filtering of detected mutations by exome sequencing the partly stochastic nature of the signal is evident (fig. 1A). An exponential-like continuum of non-rejected mutations is found when these are sorted according to frequencies (black points), making it difficult to deduce any clonal nature of the malignancy, and to evaluate the validity of possibly relevant mutations found. Using a minimum depth of coverage threshold of 30 the signal-to-noise ratio is increased (fig. 1B), and, subsequently, two clones were resolved at diagnosis (fig. 1C) by cluster analysis. This resolution decreases at lower tumor burden showing expected sensitivity towards lower allelic reads (fig. 1D), although driver mutations of the persistent clone could be detected. At second relapse only one distinct sAML phenotyoe clone and, now homozygous, CDKN2 AR80* could be detected (fig. 1E) with the mutations of secondary clone present at diagnosis (cluster 2) lost due to selective pressure from treatment, i.e. FLT3D835Y. The observations are backed by another diagnostic sample from a patient with AML with NRAS and BCOR mutations (fig. 1F), also with a distinct clone. Here, too much emphasis could easily be given to NRASG12D if the frequency had not been assessed. Conclusion We have addressed the pertinent question regarding false positive observations arising from deep sequencing or emphasizing mutations found in the low allele frequency fractions. From this dataset we have, despite the low number of samples, accomplished to suggest a formalized approach for single sample mutational analysis. As a consequence, we can now show that malignant clones with high tumor burden can be resolved semi-spatially by sequencing, generally applicable to a wide range of clinical settings. We conclude that this approach is amenable in single patient situations. While further studies are needed to ultimately test the applicability of this approach in the clinical settings, the perspectives to this observation become evident as sequencing depth and cost continue to develop in an inversely correlated manner. Disclosures No relevant conflicts of interest to declare.

Background Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) metabolic genes encode cytosolic and mitochondrial enzymes that catalyze the conversion of isocitrate to α-ketoglutarate. Acquired somatic mutations of IDH1 and IDH2 have recently been reported in some types of brain tumors and a small proportion of acute myeloid leukemia (AML) cases. Methods Two-hundred and thirty newly diagnosed AML patients were analyzed for the presence of IDH1 and IDH2 heterozygous mutations by polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) followed by direct sequencing. Clinical and biological characteristics were analyzed and correlated to the IDH mutational status. Coexisting mutations such as FLT3, PML- RARA, RAS, AML1, and NPM1 mutations were additionally explored. Results The prevalence of IDH1 and IDH2 mutations was 8.7% (20/230) and 10.4% (24/230), respectively. Six missense mutations were identified among IDH1-mutated cases; p.R132H (n = 8), p.R132C (n = 6), p.R132S (n = 2), p.R132G (n = 2), p.R132L (n = 1), and p.I99M (n = 1). Two missense mutations were found in IDH2-mutated cases; p.R140Q (n = 20) and p.R172K (n = 4). No patients had dual IDH1 and IDH2 mutations. About 18% of AML with normal cytogenetics and 31% of acute promyelocytic leukemia had IDH mutations. Half of the IDH-mutated cohort had normal karyotype and the major FAB subtype was AML-M2. Interestingly, IDH1- and IDH2-mutated cases predominantly had NPM1 mutations (60-74%) as compared to the wild type (P < 0.001). Very few IDH-mutated cases had FLT3 and/or RAS abnormalities and none of them had AML1 mutations. Older age and higher median platelet counts were significantly associated with IDH2 mutations although the clinical impact of either IDH1 or IDH2 mutations on patients' overall survival could not be observed. Conclusion Overall, 19% of newly diagnosed AML patients had alterations of IDH genes. No patients concurrently carried both IDH1 and IDH2 mutations suggesting that these mutations were mutually exclusive. NPM1 mutation appears as a major coexisting genetic mutation in IDH-mutated patients. Our present data failed to support the prognostic relevance of IDH mutations although alterations of these metabolic genes potentially have an important role in leukemia development.