Impact of Drug Pressure versus Limited Access to Drug in Malaria Control: The Dilemma

Malaria burden has severe impact on the world. Several arsenals, including the use of antimalarials, are in place to curb the malaria burden. However, the application of these antimalarials has two extremes, limited access to drug and drug pressure, which may have similar impact on malaria control, leading to treatment failure through divergent mechanisms. Limited access to drugs ensures that patients do not get the right doses of the antimalarials in order to have an effective plasma concentration to kill the malaria parasites, which leads to treatment failure and overall reduction in malaria control via increased transmission rate. On the other hand, drug pressure can lead to the selection of drug resistance phenotypes in a subpopulation of the malaria parasites as they mutate in order to adapt. This also leads to a reduction in malaria control. Addressing these extremes in antimalarial application can be essential in maintaining the relevance of the conventional antimalarials in winning the war against malaria.

Transcriptional Shift and Metabolic Adaptations during Leishmania Quiescence Using Stationary Phase and Drug Pressure as Models

Microorganisms can adopt a quiescent physiological condition which acts as a survival strategy under unfavorable conditions. Quiescent cells are characterized by slow or non-proliferation and a deep downregulation of processes related to biosynthesis. Although quiescence has been described mostly in bacteria, this survival skill is widespread, including in eukaryotic microorganisms. In Leishmania, a digenetic parasitic protozoan that causes a major infectious disease, quiescence has been demonstrated, but the molecular and metabolic features enabling its maintenance are unknown. Here, we quantified the transcriptome and metabolome of Leishmania promastigotes and amastigotes where quiescence was induced in vitro either, through drug pressure or by stationary phase. Quiescent cells have a global and coordinated reduction in overall transcription, with levels dropping to as low as 0.4% of those in proliferating cells. However, a subset of transcripts did not follow this trend and were relatively upregulated in quiescent populations, including those encoding membrane components, such as amastins and GP63, or processes like autophagy. The metabolome followed a similar trend of overall downregulation albeit to a lesser magnitude than the transcriptome. It is noteworthy that among the commonly upregulated metabolites were those involved in carbon sources as an alternative to glucose. This first integrated two omics layers afford novel insight into cell regulation and show commonly modulated features across stimuli and stages.

Antimalarial potential of homeopathic medicines against schizont maturation of Plasmodium berghei in short-term in vitro culture

In vitro assessment of antimalarial drug susceptibility of Plasmodium has been a major research success, which has paved the way for the understanding of parasite and rapid screening of antimalarial drugs for their effectiveness. In the present study a preliminary screening to check the antiplasmodial activity of mother tincture (ϕ) and various potencies (6C, 30C, 200C) of homeopathic medicines Cinchona officinalis/china (Chin.), Chelidonium majus (Chel.) and Arsenicum album (Ars.) were done by assessing the in vitro schizont maturation inhibition assay. A significant reduction in the growth of intraerythrocytic stages of P. berghei was observed with decreasing dilution of ϕ and various potencies of Chin., Chel. and Ars. exhibiting a dose dependent effect. Maximum schizont maturation inhibition was observed by Chin. ϕ (1:1), Chin. 30 (1:1, 1:2) and Chel. 30 (1:1) i.e. 80%. The standard drug CQ at 10 µM concentration exhibited 95.4±1.6% inhibition of schizont maturation. Ars. 30 (1:1) also have been found to possess strong antiplasmodial efficacy with 75.5±2.6% schizont inhibition. The presence of free merozoites in Ars. 200 with weak schizonticidal inhibition activity (40-45%) also pointed towards the ability of parasite to survive in the given drug pressure.

Transcriptional shift and metabolic adaptations during Leishmania quiescence using stationary-phase and drug pressure as models

Microorganisms can adopt a quiescent physiological condition which acts as a survival strategy under unfavourable conditions. Quiescent cells are characterized by slow or non-proliferation and deep down-regulation of processes related to biosynthesis. Although quiescence has been described mostly in bacteria, this survival skill is widespread, including in eukaryotic microorganisms. In Leishmania, a digenetic parasitic protozoan that causes a major infectious disease, quiescence has been demonstrated, but molecular and metabolic features enabling its maintenance are unknown. Here we quantified the transcriptome and metabolome of Leishmania promastigotes and amastigotes where quiescence was induced in vitro either through drug pressure or by stationary phase. Quiescent cells have a global and coordinated reduction in overall transcription, with levels dropping to as low as 0.4% of those in proliferating cells. However, a subset of transcripts did not follow this trend and were relatively upregulated in quiescent populations, including those encoding membrane components such as amastins and GP63 or processes like autophagy. The metabolome followed a similar trend of overall downregulation albeit to a lesser magnitude than the transcriptome. Noteworthy, among the commonly upregulated metabolites were those involved in carbon sources as an alternative to glucose. This first integrated two omics layers affords novel insights into cell regulation and shows commonly modulated features across stimuli and stages.

