High Drug Pressure Research Articles

Single nucleotide polymorphisms in the β-tubulin gene family of Ascaris lumbricoides and their potential role in benzimidazole resistance: a systematic review

IntroductionThe most common soil-transmitted helminthic infection is caused by Ascaris lumbricoides (A. lumbricoides). Approximately 4 billion people are at risk of infection globally. The World Health Organisation recommends the administration of benzimidazole- containing deworming drugs (Albendazole and Mebendazole) to all susceptible populations. Due to this high drug pressure, these parasites may develop resistance to current benzimidazole drugs. The β-tubulin gene family is the target gene for benzimidazole deworming drugs. This systematic review aimed to highlight work that explored the genetic mutations in the β-tubulin gene family of A. lumbricoides that are associated with potential benzimidazole resistance.MethodsAn electronic search of several online databases was used to extract eligible articles using specific keywords related to the topic of interest.ResultsThe majority of ascariasis infections occur in the subtropical and tropical regions of sub-Saharan Africa, the Americas and East Asia, although not enough studies were done to extensively cover this geographical range. In the β-tubulin gene family of A. lumbricoides the mutations at codons F200Y (TTC/Phenylalanine to TAC/Tyrosine), E198A (GAG, GAA/Glutamic acid to GCG, GCA/Alanine) and F167Y (TTC, TTT/Phenylalanine to TAC, TAT/Tyrosine) were associated with potential benzimidazole resistance.DiscussionResistant mutations were found in A. lumbricoides samples at codon F167Y from Haiti, Kenya and Panama. The first evidence of the mutation at codon F200Y was observed in Brazil. The codon E198A mutation was the least prevalent and most undetected.ConclusionThere is a serious shortage of studies investigating the prevalence of β-tubulin gene family mutations in A. lumbricoides populations from endemic areas; this is a serious concern as resistance will negatively impact current mass drug administration programmes.

The adaptive roles of aneuploidy and polyclonality in Leishmania in response to environmental stress.

Aneuploidy is generally considered harmful, but in some microorganisms, it can act as an adaptive mechanism against environmental stress. Here, we use Leishmania-a protozoan parasite with remarkable genome plasticity-to study the early steps of aneuploidy evolution under high drug pressure (using antimony or miltefosine as stressors). By combining single-cell genomics, lineage tracing with cellular barcodes, and longitudinal genome characterization, we reveal that aneuploidy changes under antimony pressure result from polyclonal selection of pre-existing karyotypes, complemented by further and rapid de novo alterations in chromosome copy number along evolution. In the case of miltefosine, early parasite adaptation is associated with independent point mutations in a miltefosine transporter gene, while aneuploidy changes only emerge later, upon exposure to increased drug levels. Therefore, polyclonality and genome plasticity are hallmarks of parasite adaptation, but the scenario of aneuploidy dynamics depends on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.

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

BackgroundThe 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 findingsFirst, 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.SignificanceThis 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.

Comprehensive evaluation of stool-based diagnostic methods and benzimidazole resistance markers to assess drug efficacy and detect the emergence of anthelmintic resistance: A Starworms study protocol

