Going Asexual: A Survey of Mites of the Genus Thyreophagus (Acari: Acaridae) Revealing a Large Number of New Parthenogenetic Species in the Holarctic Region.

Mites of the genus Thyreophagus (Acari: Acaridae) are distributed worldwide; they inhabit concealed habitats and include several beneficial and economically important species. However, species identification is difficult because many species are poorly described or delimited and their phoretic stages are unknown or uncorrelated. Furthermore, Thyreophagus is interesting because it includes entirely asexual (parthenogenetic) species. However, among the 34 described species of Thyreophagus, the asexual status is confirmed through laboratory rearing for only two species. Here, we provide detailed descriptions of five new species from North America (four) and Europe (one) based on adults and phoretic heteromorphic deutonymphs. Four of these species were asexual, while one was sexual. For most of these mites, the asexual status was confirmed and phoretic deutonymphs were obtained through rearing in the lab. We show that asexual mites retain seemingly functional copulatory and sperm storage systems, indicating that these lineages have relatively short evolutionary lifespans. One North American species, Thyreophagus ojibwe, was found in association with the native American chestnut Castanea dentata, suggesting a possibility that this mite can be used to control chestnut blight in North America. We also provide a diagnostic key to females, males, and heteromorphic deutonymphs of the Thyreophagus species in the world.

Global Analysis of Plasmodium falciparum Dihydropteroate Synthase Variants Associated with Sulfadoxine Resistance Reveals Variant Distribution and Mechanisms of Resistance: A Computational-Based Study.

The continual rise in sulfadoxine (SDX) resistance affects the therapeutic efficacy of sulfadoxine-pyrimethamine; therefore, careful monitoring will help guide its prolonged usage. Mutations in Plasmodium falciparum dihydropteroate synthase (Pfdhps) are being surveilled, based on their link with SDX resistance. However, there is a lack of continuous analyses and data on the potential effect of molecular markers on the Pfdhps structure and function. This study explored single-nucleotide polymorphisms (SNPs) in Pfdhps that were isolated in Africa and other countries, highlighting the regional distribution and its link with structure. In total, 6336 genomic sequences from 13 countries were subjected to SNPs, haplotypes, and structure-based analyses. The SNP analysis revealed that the key SDX resistance marker, A437G, was nearing fixation in all countries, peaking in Malawi. The mutation A613S was rare except in isolates from the Democratic Republic of Congo and Malawi. Molecular docking revealed a general loss of interactions when comparing mutant proteins to the wild-type protein. During MD simulations, SDX was released from the active site in mutants A581G and A613S before the end of run-time, whereas an unstable binding of SDX to mutant A613S and haplotype A437A/A581G/A613S was observed. Conformational changes in mutant A581G and the haplotypes A581G/A613S, A437G/A581G, and A437G/A581G/A613S were seen. The radius of gyration revealed an unfolding behavior for the A613S, K540E/A581G, and A437G/A581G systems. Overall, tracking such mutations by the continuous analysis of Pfdhps SNPs is encouraged. SNPs on the Pfdhps structure may cause protein-drug function loss, which could affect the applicability of SDX in preventing malaria in pregnant women and children.

Immediate pools of malaria infections at diagnosis combined with targeted deep sequencing accurately quantifies frequency of drug resistance mutations.

Antimalarial resistance surveillance in sub-Saharan Africa is often constrained by logistical and financial challenges limiting its breadth and frequency. At two sites in Ghana, we have piloted a streamlined sample pooling process created immediately by sequential addition of positive malaria cases at the time of diagnostic testing. This streamlined process involving a single tube minimized clinical and laboratory work and provided accurate frequencies of all known drug resistance mutations after high-throughput targeted sequencing using molecular inversion probes. Our study validates this method as a cost-efficient, accurate and highly-scalable approach for drug resistance mutation monitoring that can potentially be applied to other infectious diseases such as tuberculosis.

Assessment of antimalarial drug resistant markers in asymptomatic Plasmodium falciparum infections after 4 years of indoor residual spraying in Northern Ghana.

BACKGROUND: Drug resistance remains a concern for malaria control and elimination. The effect of interventions on its prevalence needs to be monitored to pre-empt further selection. We assessed the prevalence of Plasmodium falciparum gene mutations associated with resistance to the antimalarial drugs: sulfadoxine-pyrimethamine (SP), chloroquine (CQ) and artemisinin combination therapy (ACTs) after the scale-up of a vector control activity that reduced transmission. METHODS: A total of 400 P. falciparum isolates from children under five years were genotyped for seventeen single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13 genes using polymerase chain reaction (PCR) and high resolution melting (HRM) analysis. These included 80 isolates, each randomly selected from cross-sectional surveys of asymptomatic infections across 2010 (baseline), 2011, 2012, 2013 (midline: post-IRS) and 2014 (endline: post-IRS) during the peak transmission season, when IRS intervention was rolled out in Bunkpurugu Yunyoo (BY) District, Ghana. The proportions of isolates with drug resistant alleles were assessed over this period. RESULTS: There were significant decreases in the prevalence of pfdhfr- I51R59N108 haplotype from 2010 to 2014, while the decline in pfdhfr/pfdhps- I51R59N108G437 during the same period was not significant. The prevalence of lumefantrine (LM), mefloquine (MQ) and amodiaquine (AQ) resistance-associated haplotypes pfmdr1-N86F184D1246 and pfmdr1-Y86Y184Y1246 showed decreasing trends (z = -2.86, P = 0.004 and z = -2.71, P = 0.007, respectively). Each of pfcrt-T76 and pfmdr1-Y86 mutant alleles also showed a declining trend in the asymptomatic reservoir, after the IRS rollout in 2014 (z = -2.87, P = 0.004 and z = -2.65, P = 0.008, respectively). Similarly, Pyrimethamine resistance mediating polymorphisms pfdhfr-N108, pfdhfr-I51 and pfdhfr-R59 also declined (z = -2.03, P = 0.042, z = -3.54, P<0.001 and z = -4.63, P<0.001, respectively), but not the sulphadoxine resistance mediating pfdhps-G437 and pfdhps-F436 (z = -0.36, P = 0.715 and z = 0.41, P = 0.684, respectively). No mutant pfk13-Y580 were detected during the study period. CONCLUSION: The study demonstrated declining trends in the prevalence of drug resistant mutations in asymptomatic P. falciparum infections following transmission reduction after an enhanced IRS intervention in Northern Ghana.