Comparative Proteomics Demonstrates Altered Metabolism Pathways in Cotrimoxazole- Resistant and Amikacin-Resistant Klebsiella pneumoniae Isolates

Antibiotic resistance (AMR) has always been a hot topic all over the world and its mechanisms are varied and complicated. Previous evidence revealed the metabolic slowdown in resistant bacteria, suggesting the important role of metabolism in antibiotic resistance. However, the molecular mechanism of reduced metabolism remains poorly understood, which inspires us to explore the global proteome change during antibiotic resistance. Here, the sensitive, cotrimoxazole-resistant, amikacin-resistant, and amikacin/cotrimoxazole -both-resistant KPN clinical isolates were collected and subjected to proteome analysis through liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). A deep coverage of 2,266 proteins were successfully identified and quantified in total, representing the most comprehensive protein quantification data by now. Further bioinformatic analysis showed down-regulation of tricarboxylic acid cycle (TCA) pathway and up-regulation of alcohol metabolic or glutathione metabolism processes, which may contribute to ROS clearance and cell survival, in drug-resistant isolates. These results indicated that metabolic pathway alteration was directly correlated with antibiotic resistance, which could promote the development of antibacterial drugs from “target” to “network.” Moreover, combined with minimum inhibitory concentration (MIC) of cotrimoxazole and amikacin on different KPN isolates, we identified nine proteins, including garK, uxaC, exuT, hpaB, fhuA, KPN_01492, fumA, hisC, and aroE, which might contribute mostly to the survival of KPN under drug pressure. In sum, our findings provided novel, non-antibiotic-based therapeutics against resistant KPN.

Investigation of the Heterogeneity Evolution and Drug Resistance Mechanism of Diffuse Large B-Cell Lymphoma Under the Treatment of a Novel HDAC Inhibitor LAQ824

Aims: Diffuse large B-cell lymphoma (DLBCL), the most frequent malignant lymphoma subtype, is a group of highly invasive diseases with great heterogeneity in genomic alterations, clinical characteristics, morphological manifestations, treatment response and prognosis. Although most DLBCL patients can be cured by immunochemotherapy, nearly 40% of DLBCL patients still develop drug resistance and relapse. For relapsed/ refractory (R/R) DLBCL patients, there is still no optimal treatment. The heterogeneity and clonal evolution of tumor cells are the core driving forces for the occurrence and development of DLBCL, and the root causes for their refractory, recurrence and drug resistance. In this study, we screened out a novel small molecule compound effectively killing DLBCL cells, and analyzed its potential mechanism of anti-tumor. Meanwhile, by using single cell sequencing technology, we try to further investigate the heterogeneity and clona evolution and drug resistance mechanism of DLBCL under different drug pressure, explore core driver factors of drug resistance, evaluate and develop new treatment strategies. Methods: In this study, GEXSCOPE microfluidic platform was used for single-cell transcriptome sequencing. Seruat software was used for cell type recognition and clustering analysis. In order to further investigate the molecular mechanism of LAQ824 inducing the apoptosis of DLBCL cells and explore the target of LAQ824, antibody chip was performed to detect the phosphorylation of related signaling pathway. Gene expression was detected by real-time qPCR and Western blot. ChIP, RNA interfering (RNAi) and dual-luciferase activity assay were performed to validate the potential drug resistance targets for LAQ824. Moreover, WES of 21 DLBCL cell lines were performed to map mutations and analyze the correlation between related mutations and LAQ824 resistance. In this study, we established DLBCL animal models using NOD SCID mice transplanted with DLBCL cell lines, by which we could evaluate the tumor inhibition efficiency of LAQ824 alone and/or combination with other small molecular inhibitors. Results: Using GDSC database, we screened out Dacinostat (LAQ824), a novel HDAC inhibitor, was highly sensitive that could effectively induce the apoptosis of most DLBCL cells at low concentrations. Functional assay showed that LAQ824 could inhibit cell proliferation and promote apoptosis of tumor cells. LAQ824 treatment could significantly upregulate the acetylation level of histone H3 within a certain concentration range, and the DNA damage repair function of DLBCL cells was supressed by inhibiting Chk2 expression, thus significantly inducing cell apoptosis and effectively killing DLBCL cells. Meanwhile, through single-cell sequencing analysis, it was found that c-Fos could be activated under certain drug pressure of LAQ824. As a potential drug-resistant core driver gene, the expression level of c-Fos is highly correlated with IC50 of LAQ824 and the prognosis of patients with DLBCL, which can be used as a sensitivity indicator of LAQ824. Treatment with c-Fos inhibitor combined with LAQ824 can significantly improve the tumor inhibition rate, validated both in vitro and in vivo, which is expected to alleviate the recurrence and drug resistance of DLBCL patients. Conclusions: In general, we explores potential therapeutic drugs for DLBCL parients, adjusts and explores new clinical treatment strategies on this basis, and provides theoretical basis and data support for the realization of individualized precise treatment and the solution of DLBCL recurrence and drug resistance. Disclosures No relevant conflicts of interest to declare.

