Seong-Kyun Lee

Comparison of Microscopy, Nested-PCR, and Real-Time-PCR Assays Using High-Throughput Screening of Pooled Samples for Diagnosis of Malaria in Asymptomatic Carriers from Areas of Endemicity in Myanmar

ABSTRACT Asymptomatic infection is an important obstacle for controlling disease in countries where malaria is endemic. Because asymptomatic carriers do not seek treatment for their infections, they can have high levels of gametocytes and constitute a reservoir available for new infection. We employed a sample pooling/PCR-based molecular detection strategy for screening malaria infection in residents from areas of Myanmar where malaria is endemic. Blood samples (n = 1,552) were collected from residents in three areas of malaria endemicity (Kayin State, Bago, and Tanintharyi regions) of Myanmar. Two nested PCR and real-time PCR assays showed that asymptomatic infection was detected in about 1.0% to 9.4% of residents from the surveyed areas. The sensitivities of the two nested PCR and real-time PCR techniques were higher than that of microscopy examination (sensitivity, 100% versus 26.4%; kappa values, 0.2 to 0.5). Among the three regions, parasite-positive samples were highly detected in subjects from the Bago and Tanintharyi regions. Active surveillance of residents from regions of intense malaria transmission would reduce the risk of morbidity and mitigate transmission to the population in these areas of endemicity. Our data demonstrate that PCR-based molecular techniques are more efficient than microscopy for nationwide surveillance of malaria in countries where malaria is endemic.

Identification of a reticulocyte-specific binding domain of Plasmodium vivax reticulocyte-binding protein 1 that is homologous to the PfRh4 erythrocyte-binding domain

The Plasmodium vivax reticulocyte-binding protein (RBP) family was identified based on the annotation of adhesive ligands in the P. vivax genome. Reticulocyte-specific interactions with the PvRBPs (PvRBP1 and PvRBP2) were previously reported. Plasmodium falciparum reticulocyte-binding protein homologue 4 (PfRh4, a homologue of PvRBP1) was observed to possess erythrocyte-binding activity via complement receptor 1 on the erythrocyte surface. However, the reticulocyte-binding mechanisms of P. vivax are unclear because of the large molecular mass of PvRBP1 (>326 kDa) and the difficulty associated with in vitro cultivation. In the present study, 34 kDa of PvRBP1a (PlasmoDB ID: PVX_098585) and 32 kDa of PvRBP1b (PVX_098582) were selected from a 30 kDa fragment of PfRh4 for reticulocyte-specific binding activity analysis. Both PvRBP1a and PvRBP1b were found to be localized at the microneme in the mature schizont-stage parasites. Naturally acquired immune responses against PvRBP1a-34 and PvRBP1b-32 were observed lower than PvDBP-RII. The reticulocyte-specific binding activities of PvRBP1a-34 and PvRBP1b-32 were significantly higher than normocyte binding activity and were significantly reduced by chymotrypsin treatment. PvRBP1a and 1b, bind to reticulocytes and that this suggests that these ligands may have an important role in P. vivax merozoite invasion.

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.

Immunoprofiling of the Tryptophan-Rich Antigen Family in Plasmodium vivax

ABSTRACT Tryptophan-rich antigens (TRAgs) are an antigen family that has been identified in human and rodent malaria parasites. TRAgs have been proposed as candidate antigens for potential vaccines. The Plasmodium vivax TRAg (PvTRAg) family includes 36 members. Each PvTRAg contains a tryptophan-rich (TR) domain in the C-terminal region. In this study, we recombinantly expressed all 36 PvTRAgs using a cell-free expression system, and, for the first time, profiled the IgG antibody responses against all PvTRAgs in the sera from 96 vivax malaria patients and 40 healthy individuals using protein microarray technology. The mean seropositive rate for all PvTRAgs was 60.3%. Among them, nine PvTRAgs were newly identified in this study and showed a seropositive rate of >50%. Five of them, PvTRAg_13, PvTRAg_15, PvTRAg_16, PvTRAg_26, and PvTRAg_29, produced higher levels of IgG antibody, even in low-endemicity countries. In addition, the results of an immunofluorescence analysis suggest that PvTRAgs are, at least in part, associated with caveola-vesicle complexes, a unique structure of P. vivax-infected erythrocytes. The mechanism of formation and the function of these abundant membrane structures are not known. Further investigation aimed at determining the functions of these proteins would lead to a better understanding of the blood-stage biology of P. vivax.