BackgroundTo work towards reaching the WHO goal of eliminating soil-transmitted helminth (STH) infections as a public health problem, the total number of children receiving anthelmintic drugs has strongly increased over the past few years. However, as drug pressure levels rise, the development of anthelmintic drug resistance (AR) is more and more likely to appear. Currently, any global surveillance system to monitor drug efficacy and the emergence of possible AR is lacking. Consequently, it remains unclear to what extent the efficacy of drugs may have dropped and whether AR is already present. The overall aim of this study is to recommend the best diagnostic methods to monitor drug efficacy and molecular markers to assess the emergence of AR in STH control programs.MethodsA series of drug efficacy trials will be performed in four STH endemic countries with varying drug pressure (Ethiopia and Brazil: low drug pressure, Lao PDR: moderate drug pressure and Tanzania: high drug pressure). These trials are designed to assess the efficacy of a single oral dose of 400 mg albendazole (ALB) against STH infections in school-aged children (SAC) by microscopic (duplicate Kato-Katz thick smear, Mini-FLOTAC and FECPAKG2) and molecular stool-based diagnostic methods (quantitative PCR (qPCR)). Data will be collected on the cost of the materials used, as well as the time required to prepare and examine stool samples for the different diagnostic methods. Following qPCR, DNA samples will also be submitted for pyrosequencing to assess the presence and prevalence of single nucleotide polymorphisms (SNPs) in the β-tubulin gene. These SNPs are known to be linked to AR in animal STHs.DiscussionThe results obtained by these trials will provide robust evidence regarding the cost-efficiency and diagnostic performance of the different stool-based diagnostic methods for the assessment of drug efficacy in control programs. The assessment of associations between the frequency of SNPs in the β-tubulin gene and the history of drug pressure and drug efficacy will allow the validation of these SNPs as a marker for AR in human STHs.Trial registrationThe trial was retrospectively registered the 7th of March 2018 on Clinicaltrials.gov (ID: NCT03465488).

Efficacy and Safety of a Single Dose versus a Multiple Dose Regimen of Mebendazole against Hookworm Infections in Children: A Randomised, Double-blind Trial

BackgroundSingle-dose mebendazole is widely used in preventive chemotherapy against the soil-transmitted helminths (STHs) Ascaris lumbricoides, hookworm and Trichuris trichiura, yet it shows limited efficacy against hookworm and T. trichiura infections. The use of adapted treatment regimens might provide a strategy to control and eliminate STH infections in STH-persistent settings. We evaluated the safety and efficacy of the multiple dose mebendazole regimen (3 days 100 mg bid) versus a single dose of 500 mg mebendazole in a setting with high STH prevalence and high drug pressure. MethodsThis randomised, double-blind clinical trial took place in a primary school on Pemba Island, Tanzania, in school-aged children (6–12 years). Using a computer random number generator (block size 10), hookworm-positive children were randomly assigned (1:1) to either a single or multiple dose regimen of mebendazole by an independent statistician. Two stool samples were collected at baseline and follow-up (18 to 22 days after treatment) for Kato-Katz analysis. The primary outcome was cure rate (CR) against hookworm. Secondary outcomes were egg reduction rate (ERR) against hookworm, CRs and ERRs against A. lumbricoides and T. trichiura, and tolerability assessed 3, 24 and 48 h post-treatment. Participants, investigators, caregivers, outcome assessors and the trial statistician were blinded. This trial is registered with ClinicalTrials.gov, number NCT03245398. Findings93 children were assigned to each treatment arm. 185 children completed treatment and provided follow-up stool samples. CR against hookworm was significantly higher in the multiple dose (98%) than in the single dose arm (13%, OR 389.1, 95% CI 95.2 to 2885.7%, p < 0.001). 34 and 42 children reported mild adverse events in the single and multiple dose arms, respectively. The most common events were abdominal pain, headache and diarrhoea. InterpretationThe poor performance of single dose mebendazole against hookworm infections was confirmed, but the multiple dose treatment regimen of mebendazole showed high efficacy. Hence, multiple dose mebendazole might provide a treatment strategy in given epidemiological situations to boost control and elimination of STH infections. FundingPATH.

Polymorphism in drug resistance genes dihydrofolate reductase and dihydropteroate synthase in Plasmodium falciparum in some states of India.

