The Impact of Preparedness in Defying COVID-19 Pandemic Expectations in the Lower Mekong Region: A Case Study

Dire COVID-19 expectations in the Lower Mekong Region (LMR) can be understood as Cambodia, the Lao PDR, Myanmar, Thailand, and Vietnam have stared down a succession of emerging infectious disease (EID) threats from neighboring China. Predictions that the LMR would be overwhelmed by a coming COVID-19 tsunami were felt well before the spread of the COVID-19 pandemic had been declared. And yet, the LMR, excepting Myanmar, has proved surprisingly resilient in keeping COVID-19 contained to mostly sporadic cases. Cumulative case rates (per one million population) for the LMR, including or excluding Myanmar, from January 1 to October 31 2020, are 1,184 and 237, respectively. More telling are the cumulative rates of COVID-19–attributable deaths for the same period of time, 28 per million with and six without Myanmar. Graphics demonstrate a flattening of pandemic curves in the LMR, minus Myanmar, after managing temporally and spatially isolated spikes in case counts, with negligible follow-on community spread. The comparable success of the LMR in averting pandemic disaster can likely be attributed to years of preparedness investments, triggered by avian influenza A (H5N1). Capacity building initiatives applied to COVID-19 containment included virological (influenza-driven) surveillance, laboratory diagnostics, field epidemiology training, and vaccine preparation. The notable achievement of the LMR in averting COVID-19 disaster through to October 31, 2020 can likely be credited to these preparedness measure

Validation of Plasmodium vivax centromere and promoter activities using Plasmodium yoelii

Plasmodium vivax is the leading cause of malaria outside Africa and represents a significant health and economic burden on affected countries. A major obstacle for P. vivax eradication is the dormant hypnozoite liver stage that causes relapse infections and the limited antimalarial drugs that clear this stage. Advances in studying the hypnozoite and other unique biological aspects of this parasite are hampered by the lack of a continuous in vitro laboratory culture system and poor availability of molecular tools for genetic manipulation. In this study, we aim to develop molecular tools that can be used for genetic manipulation of P. vivax. A putative P. vivax centromere sequence (PvCEN) was cloned and episomal centromere based plasmids expressing a GFP marker were constructed. Centromere activity was evaluated using a rodent malaria parasite Plasmodium yoelii. A plasmid carrying PvCEN was stably maintained in asexual-stage parasites in the absence of drug pressure, and approximately 45% of the parasites retained the plasmid four weeks later. The same retention rate was observed in parasites possessing a native P. yoelii centromere (PyCEN)-based control plasmid. The segregation efficiency of the plasmid per nuclear division was > 99% in PvCEN parasites, compared to ?90% in a control parasite harboring a plasmid without a centromere. In addition, we observed a clear GFP signal in both oocysts and salivary gland sporozoites isolated from mosquitoes. In blood-stage parasites after liver stage development, GFP positivity in PvCEN parasites was comparable to control PyCEN parasites. Thus, PvCEN plasmids were maintained throughout the parasite life cycle. We also validated several P. vivax promoter activities and showed that hsp70 promoter (?1 kb) was active throughout the parasite life cycle. This is the first data for the functional characterization of a P. vivax centromere that can be used in future P. vivax biological research

Low seroprevalence of COVID-19 in Lao PDR, late 2020

Background In 2020 Lao PDR had low reported COVID-19 cases but it was unclear whether this masked silent transmission. A seroprevalence study was done August - September 2020 to determine SARS-CoV-2 exposure. Methods Participants were from the general community (n=2433) or healthcare workers (n=666) in five provinces and bat/wildlife contacts (n=74) were from Vientiane province. ELISAs detected anti- SARS-CoV-2 Nucleoprotein (N; n=3173 tested) and Spike (S; n=1417 tested) antibodies. Double-positive samples were checked by IgM/IgG rapid tests. Controls were confirmed COVID-19 cases (n=15) and pre-COVID-19 samples (n=265). Seroprevalence for the general community was weighted to account for complex survey sample design, age and sex. Findings In pre-COVID-19 samples, 5·3%, [95% CI=3·1-8·7%] were anti-N antibody single-positive and 1·1% [0·3-3·5%] were anti-S antibody single positive. None were double positive. Anti-N and anti-S antibodies were detected in 5·2% [4·2-6·5%] and 2·1% [1·1-3·9%] of the general community, 2·0% [1·1-3·3%] and 1·4% [0·5-3·7%] of healthcare workers and 20·3% [12·6-31·0%] and 6·8% [2·8-15·3%] of bat/wildlife contacts. 0·1% [0·02-0·3%] were double positive for anti-N and anti-S antibodies (rapid test negative). Interpretation We find no evidence for significant SARS-CoV-2 circulation in Lao PDR before September 2020. This likely results from early decisive measures taken by the government, social behavior, and low population density. High anti-N /low anti-S seroprevalence in bat/wildlife contacts may indicate exposure to cross-reactive animal coronaviruses with threat of emerging novel viruses. Funding Agence Française de Développement. Additional; Institut Pasteur du Laos, Institute Pasteur, Paris and Luxembourg Ministry of Foreign and European Affairs (“PaReCIDS II”)

Pooling samples to increase testing capacity with Xpert Xpress SARS-CoV-2 during the Covid-19 pandemic in Lao People's Democratic Republic.

