Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum.

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an "irresistible" compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this "mutator" parasite can be leveraged to drive P. falciparum resistome discovery.

SARS-CoV-2 RT-PCR positivity of individuals subsequent to completing quarantine upon entry into a country during a transmission-free period.

BACKGROUND: During the current coronavirus disease 2019 (COVID-19) pandemic, many countries require travellers to undergo a reverse transcription-polymerase chain reaction (RT-PCR) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before travelling across borders. However, in persons having recovered from COVID-19, RT-PCR positivity can persist for an extended period. MATERIALS AND METHODS: We describe three cases who sought fit-to-fly certificates in Thailand during the period free of local transmission but were tested positive for RT-PCR for SARS-CoV-2. All had returned from a country with an active outbreak of COVID-19. Their clinical courses are described; positive nasopharyngeal swab samples were processed for viral isolation and whole-genome sequencing (WGS); and serology as well as neutralizing antibody were assessed. The contact tracing was carried out for determining evidence of indigenous transmission among close contacts of those three cases. RESULTS: All three cases were completely asymptomatic. Chest computerized tomography was not compatible with COVID-19 pneumonia; cell cultures failed to rescue replication-competent virus; WGS revealed fragmented viral genetic material from nasopharyngeal swab samples; and serological tests demonstrated stable levels of antibodies, together with the presence of neutralizing antibody, suggesting past infection with negligible transmission risk. Contact tracing identified no transmission in high-risk close contact individuals. CONCLUSION: RT-PCR positivity for SARS-CoV-2 might detect fragmented viral genome. Issuance of a travel certificate in these circumstances is problematic. Serology tests can help to define past infection. A practical acceptable set of guidelines for issuance of a COVID-19 safety travel certification is a necessity.

COVID-19 Transmission among Healthcare Workers at a Quarantine Facility in Thailand: Genomic and Outbreak Investigations.

During the COVID-19 pandemic, Thailand implemented a quarantine program at approved quarantine facilities for every international traveler. Here, we report an epidemiological and genomic investigation of a COVID-19 cluster consisting of seven healthcare workers (HCWs) at a quarantine facility and its partnered hospital in Thailand. Outbreak investigations were implemented to obtain contact tracing data and to establish chains of transmission. Genomic sequencing of SARS-CoV-2 with samples within the cohort was performed. Investigations of 951 HCWs and staff with quarantined travelers were implemented to determine the chain of transmission. Genomic and outbreak investigations identified the international travelers infected with the B.1.1.31 SARS-CoV-2 lineage as the source of this outbreak. The genomic data and the investigated timeline revealed a putative transmission chain among HCWs, pointing toward the transmission via the use of common living quarters at the investigated quarantine site. The evaluation of this cohort has led to a policy recommendation on quarantine facility management. International travel quarantine is an important strategy to contain importation of COVID-19 cases. However, a quarantine facility is likely to become a potential hotspot, requiring thorough preventive measures. Reducing the exposure risk by providing private living quarters and scheduling clinical duties at a quarantine site separated from the conventional healthcare workforce have been implemented.

Genomic surveillance of SARS-CoV-2 in Thailand reveals mixed imported populations, a local lineage expansion and a virus with truncated ORF7a.

Coronavirus Disease 2019 (COVID-19) is a global public health threat. Genomic surveillance of SARS-CoV-2 was implemented in March of 2020 at a major diagnostic hub in Bangkok, Thailand. Several virus lineages supposedly originated in many countries were found, and a Thai-specific lineage, designated A/Thai-1, has expanded to be predominant in Thailand. A virus sample in the SARS-CoV-2 A/Thai-1 lineage contains a frame-shift deletion at ORF7a, encoding a putative host antagonizing factor of the virus.

The resistome and genomic reconnaissance in the age of malaria elimination.

Malaria is an infectious disease caused by parasitic protozoa in the Plasmodium genus. A complete understanding of the biology of these parasites is challenging in view of their need to switch between the vertebrate and insect hosts. The parasites are also capable of becoming highly motile and of remaining dormant for decades, depending on the stage of their life cycle. Malaria elimination efforts have been implemented in several endemic countries, but the parasites have proven to be resilient. One of the major obstacles for malaria elimination is the development of antimalarial drug resistance. Ineffective treatment regimens will fail to remove the circulating parasites and to prevent the local transmission of the disease. Genomic epidemiology of malaria parasites has become a powerful tool to track emerging drug-resistant parasite populations almost in real time. Population-scale genomic data are instrumental in tracking the hidden pockets of Plasmodium in nationwide elimination efforts. However, genomic surveillance data can be useful in determining the threat only when combined with a thorough understanding of the malarial resistome - the genetic repertoires responsible for causing and potentiating drug resistance evolution. Even though long-term selection has been a standard method for drug target identification in laboratories, its implementation in large-scale exploration of the druggable space in Plasmodium falciparum, along with genome-editing technologies, have enabled mapping of the genetic repertoires that drive drug resistance. This Review presents examples of practical use and describes the latest technology to show the power of real-time genomic epidemiology in achieving malaria elimination.