BackgroundSulfadoxine-pyrimethamine (SP) combination drug is currently being used in India for the treatment of Plasmodium falciparum as partner drug in artemisinin-based combination therapy (ACT). Resistance to sulfadoxine and pyrimethamine in P. falciparum is linked with mutations in dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes respectively. This study was undertaken to estimate the prevalence of such mutations in pfdhfr and pfdhps genes in four states of India.MethodsPlasmodium falciparum isolates were collected from two states of India with high malaria incidence i.e., Jharkhand and Odisha and two states with low malaria incidence i.e., Andhra Pradesh and Uttar Pradesh between years 2006 to 2012. Part of sulfadoxine-pyrimethamine (SP) drug resistance genes, pfdhfr and pfdhps were PCR-amplified, sequenced and analyzed.ResultsA total of 217 confirmed P. falciparum isolates were sequenced for both Pfdhfr and pfdhps gene. Two pfdhfr mutations 59R and 108N were most common mutations prevalent in all localities in 77 % of isolates. Additionally, I164L was found in Odisha and Jharkhand only (4/70 and 8/84, respectively). Another mutation 51I was found in Odisha only (3/70). The pfdhps mutations 436A, 437G, 540E and 581G were found in Jharkhand and Odisha only in 13, 26, 14 and 13 % isolates respectively, and was absent in Uttar Pradesh and Andhra Pradesh. Combined together for pfdhps and pfdhfr locus, triple, quadruple, quintuple and sextuple mutations were present in Jharkhand and Odisha while absent in Uttar Pradesh and Andhra Pradesh.ConclusionWhile only double mutants of pfdhfr was present in low transmission area (Uttar Pradesh and Andhra Pradesh) with total absence of pfdhps mutants, up to sextuple mutations were present in high transmission areas (Odisha and Jharkhand) for both the genes combined. Presence of multiple mutations in pfdhfr and pfdhps genes linked to SP resistance in high transmission area may lead to fixation of multiple mutations in presence of high drug pressure and high recombination rate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1080-2) contains supplementary material, which is available to authorized users.

Five Years of Antimalarial Resistance Marker Surveillance in Gaza Province, Mozambique, Following Artemisinin-Based Combination Therapy Roll Out

Antimalarial drug resistance is a major obstacle to malaria control and eventual elimination. The routine surveillance for molecular marker of resistance is an efficient way to assess drug efficacy, which remains feasible in areas where malaria control interventions have succeeded in substantially reducing malaria transmission. Community based asexual parasite prevalence surveys were conducted annually in sentinel sites in Gaza Province, Mozambique from 2006 until 2010, before, during and after antimalarial policy changes to artesunate plus sulfadoxine-pyrimethamine in 2006 and to artemether-lumefantrine in 2008. Genetic analysis of dhfr, dhps, crt, and mdr1 resistant genes was conducted on 3 331 (14.4%) Plasmodium falciparum PCR positive samples collected over the study period from 23 229 children aged 2 to 15 years. The quintuple dhfr/dhps mutation associated with sulfadoxine-pyrimethamine resistance increased from 56.2% at baseline to 75.8% by 2010. At baseline the crt76T and mdr186Y mutants were approaching fixation, 96.1% and 74.7%, respectively. Following the deployment of artemisinin-based combination therapy, prevalence of both these chloroquine-resistance markers began declining, reaching 32.4% and 30.9%, respectively, by 2010. All samples analysed over the 5-year period possessed a single copy of the mdr1 gene. The high and increasing prevalence of the quintuple mutation supports the change in drug policy from artesunate plus sulfadoxine-pyrimethamine to artemether-lumefantrine in Mozambique. As chloroquine related drug pressure decreased in the region, so did the molecular markers associated with chloroquine resistance (crt76T and mdr186Y). However, this reversion to the wild-type mdr186N predisposes parasites towards developing lumefantrine resistance. Close monitoring of artemether-lumefantrine efficacy is therefore essential, particularly given the high drug pressure within the region where most countries now use artemether-lumefantrine as first line treatment.

Polymorphism of Plasmodium falciparum Na+/H+ exchanger is indicative of a low in vitro quinine susceptibility in isolates from Viet Nam

