In vitro activity of rhinacanthin analogues against drug resistant Plasmodium falciparum isolates from Northeast Thailand.

BACKGROUND: New anti-malarial drugs are needed urgently to address the increasing challenges of drug-resistant falciparum malaria. Two rhinacanthin analogues containing a naphthoquinone moiety resembling atovaquone showed promising in-vitro activity against a P. falciparum laboratory reference strain (K1). The anti-malarial activity of these 2 compounds was further evaluated for P. falciparum field isolates from an area of multi-drug resistance in Northeast Thailand. METHODS: Using a pLDH enzyme-linked immunosorbent assay, four P. falciparum isolates from Northeast Thailand in 2018 were tested for in vitro sensitivity to the two synthetic rhinacanthin analogues 1 and 2 as well as established anti-malarials. Mutations in the P. falciparum cytochrome b gene, a marker for atovaquone (ATQ) resistance, were genotyped in all four field isolates as well as 100 other clinical isolates from the same area using PCR-artificial Restriction Fragment Length Polymorphisms. Pfkelch13 mutations, a marker for artemisinin (ART) resistance, were also examined in all isolates. RESULTS: The 50% inhibitory concentrations (IC50) of P. falciparum field isolates for rhinacanthin analogue 1 was 321.9-791.1 nM (median = 403.1 nM). Parasites were more sensitive to analogue 2: IC50 48.6-63.3 nM (median = 52.2 nM). Similar results were obtained against P. falciparum reference laboratory strains 3D7 and W2. The ART-resistant IPC-5202 laboratory strain was more sensitive to these compounds with a median IC50 45.9 and 3.3 nM for rhinacanthin analogues 1 and 2, respectively. The ATQ-resistant C2B laboratory strain showed high-grade resistance towards both compounds (IC50 > 15,000 nM), and there was a strong positive correlation between the IC50 values for these compounds and ATQ (r = 0.83-0.97, P < 0.001). There were no P. falciparum cytochrome b mutations observed in the field isolates, indicating that P. falciparum isolates from this area remained ATQ-sensitive. Pfkelch13 mutations and the ring-stage survival assay confirmed that most isolates were resistant to ART. CONCLUSIONS: Two rhinacanthin analogues showed parasiticidal activity against multi-drug resistant P. falciparum isolates, although less potent than ATQ. Rhinacanthin analogue 2 was more potent than analogue 1, and can be a lead compound for further optimization as an anti-malarial in areas with multidrug resistance.

Comparative analysis of targeted next-generation sequencing for Plasmodium falciparum drug resistance markers.

Well-defined molecular resistance markers are available for a range of antimalarial drugs, and molecular surveillance is increasingly important for monitoring antimalarial drug resistance. Different genotyping platforms are available, but these have not been compared in detail. We compared Targeted Amplicon Deep sequencing (TADs) using Ion Torrent PGM with Illumina MiSeq for the typing of antimalarial drug resistance genes. We developed and validated protocols to type the molecular resistance markers pfcrt, pfdhfr, pfdhps, pfmdr1, pfkelch, and pfcytochrome b, in Plasmodium falciparum for the Ion Torrent PGM and Illumina MiSeq sequencing platforms. With P. falciparum 3D7 and K1 as reference strains, whole blood samples (N = 20) and blood spots from Rapid Diagnostic Test (RDT) samples (N = 5) from patients with uncomplicated falciparum malaria from Ubon Ratchathani were assessed on both platforms and compared for coverage (average reads per amplicon), sequencing accuracy, variant accuracy, false positive rate, false negative rate, and alternative allele detection, with conventional Sanger sequencing as the reference method for SNP calling. Both whole blood and RDT samples could be successfully sequenced using the Ion Torrent PGM and Illumina MiSeq platforms. Coverage of reads per amplicon was higher with Illumina MiSeq (28,886 reads) than with Ion Torrent PGM (1754 reads). In laboratory generated artificial mixed infections, the two platforms could detect the minor allele down to 1% density at 500X coverage. SNPs calls from both platforms were in complete agreement with conventional Sanger sequencing. The methods can be multiplexed with up to 96 samples per run, which reduces cost by 86% compared to conventional Sanger sequencing. Both platforms, using the developed TAD protocols, provide an accurate method for molecular surveillance of drug resistance markers in P. falciparum, but Illumina MiSeq provides higher coverage than Ion Torrent PGM.

