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.

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Sideways: road to gene-by-gene functional screening in malaria parasites.

Genome-wide screening in apicomplexan species has transformed our understanding of these parasitic protozoa. Kimmel et al. report a 'knock sideways' system and provide a powerful use case for its feasibility in a gene-by-gene screening in Plasmodium falciparum. Carefully deployed, a novel toolkit helps to dissect the biological uniqueness of an important parasite.

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.

Lumefantrine attenuates Plasmodium falciparum artemisinin resistance during the early ring stage.

Emerging artemisinin resistance in Plasmodium falciparum malaria has the potential to become a global public health crisis. In Southeast Asia, this phenomenon clinically manifests in the form of delayed parasite clearance following artemisinin treatment. Reduced artemisinin susceptibility is limited to the early ring stage window, which is sufficient to allow parasites to survive the short half-life of artemisinin exposure. A screen of known clinically-implemented antimalarial drugs was performed to identify a drug capable of enhancing the killing activity of artemisinins during this critical resistance window. As a result, lumefantrine was found to increase the killing activity of artemisinin against an artemisinin-resistant clinical isolate harboring the C580Y kelch13 mutation. Isobologram analysis revealed synergism during the early ring stage resistance window, when lumefantrine was combined with artemether, an artemisinin derivative clinically partnered with lumefantrine. These findings suggest that lumefantrine should be clinically explored as a partner drug in artemisinin-based combination therapies to control emerging artemisinin resistance.

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

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.

Role of a Concentration Gradient in Malaria Drug Resistance Evolution: A Combined within- and between-Hosts Modelling Approach.

Resistance to antimalarial drugs is currently a growing public health problem, resulting in more cases with treatment failure. Although previous studies suggested that a concentration gradient facilitates the antibiotic resistance evolution in bacteria, no attempt has been made to investigate the roles of a concentration gradient in malaria drug resistance. Unlike the person-to-person mode of transmission of bacteria, the malaria parasites need to switch back and forth between the human and mosquito hosts to complete the life cycle and to spread the resistant alleles. Here we developed a stochastic combined within- and between-hosts evolutionary dynamics model specific to malaria parasites in order to investigate the influence of an antimalarial concentration gradient on the evolutionary dynamics of malaria drug resistance. Every stage of malaria development in both human and mosquito hosts are individually modelled using the tau-leaping algorithm. We found that the concentration gradient can accelerate antimalarial resistance evolution. The gain in resistance evolution was improved by the increase in the parasite mutation rate and the mosquito biting rate. In addition, even though the rate of resistance evolution is not sensitive to the changes in parasite reduction ratios (PRRs) of antimalarial drugs, the probability of finding the antimalarial drug resistant parasites decreases when the PRR increases.

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.

Plasmodium falciparum Falcipain-2a Polymorphisms in Southeast Asia and Their Association With Artemisinin Resistance.

Background: Falcipain-2a ([FP2a] PF3D7_1115700) is a Plasmodium falciparum cysteine protease and hemoglobinase. Functional FP2a is required for potent activity of artemisinin, and in vitro selection for artemisinin resistance selected for an FP2a nonsense mutation. Methods: To investigate associations between FP2a polymorphisms and artemisinin resistance and to characterize the diversity of the enzyme in parasites from the China-Myanmar border, we sequenced the full-length FP2a gene in 140 P falciparum isolates collected during 2004-2011. Results: The isolates were grouped into 8 different haplotype groups. Haplotype group I appeared in samples obtained after 2008, coinciding with implementation of artemisinin-based combination therapy in this region. In functional studies, compared with wild-type parasites, the FP2a haplotypes demonstrated increased ring survival, and all haplotype groups exhibited significantly reduced FP2a activity, with group I showing the slowest protease kinetics and reduced parasite fitness. Conclusions: These results suggest that altered hemoglobin digestion due to FP2a mutations may contribute to artemisinin resistance.

How to combat emerging artemisinin resistance: Lessons from "The Three Little Pigs".