BackgroundThe Plasmodium falciparum NA+/H+ exchanger (pfnhe1, gene PF13_0019) has recently been proposed to influence quinine (QN) susceptibility. However, its contribution to QN resistance seems to vary geographically depending on the genetic background of the parasites. Here, the role of this gene was investigated in in vitro QN susceptibility of isolates from Viet Nam.MethodNinety-eight isolates were obtained from three different regions of the Binh Phuoc and Dak Nong bordering Cambodia provinces during 2006-2008. Among these, 79 were identified as monoclonal infection and were genotyped at the microsatellite pfnhe1 ms4760 locus and in vitro QN sensitivity data were obtained for 51 isolates. Parasite growth was assessed in the field using the HRP2 immunodetection assay.ResultsSignificant associations were found between polymorphisms at pfnhe1 microsatellite ms4760 and susceptibility to QN. Isolates with two or more DNNND exhibited much lower susceptibility to QN than those harbouring zero or one DNNND repeats (median IC50 of 682 nM versus median IC50 of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC50 of 704 nM versus median IC50 of 375 nM; p < 0.01). These QNR associated genotype features were mainly due to the over representation of profile 7 among isolates (76.5%). The majority of parasites had pfcrt76T and wild-type pfmdr1 (> 95%) thus preventing analysis of associations with these mutations. Interestingly, area with the highest median QN IC50 showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7.ConclusionsThe haplotype 7 which is the typical Asian profile is likely well-adapted to high drug pressure in this area and may constitute a good genetic marker to evaluate the dissemination of QNR in this part of the world.

Residual Antimalarial Concentrations before Treatment in Patients with Malaria from Cambodia: Indication of Drug Pressure

The Thai-Cambodian border has been known as the origin of antimalarial drug resistance for the past 30 years. There is a highly diverse market for antimalarials in this area, and improved knowledge of drug pressure would be useful to target interventions aimed at reducing inappropriate drug use. Baseline samples from 125 patients with falciparum malaria recruited for 2 in vivo studies (in Preah Vihear and Pursat provinces) were analyzed for the presence of 14 antimalarials in a single run, by means of a liquid chromatography-tandem mass spectrometry assay. Half of the patients had residual drug concentrations above the lower limit of calibration for at least 1 antimalarial at admission. Among the drugs detected were the currently used first-line drugs mefloquine (25% and 35% of patients) and piperaquine (15% of patients); the first-line drug against vivax malaria, chloroquine (25% and 41% of patients); and the former first-line drug, quinine (5% and 34% patients). The findings demonstrate that there is high drug pressure and that many people still seek treatment in the private and informal sector, where appropriate treatment is not guaranteed. Promotion of comprehensive behavioral change, communication, community-based mobilization, and advocacy are vital to contain the emergence and spread of parasite resistance against new antimalarials.

Public health impact of drug resistant Plasmodium falciparum malaria

The alarming increase in Plasmodium falciparum resistance to commonly used anti-malarial drugs represents a major public health threat. The impact is however difficult to quantify. In low transmission areas, an increase in acute manifestations (“epidemic”) is often quickly apparent and resistance is rapidly propagated due to high drug pressure on existing parasite populations. In high transmission areas, the clinical effects are mainly prolonged/chronic infections with increasing risk of severe anemia. Mortality estimates from public health records in Africa generally suggest significant increases (from 2- to 11-fold) in malaria-associated mortality among children when resistance develops and spreads. Hospital attendances and admissions show similar trends. Change of policy to alternative efficacious treatment with radical cure is necessary at an earlier stage (from 10% treatment failure) than previously assumed in order to prevent deaths in millions of African children. Early switch to artemisinin based combination therapy (ACT) represents such a critical and urgent strategy.

An epidemic of Plasmodium falciparum malaria in Balcad, Somalia, and its causation

The causative factors of an epidemic of falciparum malaria were investigated in Balcad, Somalia, a town with previously low malaria transmission, where malaria incidence rose more than twenty-fold between 1986 and 1988. The emergence of chloroquine resistance, accelerated by high drug pressure, low herd immunity and favourable meteorological conditions were identified as major causes of the epidemic. Chloroquine resistance of grades RII and RIII was first observed in Balcad in 1987 and rapidly increased to 72% of the Plasmodium falciparum infections in 1988. In the absence of alternative treatment, resistance resulted in the accumulation of a massive infective reservoir and therefore increased malaria transmission, associated with intensive clinical symptomatology. The advent of chloroquine resistance was less violent in the area of Malable, where malaria is stable and communal immunity higher than in Balcad.