Genetic population of Plasmodium knowlesi during pre-malaria elimination in Thailand.

BACKGROUND: Thailand is committed to eliminating malaria by 2024. From 2013 to 2020, the total number of malaria cases have decreased, from 37,741 to 4474 (an 88.1% reduction). However, infections with Plasmodium knowlesi, a monkey malarial pathogen that can also infect humans, have been increasingly observed. This study focused on the molecular analysis of P. knowlesi parasites causing malaria in Thailand. METHODS: Under Thailand's integrated Drug Efficacy Surveillance (iDES), which includes drug-resistance monitoring as part of routine case-based surveillance and responses, specimens were collected from malaria patients (n = 966) between 2018 and 2020. Thirty-one mono P. knowlesi infections (3.1%), most of which were from eastern and southern Thailand, were observed and confirmed by nested PCR assay and DNA sequencing. To evaluate whether these pathogens were from different lineages, cluster analysis based on seven microsatellite genotyping markers and the merozoite surface protein 1 (pkmsp1) gene was carried out. The P. knowlesi pyrimethamine resistance gene dihydrofolate reductase (pkdhfr) was sequenced and homology modelling was constructed. RESULTS: The results of analysing the seven microsatellite markers and pkmsp1 sequence demonstrated that P. knowlesi parasites from eastern Thailand were of the same lineage as those isolated in Cambodia, while the parasites causing malaria in southern Thailand were the same lineage as those isolated from Malaysia. The sequencing results for the pkdhfr genes indicated the presence of two mutations, Arg34Leu and a deletion at position 105. On analysis with homology modelling, the two mutations were not associated with anti-malarial drug resistance. CONCLUSIONS: This report compared the genetic populations of P. knowlesi parasites in Thailand from 2018 to 2020 and have shown similar lineages as those isolated in Cambodia and Malaysia of P. knowlesi infection in Thailand and demonstrated that the P. knowlesi parasites were of the same lineages as those isolated in Cambodia and Malaysia. The parasites were also shown to be sensitive to pyrimethamine.

Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study.

BACKGROUND: The Greater Mekong subregion is a recurrent source of antimalarial drug resistance in Plasmodium falciparum malaria. This study aimed to characterise the extent and spread of resistance across this entire region between 2007 and 2018. METHODS: P falciparum isolates from Myanmar, Thailand, Laos, and Cambodia were obtained from clinical trials and epidemiological studies done between Jan 1, 2007, and Dec 31, 2018, and were genotyped for molecular markers (pfkelch, pfcrt, pfplasmepsin2, and pfmdr1) of antimalarial drug resistance. Genetic relatedness was assessed using microsatellite and single nucleotide polymorphism typing of flanking sequences around target genes. FINDINGS: 10 632 isolates were genotyped. A single long pfkelch Cys580Tyr haplotype (from -50 kb to +31·5 kb) conferring artemisinin resistance (PfPailin) now dominates across the eastern Greater Mekong subregion. Piperaquine resistance associated with pfplasmepsin2 gene amplification and mutations in pfcrt downstream of the Lys76Thr chloroquine resistance locus has also developed. On the Thailand-Myanmar border a different pfkelch Cys580Tyr lineage rose to high frequencies before it was eliminated. Elsewhere in Myanmar the Cys580Tyr allele remains widespread at low allele frequencies. Meanwhile a single artemisinin-resistant pfkelch Phe446Ile haplotype has spread across Myanmar. Despite intense use of dihydroartemisinin-piperaquine in Kayin state, eastern Myanmar, both in treatment and mass drug administrations, no selection of piperaquine resistance markers was observed. pfmdr1 amplification, a marker of resistance to mefloquine, remains at low prevalence across the entire region. INTERPRETATION: Artemisinin resistance in P falciparum is now prevalent across the Greater Mekong subregion. In the eastern Greater Mekong subregion a multidrug resistant P falciparum lineage (PfPailin) dominates. In Myanmar a long pfkelch Phe446Ile haplotype has spread widely but, by contrast with the eastern Greater Mekong subregion, there is no indication of artemisinin combination therapy (ACT) partner drug resistance from genotyping known markers, and no evidence of spread of ACT resistant P falciparum from the east to the west. There is still a window of opportunity to prevent global spread of ACT resistance. FUNDING: Thailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.