The impact of antimalarial resistance on the genetic structure of Plasmodium falciparum in the DRC.

The Democratic Republic of the Congo (DRC) harbors 11% of global malaria cases, yet little is known about the spatial and genetic structure of the parasite population in that country. We sequence 2537 Plasmodium falciparum infections, including a nationally representative population sample from DRC and samples from surrounding countries, using molecular inversion probes - a high-throughput genotyping tool. We identify an east-west divide in haplotypes known to confer resistance to chloroquine and sulfadoxine-pyrimethamine. Furthermore, we identify highly related parasites over large geographic distances, indicative of gene flow and migration. Our results are consistent with a background of isolation by distance combined with the effects of selection for antimalarial drug resistance. This study provides a high-resolution view of parasite genetic structure across a large country in Africa and provides a baseline to study how implementation programs may impact parasite populations.

Antimalarial Drug Resistance Profiling of Plasmodium falciparum Infections in Ghana Using Molecular Inversion Probes and Next-Generation Sequencing.

A key drawback to monitoring the emergence and spread of antimalarial drug resistance in sub-Saharan Africa is early detection and containment. Next-generation sequencing methods offer the resolution, sensitivity, and scale required to fill this gap by surveilling for molecular markers of drug resistance. We performed targeted sequencing using molecular inversion probes to interrogate five Plasmodium falciparum genes (pfcrt, pfmdr1, pfdhps, pfdhfr, and pfk13) implicated in chloroquine, sulfadoxine-pyrimethamine (SP), and artemisinin resistance in two sites in Ghana. A total of 803 dried blood spots from children aged between 6 months and 14 years presenting with uncomplicated P. falciparum malaria at the Begoro District Hospital in Begoro and the Ewim Polyclinic in Cape Coast, Ghana, from 2014 to 2017 were prepared on filter paper. Thirteen years after the removal of drug pressure, chloroquine-sensitive parasite strains with pfcrt K76 have increased nearly to fixation in Begoro, in the forest area (prevalence = 95%), but at a lower rate in Cape Coast, in the coastal region (prevalence = 71%, Z = -3.5, P < 0.001). In addition, pfmdr1 184F-bearing parasites are under strong selection. The pfdhfr/pfdhps quadruple genotype ( IRNG K), associated with SP resistance, is near saturation. Our study identified at a 2 to 10% prevalence pfdhps 581G, which is a sulfadoxine resistance marker that correlates with the failure of SP prophylaxis in pregnancy and which has not been observed in Ghana. The differences in the reexpansion of chloroquine-sensitive strains observed at the two study sites, the stronger SP resistance, and the high prevalence of pfmdr1 184F should be further monitored to inform malaria control strategies in Ghana.

Efficacy of Artesunate/Amodiaquine in the Treatment of Uncomplicated Malaria among Children in Ghana.

The declining efficacy of chloroquine in the early 2000s in Ghana led to its replacement with artesunate/amodiaquine (AS/AQ) combination as first-line drug for treating uncomplicated malaria in 2005. Since then efficacy studies have been ongoing in the country to provide continuous data on the efficacy of AS/AQ and other alternative antimalarials (artemether/lumefantrine and dihyroartemisinin/piperaquine combinations) introduced in 2008. In vivo AS/AQ efficacy studies were conducted between June and October 2014 among children aged 6 months to 14 years, in two sentinel sites representing the forest and coastal zones of the country. The 2009 World Health Organization protocol for monitoring antimalarial drug efficacy was used in these studies. The studies showed an overall cumulative polymerase chain reaction-corrected day 28 cure rate of 97.2% (95% confidence interval [CI]: 93.6-99.1): 97.7% (95% CI: 92.0-99.7) within the forest zone and 96.7% (95% CI: 90.7-99.3) within the coastal zone (P = 0.686). Prevalence of fever declined from 100% to < 4% after first day of treatment in both ecological zones. All children in the coastal zone had cleared parasites by day 2. Three children (3.2%) in the forest zone were parasitemic on day 2, whereas one child was parasitemic on day 3. Gametocytemia was absent in both zones after day 14, and mean hemoglobin concentration significantly increased from 10.3 g/dL (95% CI: 10.1-10.5) on day 0 to 11.8 g/dL (95% CI: 11.6-12.0) on day 28. We conclude that AS/AQ combination remains efficacious in the treatment of uncomplicated malaria in Ghana.