The COVID-19 pandemic created the need for large-scale testing of populations. However, most laboratories do not have sufficient testing capacity for mass screening. We evaluated pooled testing of samples, as a strategy to increase testing capacity in Lao PDR. Samples of consecutive patients were tested in pools of four using the Xpert Xpress SARS CoV-2 assay. Positive pools were confirmed by individual testing, and we describe the performance of the test and savings achieved. We also diluted selected positive samples to describe its effect on the assays CT values. 1,568 patients were tested in 392 pools of four. 361 (92.1%) pools were negative and 31 (7.9%) positive. 29/31 (93.5% (95%CI 77-99%) positive pools were confirmed by individual testing of the samples but, in 2/31 (6.5%) the four individual samples were negative, suggesting contamination. Pools with only one positive sample had higher CT values (lower RNA concentrations) than the respective individual samples, indicating a dilution effect, which suggested an increased risk of false negative results with dilutions >1:10. However, this risk may be low if the prevalence of infection is high, when pools are more likely to contain more than one positive sample. Pooling saved 67% of cartridges and substantially increased testing capacity. Pooling samples increased SARS-CoV-2 testing capacity and resulted in considerable cartridge savings. Given the need for high-volume testing, countries may consider implementation of pooling for SARS-CoV-2 screening

Stable allele frequency distribution of the polymorphic region of SURFIN4.2 in Plasmodium falciparum isolates from Thailand

Plasmodium falciparum SURFIN 4.2 (PFD1160w) is a polymorphic protein expressed on the surface of parasite-infected erythrocytes. Such molecules are expected to be under strong host immune pressure, thus we analyzed the nucleotide diversity of the N-terminal extracellular region of SURFIN 4.2 using P. falciparum isolates obtained from a malaria hypoendemic area of Thailand. The extracellular region of SURFIN 4.2 was divided into four regions based on the amino acid sequence conservation among SURFIN members and the level of polymorphism among SURFIN 4.2 sequences; N-terminal segment (Nter), a cysteine-rich domain (CRD), a variable region 1 (Var1), and a variable region 2 (Var2). Comparison between synonymous and non-synonymous substitutions, Tajima\u27s D test, and Fu and Li\u27s D* and F* tests detected signatures of positive selection on Var2 and to a lesser extent Var1, suggesting that these regions were likely under host immune pressure. Strong linkage disequilibrium was detected for nucleotide pairs separated by a distance of more than 1.5 kb, and 7 alleles among 19 alleles detected in 1988-1989 still circulated 14 years later, suggesting low recombination of the analyzed surf 4.2 sequence region in Thailand. The allele frequency distribution of polymorphic areas in Var2 did not differ between two groups collected in different time points, suggesting the allele frequency distribution of this region was stable for 14 years. The observed allele frequency distribution of SURFIN 4.2 Var2 may be fixed in Thai P. falciparum population as similar to the observation for P. falciparum merozoite surface protein 1, for which a stable allele frequency distribution was reported

Genetic Diversity and Lack of Artemisinin Selection Signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca2+-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average dN/dS ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance

Rapid identification of genes controlling virulence and immunity in malaria parasites

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.This work was supported by the JSPS (project numbers Nos. JP25870525, JP24255009 and JP16K21233) (to RCu), A Royal Society Bilateral Grant for Co-operative Research (to RCa and RCu) and a Sasakawa Foundation Butterfield Award (to RCu), faculty baseline fund (BAS/1/1020-01-01) from the King Abdullah University of Science and Technology (KAUST) to AP, and Grants-in-Aid for Scientific Research on Innovative Areas JR23117008 (to OK). CJRI was supported by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (101239/Z/13/Z)

熱帯熱マラリア原虫多型抗原のアミノ酸型頻度分布の経時的ダイナミクス

長崎大学学位論文 [学位記番号]博（医歯薬）甲第540号 [学位授与年月日]平成24年9月20

Validation of Plasmodium vivax centromere and promoter activities using Plasmodium yoelii

Plasmodium vivax is the leading cause of malaria outside Africa and represents a significant health and economic burden on affected countries. A major obstacle for P. vivax eradication is the dormant hypnozoite liver stage that causes relapse infections and the limited antimalarial drugs that clear this stage. Advances in studying the hypnozoite and other unique biological aspects of this parasite are hampered by the lack of a continuous in vitro laboratory culture system and poor availability of molecular tools for genetic manipulation. In this study, we aim to develop molecular tools that can be used for genetic manipulation of P. vivax. A putative P. vivax centromere sequence (PvCEN) was cloned and episomal centromere based plasmids expressing a GFP marker were constructed. Centromere activity was evaluated using a rodent malaria parasite Plasmodium yoelii. A plasmid carrying PvCEN was stably maintained in asexual-stage parasites in the absence of drug pressure, and approximately 45% of the parasites retained the plasmid four weeks later. The same retention rate was observed in parasites possessing a native P. yoelii centromere (PyCEN)-based control plasmid. The segregation efficiency of the plasmid per nuclear division was > 99% in PvCEN parasites, compared to ∼90% in a control parasite harboring a plasmid without a centromere. In addition, we observed a clear GFP signal in both oocysts and salivary gland sporozoites isolated from mosquitoes. In blood-stage parasites after liver stage development, GFP positivity in PvCEN parasites was comparable to control PyCEN parasites. Thus, PvCEN plasmids were maintained throughout the parasite life cycle. We also validated several P. vivax promoter activities and showed that hsp70 promoter (∼1 kb) was active throughout the parasite life cycle. This is the first data for the functional characterization of a P. vivax centromere that can be used in future P. vivax biological research