Molecular characterization of Plasmodium falciparum antifolate resistance markers in Thailand between 2008 and 2016.

BACKGROUND: Resistance to anti-malarials is a major threat to the control and elimination of malaria. Sulfadoxine-pyrimethamine (SP) anti-malarial treatment was used as a national policy for treatment of uncomplicated falciparum malaria in Thailand from 1973 to 1990. In order to determine whether withdrawal of this antifolate drug has led to restoration of SP sensitivity, the prevalence of genetic markers of SP resistance was assessed in historical Thai samples. METHODS: Plasmodium falciparum DNA was collected from the Thailand-Myanmar, Thailand-Malaysia and Thailand-Cambodia borders during 2008-2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated. RESULTS: Mutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr-pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr-pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand-Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr-pfdhps haplotypes. In particular, the prevalence of pfdhfr-pfdhps, septuple mutation was observed in the Thailand-Myanmar (50%, 73/146) and Thailand-Cambodia (65%, 26/40) border. In Thailand-Malaysia border, majority of the pfdhfr-pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008-2016. Within the pfdhfr-pfdhps haplotypes, during 2008-2013 the pfdhps A/S436F mutation was observed only in Thailand-Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification. CONCLUSIONS: Despite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation.

Asymptomatic Natural Human Infections With the Simian Malaria Parasites Plasmodium cynomolgi and Plasmodium knowlesi.

BACKGROUND: In Southeast Asia, Plasmodium knowlesi, a parasite of long-tailed macaques (Macaca fascicularis), is an important cause of human malaria. Plasmodium cynomolgi also commonly infects these monkeys, but only one naturally acquired symptomatic human case has been reported previously. METHODS: Malariometric studies involving 5422 subjects (aged 6 months to 65 years) were conducted in 23 villages in Pailin and Battambang, western Cambodia. Parasite detection and genotyping was conducted on blood samples, using high-volume quantitative PCR (uPCR). RESULTS: Asymptomatic malaria parasite infections were detected in 1361 of 14732 samples (9.2%). Asymptomatic infections with nonhuman primate malaria parasites were found in 21 individuals living close to forested areas; P. cynomolgi was found in 11, P. knowlesi was found in 8, and P. vivax and P. cynomolgi were both found in 2. Only 2 subjects were female, and 14 were men aged 20-40 years. Geometric mean parasite densities were 3604 parasites/mL in P. cynomolgi infections and 52488 parasites/mL in P. knowlesi infections. All P. cynomolgi isolates had wild-type dihydrofolate reductase genes, in contrast to the very high prevalence of mutations in the human malaria parasites. Asymptomatic reappearance of P. cynomolgi occurred in 2 subjects 3 months after the first infection. CONCLUSIONS: Asymptomatic naturally acquired P. cynomolgi and P. knowlesi infections can both occur in humans. CLINICAL TRIALS REGISTRATION: NCT01872702.

The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion: a molecular epidemiology observational study.