Overexpression of plasmepsin II and plasmepsin III does not directly cause reduction in Plasmodium falciparum sensitivity to artesunate, chloroquine and piperaquine.

Artemisinin derivatives and their partner drugs in artemisinin combination therapies (ACTs) have played a pivotal role in global malaria mortality reduction during the last two decades. The loss of artemisinin efficacy due to evolving drug-resistant parasites could become a serious global health threat. Dihydroartemisinin-piperaquine is a well tolerated and generally highly effective ACT. The implementation of a partner drug in ACTs is critical in the control of emerging artemisinin resistance. Even though artemisinin is highly effective in parasite clearance, it is labile in the human body. A partner drug is necessary for killing the remaining parasites when the pulses of artemisinin have ceased. A population of Plasmodium falciparum parasites in Cambodia and adjacent countries has become resistant to piperaquine. Increased copy number of the genes encoding the haemoglobinases Plasmepsin II and Plasmepsin III has been linked with piperaquine resistance by genome-wide association studies and in clinical trials, leading to the use of increased plasmepsin II/plasmepsin III copy number as a molecular marker for piperaquine resistance. Here we demonstrate that overexpression of plasmepsin II and plasmepsin III in the 3D7 genetic background failed to change the susceptibility of P. falciparum to artemisinin, chloroquine and piperaquine by both a standard dose-response analysis and a piperaquine survival assay. Whilst plasmepsin copy number polymorphism is currently implemented as a molecular surveillance resistance marker, further studies to discover the molecular basis of piperaquine resistance and potential epistatic interactions are needed.

Fitness Loss under Amino Acid Starvation in Artemisinin-Resistant Plasmodium falciparum Isolates from Cambodia.

Artemisinin is the most rapidly effective drug for Plasmodium falciparum malaria treatment currently in clinical use. Emerging artemisinin-resistant parasites pose a great global health risk. At present, the level of artemisinin resistance is still relatively low with evidence pointing towards a trade-off between artemisinin resistance and fitness loss. Here we show that artemisinin-resistant P. falciparum isolates from Cambodia manifested fitness loss, showing fewer progenies during the intra-erythrocytic developmental cycle. The loss in fitness was exacerbated under the condition of low exogenous amino acid supply. The resistant parasites failed to undergo maturation, whereas their drug-sensitive counterparts were able to complete the erythrocytic cycle under conditions of amino acid deprivation. The artemisinin-resistant phenotype was not stable, and loss of the phenotype was associated with changes in the expression of a putative target, Exp1, a membrane glutathione transferase. Analysis of SNPs in haemoglobin processing genes revealed associations with parasite clearance times, suggesting changes in haemoglobin catabolism may contribute to artemisinin resistance. These findings on fitness and protein homeostasis could provide clues on how to contain emerging artemisinin-resistant parasites.

Comparative genome analysis between Southeast Asian and South American Zika viruses.

OBJECTIVE: To understand the cause for the differences between potentially mild Southeast Asian and the more pathogenic ZIKV in South America. METHODS: A comparative genomic analysis was performed to determine putative causations stemming from ZIKV. RESULTS: Phylogenetic analyses integrating geographical and time factors revealed that Southeast Asian ZIKV might not be the direct source of South American outbreaks as previously speculated. Amino acid residues unique to South American ZIKV isolates at the envelope, pr and NS1 proteins are listed and shown in the structural context. These unique residues on external viral proteins are not found in Southeast Asian ZIKV and could be responsible for the ongoing outbreak either via an intrinsic property of the virus or interactions with human immunity. Only a selected few primer/probe sets currently in clinical use were identified of being capable of detecting ZIKV strains worldwide. The envelope proteins of dengue virus (DENV) and ZIKV also showed a remarkable degree of similarity especially at the surface residues. CONCLUSIONS: These findings may help explain the cross-reactivity of DENV antibodies to ZIKV. Thus, major caveats must be exercised in using existing diagnostic tools for ZIKV.