Insights from Leishmania (Viannia) guyanensis in vitro behavior and intercellular communication

Background Pentavalent antimonial-based chemotherapy is the first-line approach for leishmaniasis treatment and disease control. Nevertheless antimony-resistant parasites have been reported in some endemic regions. Treatment refractoriness is complex and is associated with patient- and parasite-related variables. Although amastigotes are the parasite stage in the vertebrate host and, thus, exposed to the drug, the stress caused by trivalent antimony in promastigotes has been shown to promote significant modification in expression of several genes involved in various biological processes, which will ultimately affect parasite behavior. Leishmania (Viannia) guyanensis is one of the main etiological agents in the Amazon Basin region, with a high relapse rate (approximately 25%). Methods Herein, we conducted several in vitro analyses with L. (V.) guyanensis strains derived from cured and refractory patients after treatment with standardized antimonial therapeutic schemes, in addition to a drug-resistant in vitro-selected strain. Drug sensitivity assessed through Sb(III) half-maximal inhibitory concentration (IC50) assays, growth patterns (with and without drug pressure) and metacyclic-like percentages were determined for all strains and compared to treatment outcomes. Finally, co-cultivation without intercellular contact was followed by parasitic density and Sb(III) IC50 measurements. Results Poor treatment response was correlated with increased Sb(III) IC50 values. The decrease in drug sensitivity was associated with a reduced cell replication rate, increased in vitro growth ability, and higher metacyclic-like proportion. Additionally, in vitro co-cultivation assays demonstrated that intercellular communication enabled lower drug sensitivity and enhanced in vitro growth ability, regardless of direct cell contact. Conclusions Data concerning drug sensitivity in the Viannia subgenus are emerging, and L. (V.) guyanensis plays a pivotal epidemiological role in Latin America. Therefore, investigating the parasitic features potentially related to relapses is urgent. Altogether, the data presented here indicate that all tested strains of L. (V.) guyanensis displayed an association between treatment outcome and in vitro parameters, especially the drug sensitivity. Remarkably, sharing enhanced growth ability and decreased drug sensitivity, without intercellular communication, were demonstrated. Graphical Abstract

Individual responses to a single oral dose of albendazole indicate reduced efficacy against soil-transmitted helminths in an area with high drug pressure

Background Albendazole (ALB) is administered annually to millions of children through global deworming programs targeting soil-transmitted helminths (STHs: Ascaris lumbricoides, Trichuris trichiura and hookworms, Necator americanus and Ancylostoma duodenale). However, due to the lack of large individual patient datasets collected using standardized protocols and the application of population-based statistical methods, little is known about factors that may affect individual responses to treatment. Methodology/Principal findings We re-analyzed 645 individual patient data from three standardized clinical trials designed to assess the efficacy of a single 400 mg oral dose of ALB against STHs in schoolchildren from different study sites, each with varying history of drug pressure based on duration of mass drug administration programs: Ethiopia, low; Lao People’s Democratic Republic (PDR), moderate; Pemba Island (Tanzania), high. Using a Bayesian statistical modelling approach to estimate individual responses (individual egg reduction rates, ERRi), we found that efficacy was lower in Pemba Island, particularly for T. trichiura. For this STH, the proportion of participants with a satisfactory response (ERRi ≥50%), was 65% in Ethiopia, 61% in Lao PDR but only 29% in Pemba Island. There was a significant correlation between ERRi and infection intensity prior to drug administration (ERRi decreasing as a function of increasing infection intensity). Individual age and sex also affected the drug response, but these were of negligible clinical significance and not consistent across STHs and study sites. Conclusions/Significance We found decreased efficacy of ALB against all the STHs analyzed in Pemba Island (Tanzania), an area with high drug pressure. This does not indicate causality, as this association may also be partially explained by differences in infection intensity prior to drug administration. Notwithstanding, our results indicate that without alternative treatment regimens, program targets will not be achievable on Pemba Island because of inadequate efficacy of ALB. Trial registration The study was registered on Clinicaltrials.gov (ID: NCT03465488) on March 7, 2018.

Mutation in Plasmodium falciparum BTB/POZ domain of K13 protein confers artemisinin resistance

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 ( pfk13 ) gene. Here, we carried out in vitro selection over a one-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA 0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

Characterization of the β-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance

Background The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs. Methodology and principal findings First, the β-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different β-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. Significance This study demonstrated the presence of at least seven β-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.