BACKGROUND: Evidence suggests that the PfKelch13 mutations that confer artemisinin resistance in falciparum malaria have multiple independent origins across the Greater Mekong subregion, which has motivated a regional malaria elimination agenda. We aimed to use molecular genotyping to assess antimalarial drug resistance selection and spread in the Greater Mekong subregion. METHODS: In this observational study, we tested Plasmodium falciparum isolates from Myanmar, northeastern Thailand, southern Laos, and western Cambodia for PfKelch13 mutations and for Pfplasmepsin2 gene amplification (indicating piperaquine resistance). We collected blood spots from patients with microscopy or rapid test confirmed uncomplicated falciparum malaria. We used microsatellite genotyping to assess genetic relatedness. FINDINGS: As part of studies on the epidemiology of artemisinin-resistant malaria between Jan 1, 2008, and Dec 31, 2015, we collected 434 isolates. In 2014-15, a single long PfKelch13 C580Y haplotype (-50 to +31·5 kb) lineage, which emerged in western Cambodia in 2008, was detected in 65 of 88 isolates from northeastern Thailand, 86 of 111 isolates from southern Laos, and 14 of 14 isolates from western Cambodia, signifying a hard transnational selective sweep. Pfplasmepsin2 amplification occurred only within this lineage, and by 2015 these closely related parasites were found in ten of the 14 isolates from Cambodia and 15 of 15 isolates from northeastern Thailand. C580Y mutated parasites from Myanmar had a different genetic origin. INTERPRETATION: Our results suggest that the dominant artemisinin-resistant P falciparum C580Y lineage probably arose in western Cambodia and then spread to Thailand and Laos, outcompeting other parasites and acquiring piperaquine resistance. The emergence and spread of fit artemisinin-resistant P falciparum parasite lineages, which then acquire partner drug resistance across the Greater Mekong subregion, threatens regional malaria control and elimination goals. Elimination of falciparum malaria from this region should be accelerated while available antimalarial drugs still remain effective. FUNDING: The Wellcome Trust and the Bill and Melinda Gates Foundation.

An outbreak of artemisinin resistant falciparum malaria in Eastern Thailand.

Artemisinin resistant falciparum malaria is an increasing problem in Southeast Asia, but has not been associated with increased transmission of the disease, yet. During a recent outbreak in 2014 in Ubon Ratchatani, Eastern Thailand, parasites from 101 patients with falciparum malaria were genotyped for antimalarial drug resistance markers. Mutations in the Kelch13 marker for artemisinin resistance were present in 93% of samples, mainly C580Y from 2 major clusters as identified by microsatellite typing. Resistance markers for antifolates and chloroquine were also highly prevalent. Most strains (91%) carried single copy number PfMDR1, suggesting sustained sensitivity to mefloquine, the partner drug in the local first-line artemisinin combination therapy (ACT). The high prevalence of artemisinin resistance in this recent malaria outbreak suggests but does not prove a causative role in increased transmission. Careful monitoring of ACT efficacy and additional genetic epidemiological studies are warranted to guide the public health response to the outbreak.

N-Linked glycosylation is essential for the yellow head virus replication cycle.

Yellow head virus (YHV) particles contain a nucleocapsid protein (p20) and two envelope glycoproteins (gp116 and gp64). The glycans attached to the two glycoproteins are N-linked and are complex and high mannose types, respectively. Here, we show that treatment with the N-linked glycosylation inhibitor tunicamycin in YHV-infected black tiger shrimp (Penaeus monodon) resulted in less severe yellow head disease and reduced mortality when compared with untreated control shrimp. Quantitative real-time reverse transcription PCR analysis also revealed lower YHV copy numbers in the haemolymph of treated than control shrimp. This was concurrent with less intense immuno-reactions in tissues of treated versus untreated shrimp using mAbs against all three YHV structural proteins. In addition, transmission electron microscopy of lymphoid organ tissue of the treated and untreated shrimp [eight collected at 36 h and eight at 48 h post-infection (p.i.)] revealed only unenveloped nucleocapsids in all but one of the treated shrimp (collected at 48 h p.i.). By contrast, all the untreated shrimp showed a mixture of many unenveloped and enveloped virions. These results were supported by purification of YHV from the cell-free haemolymph of treated and untreated shrimp followed by YHV structural protein analysis by SDS-PAGE. It revealed three expected structural protein bands (116, 64 and 20 kDa) from the untreated shrimp but no structural protein bands from the tunicamycin-treated shrimp (confirmed by Western blot analysis). Overall, the results indicated that blocking glycosylation with tunicamycin inhibited the formation of mature YHV virions and their subsequent release into shrimp haemolymph, reducing the severity of disease.