Comparative genome-wide analysis and evolutionary history of haemoglobin-processing and haem detoxification enzymes in malarial parasites.

BACKGROUND: Malaria parasites have evolved a series of intricate mechanisms to survive and propagate within host red blood cells. Intra-erythrocytic parasitism requires these organisms to digest haemoglobin and detoxify iron-bound haem. These tasks are executed by haemoglobin-specific proteases and haem biocrystallization factors that are components of a large multi-subunit complex. Since haemoglobin processing machineries are functionally and genetically linked to the modes of action and resistance mechanisms of several anti-malarial drugs, an understanding of their evolutionary history is important for drug development and drug resistance prevention. METHODS: Maximum likelihood trees of genetic repertoires encoding haemoglobin processing machineries within Plasmodium species, and with the representatives of Apicomplexan species with various host tropisms, were created. Genetic variants were mapped onto existing three-dimensional structures. Genome-wide single nucleotide polymorphism data were used to analyse the selective pressure and the effect of these mutations at the structural level. RESULTS: Recent expansions in the falcipain and plasmepsin repertoires are unique to human malaria parasites especially in the Plasmodium falciparum and P. reichenowi lineage. Expansion of haemoglobin-specific plasmepsins occurred after the separation event of Plasmodium species, but the other members of the plasmepsin family were evolutionarily conserved with one copy for each sub-group in every Apicomplexan species. Haemoglobin-specific falcipains are separated from invasion-related falcipain, and their expansions within one specific locus arose independently in both P. falciparum and P. vivax lineages. Gene conversion between P. falciparum falcipain 2A and 2B was observed in artemisinin-resistant strains. Comparison between the numbers of non-synonymous and synonymous mutations suggests a strong selective pressure at falcipain and plasmepsin genes. The locations of amino acid changes from non-synonymous mutations mapped onto protein structures revealed clusters of amino acid residues in close proximity or near the active sites of proteases. CONCLUSION: A high degree of polymorphism at the haemoglobin processing genes implicates an imposition of selective pressure. The identification in recent years of functional redundancy of haemoglobin-specific proteases makes them less appealing as potential drug targets, but their expansions, especially in the human malaria parasite lineages, unequivocally point toward their functional significance during the independent and repetitive adaptation events in malaria parasite evolutionary history.

Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I.

BACKGROUND: Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. METHODS: Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. RESULTS: Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. CONCLUSIONS: Functional assays for P. falciparum GCH1 based on enzymatic activity and genetic complementation were successfully developed. The assays in combination with a homology model characterized the enzymatic activity of P. falciparum GCH1 and the importance of its key amino acid residues. The potential to use the assay for inhibitor screening was validated by 8-oxo-GTP, a known GTP analogue inhibitor.

A nuclear targeting system in Plasmodium falciparum.

BACKGROUND: The distinct differences in gene control mechanisms acting in the nucleus between Plasmodium falciparum and the human host could lead to new potential drug targets for anti-malarial development. New molecular toolkits are required for dissecting molecular machineries in the P. falciparum nucleus. One valuable tool commonly used in model organisms is protein targeting to specific sub-cellular locations. Targeting proteins to specified locations allows labeling of organelles for microscopy, or testing of how the protein of interest modulates organelle function. In recent years, this approach has been developed for various malaria organelles, such as the mitochondrion and the apicoplast. A tool for targeting a protein of choice to the P. falciparum nucleus using an exogenous nuclear localization sequence is reported here. METHODS: To develop a nuclear targeting system, a putative nuclear localization sequence was fused with green fluorescent protein (GFP). The nuclear localization sequence from the yeast transcription factor Gal4 was chosen because of its well-defined nuclear localization signal. A series of truncated Gal4 constructs was also created to narrow down the nuclear localization sequence necessary for P. falciparum nuclear import. Transfected parasites were analysed by fluorescent and laser-scanning confocal microscopy. RESULTS: The nuclear localization sequence of Gal4 is functional in P. falciparum. It effectively transported GFP into the nucleus, and the first 74 amino acid residues were sufficient for nuclear localization. CONCLUSIONS: The Gal4 fusion technique enables specific transport of a protein of choice into the P. falciparum nucleus, and thus provides a tool for labeling nuclei without using DNA-staining dyes. The finding also indicates similarities between the nuclear transport mechanisms of yeast and P. falciparum. Since the nuclear transport system has been thoroughly studied in yeast, this could give clues to research on the same mechanism in P. falciparum.