Clinical and molecular surveillance of drug resistant vivax malaria in Myanmar (2009–2016)

BackgroundOne of the major challenges for control and elimination of malaria is ongoing spread and emergence of drug resistance. While epidemiology and surveillance of the drug resistance in falciparum malaria is being explored globally, there are few studies on drug resistance vivax malaria.MethodsTo assess the spread of drug-resistant vivax malaria in Myanmar, a multisite, prospective, longitudinal study with retrospective analysis of previous therapeutic efficacy studies, was conducted. A total of 906 from nine study sites were included in retrospective analysis and 208 from three study sites in prospective study. Uncomplicated vivax mono-infected patients were recruited and monitored with longitudinal follow-up until day 28 after treatment with chloroquine. Amplification and sequence analysis of molecular markers, such as mutations in pvcrt-O, pvmdr1, pvdhps and pvdhfr, were done in day-0 samples in prospective study.ResultsClinical failure cases were found only in Kawthaung, southern Myanmar and western Myanmar sites within 2009–2016. Chloroquine resistance markers, pvcrt-O ‘AAG’ insertion and pvmdr1 mutation (Y976F) showed higher mutant rate in southern and central Myanmar than western site: 66.7, 72.7 vs 48.3% and 26.7, 17.0 vs 1.7%, respectively. A similar pattern of significantly higher mutant rate of antifolate resistance markers, pvdhps (S382A, K512M, A553G) and pvdhfr (F57L/I, S58R, T61M, S117T/N) were noted.ConclusionsAlthough clinical failure rate was low, widespread distribution of chloroquine and antifolate resistance molecular makers alert to the emergence and spread of drug resistance vivax malaria in Myanmar. Proper strategy and action plan to eliminate and contain the resistant strain strengthened together with clinical and molecular surveillance on drug resistance vivax is recommended.

Molecular Evidence of Drug Resistance in Asymptomatic Malaria Infections, Myanmar, 2015

Artemisinin resistance containment in Myanmar was initiated in 2011 after artemisinin-resistant Plasmodium falciparum malaria was reported. Molecular evidence suggests that asymptomatic malaria infections harboring drug resistance genes are present among residents of the Myanmar artemisinin resistance containment zone. This evidence supports efforts to eliminate these hidden infections.

Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1.

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.

Cross-species analysis of apical asparagine-rich protein of Plasmodium vivax and Plasmodium knowlesi

The Plasmodium falciparum apical asparagine (Asn)-rich protein (AARP) is one of malarial proteins, and it has been studied as a candidate of malaria subunit vaccine. Basic characterization of PvAARP has been performed with a focus on its immunogenicity and localization. In this study, we further analyzed the immunogenicity of PvAARP, focusing on the longevity of the antibody response, cross-species immunity and invasion inhibitory activity by using the primate malaria parasite Plasmodium knowlesi. We found that vivax malaria patient sera retained anti-PvAARP antibodies for at least one year without re-infection. Recombinant PvAARP protein was strongly recognized by knowlesi malaria patients. Antibody raised against the P. vivax and P. knowlesi AARP N-termini reacted with the apical side of the P. knowlesi merozoites and inhibited erythrocyte invasion by P. knowlesi in a concentration-dependent manner, thereby suggesting a cross-species nature of anti-PvAARP antibody against PkAARP. These results can be explained by B cell epitopes predicted in conserved surface-exposed regions of the AARP N-terminus in both species. The long-lived anti-PvAARP antibody response, cross-reactivity, and invasion inhibitory activity of anti-PvAARP support a critical role of AARP during the erythrocyte invasion and suggest that PvAARP induces long-lived cross-species protective immunity against P. vivax and P. knowlesi.

Correction for Wang et al., “Immunoprofiling of the Tryptophan-Rich Antigen Family in Plasmodium vivax”

Volume 83, no. 8, p. 3083–3095, 2015, [https://doi.org/10.1128/IAI.03067-14][1]. Page 3090: Figure 4B should appear as shown below. The original figure contained a duplication in 26H gel and 26R gel, as the result of an error in figure preparation. ![Figure][2] [1]: /lookup/doi/10.1128/