It is rare to come across an Aesop's fable in respectable journals. It might catch scientists outside the malaria field by surprise to learn that the famous story of "The Boy Who Cried Wolf" has been repeatedly compared to the threat from artemisinin-resistant malaria parasites, including the two latest reports on the rise of a specific haplotype in Cambodia and Thailand, sensationally dubbed "Super Malaria" by the media [1, 2]. The comparison to a children's tale should not negate the fact that malaria drug resistance is one of the most pressing threats to the global public health community. Here, the findings leading to this contentious discourse will be delineated in order to provide a perspective. Possible solutions will be presented to stimulate further research and discussion to solve one of the greatest public health challenges of our lifetime.

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.

Plasmodium falciparum malaria: Convergent evolutionary trajectories towards delayed clearance following artemisinin treatment.

Malaria is a major global health challenge with 300million new cases every year. The most effective regimen for treating Plasmodium falciparum malaria is based on artemisinin and its derivatives. The drugs are highly effective, resulting in rapid clearance of parasites even in severe P. falciparum malaria patients. During the last five years, artemisinin-resistant parasites have begun to emerge first in Cambodia and now in Thailand and Myanmar. At present, the level of artemisinin resistance is relatively low with clinical presentation of delayed artemisinin clearance (a longer time to reduce parasite load) and a small decrease in artemisinin sensitivity in cultured isolates. Nevertheless, multiple genetic loci associated with delayed parasite clearance have been reported, but they cannot account for a large portion of cases. Even the most well-studied kelch 13 propeller mutations cannot always predict the outcome of artemisinin treatment in vitro and in vivo. Here we propose that delayed clearance by artemisinin could be the result of convergent evolution, driven by multiple trajectories to overcome artemisinin-induced stress, but precluded to become full blown resistance by high fitness cost. Genetic association studies by several genome-wide approaches reveal linkage disequilibrium between multiple loci and delayed parasite clearance. Genetic manipulations at some of these loci already have resulted in loss in artemisinin sensitivity. The notion presented here is by itself consistent with existing evidence on artemisinin resistance and has the potential to be explored using available genomic data. Most important of all, molecular surveillance of artemisinin resistance based on multi-genic markers could be more informative than relying on any one particular molecular marker.

Combinatorial Genetic Modeling of pfcrt-Mediated Drug Resistance Evolution in Plasmodium falciparum.

The emergence of drug resistance continuously threatens global control of infectious diseases, including malaria caused by the protozoan parasite Plasmodium falciparum A critical parasite determinant is the P. falciparum chloroquine resistance transporter (PfCRT), the primary mediator of chloroquine (CQ) resistance (CQR), and a pleiotropic modulator of susceptibility to several first-line artemisinin-based combination therapy partner drugs. Aside from the validated CQR molecular marker K76T, P. falciparum parasites have acquired at least three additional pfcrt mutations, whose contributions to resistance and fitness have been heretofore unclear. Focusing on the quadruple-mutant Ecuadorian PfCRT haplotype Ecu1110 (K76T/A220S/N326D/I356L), we genetically modified the pfcrt locus of isogenic, asexual blood stage P. falciparum parasites using zinc-finger nucleases, producing all possible combinations of intermediate pfcrt alleles. Our analysis included the related quintuple-mutant PfCRT haplotype 7G8 (Ecu1110 + C72S) that is widespread throughout South America and the Western Pacific. Drug susceptibilities and in vitro growth profiles of our combinatorial pfcrt-modified parasites were used to simulate the mutational trajectories accessible to parasites as they evolved CQR. Our results uncover unique contributions to parasite drug resistance and growth for mutations beyond K76T and predict critical roles for the CQ metabolite monodesethyl-CQ and the related quinoline-type drug amodiaquine in driving mutant pfcrt evolution. Modeling outputs further highlight the influence of parasite proliferation rates alongside gains in drug resistance in dictating successful trajectories. Our findings suggest that P. falciparum parasites have navigated constrained pfcrt adaptive landscapes by means of probabilistically rare mutational bursts that led to the infrequent emergence of pfcrt alleles in the field.