ABSTRACT
We conducted a retrospective cohort study that tested 2,000 US military personnel for Coccidioides antibodies in a disease-endemic region. The overall incidence of seroconversion was 0.5 cases/100 person-years; 12.5% of persons who seroconverted had illnesses requiring medical care. No significant association was found between demographic characteristics and seroconversion or disease.
Subject(s)
Coccidioidomycosis , Military Personnel , California , Coccidioides , Coccidioidomycosis/epidemiology , Coccidioidomycosis/etiology , Humans , Incidence , Retrospective StudiesABSTRACT
The active metabolites of primaquine, in particular 5-hydroxyprimaquine, likely responsible for the clearance of dormant hypnozoites, are produced through the hepatic CYP450 2D6 (CYP2D6) enzymatic pathway. With the inherent instability of 5-hydroxyprimaquine, a stable surrogate, 5,6-orthoquinone, can now be detected and measured in the urine as part of primaquine pharmacokinetic studies. This study performed CYP450 2D6 genotyping and primaquine pharmacokinetic testing, to include urine 5,6-orthoquinone, in 27 healthy adult Cambodians, as a preliminary step to prepare for future clinical studies assessing primaquine efficacy for Plasmodium vivax infections. The CYP2D6 *10 reduced activity allele was found in 57% of volunteers, and the CYP2D6 genotypes were dominated by *1/*10 (33%) and *10/*10 (30%). Predicted phenotypes were evenly split between Normal Metabolizer (NM) and Intermediate Metabolizer (IM) except for one volunteer with a gene duplication and unclear phenotype, classifying as either IM or NM. Median plasma primaquine (PQ) area under the curve (AUC) was lower in the NM group (460 h*ng/mL) compared to the IM group (561 h*ng/mL), although not statistically significant. Similar to what has been found in the US study, no 5,6-orthoquinone was detected in the plasma. The urine creatinine-corrected 5,6-orthoquinone AUC in the NM group was almost three times higher than in the IM group, with peak measurements (Tmax) at 4 h. Although there is variation among individuals, future studies examining the relationship between the levels of urine 5,6-orthoquinone and primaquine radical cure efficacy could result in a metabolism biomarker predictive of radical cure.
Subject(s)
Antimalarials , Malaria, Vivax , Adult , Antimalarials/pharmacokinetics , Antimalarials/therapeutic use , Asian People , Cytochrome P-450 CYP2D6/genetics , Cytochrome P-450 CYP2D6/metabolism , Humans , Malaria, Vivax/drug therapy , Plasmodium vivax/genetics , Primaquine/analogs & derivatives , Primaquine/pharmacokinetics , Primaquine/therapeutic useABSTRACT
BACKGROUND: Characterizing the longevity and quality of cellular immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enhances understanding of coronavirus disease 2019 (COVID-19) immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Analysis of the function, durability, and diversity of cellular response long after natural infection, over a range of ages and disease phenotypes, is needed to identify preventative and therapeutic interventions. METHODS: We identified participants in our multisite longitudinal, prospective cohort study 12 months after SARS-CoV-2 infection representing a range of disease severity. We investigated function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry, and compared magnitude of SARS-CoV-2-specific antibodies. RESULTS: SARS-CoV-2-specific antibodies and T cells were detected 12 months postinfection. Severe acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12 months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. CONCLUSIONS: SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12 months postinfection, with higher frequency noted in those who experienced severe disease.
Subject(s)
COVID-19/immunology , COVID-19/virology , Immunologic Memory , Memory T Cells , SARS-CoV-2/immunology , T-Lymphocyte Subsets/immunology , Adult , Antibodies, Viral , Antigens, Viral , Biomarkers , COVID-19/diagnosis , COVID-19/epidemiology , Female , Humans , Immunity, Cellular , Longitudinal Studies , Male , Middle Aged , Prospective Studies , Severity of Illness Index , T-Lymphocyte Subsets/metabolism , Time FactorsABSTRACT
BACKGROUND: Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017. METHODS: The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number. RESULTS: Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2. CONCLUSIONS: The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.
Subject(s)
Antimalarials/pharmacology , Biomarkers/metabolism , Drug Resistance/genetics , Malaria, Falciparum/drug therapy , Membrane Transport Proteins/genetics , Piperazines/therapeutic use , Protozoan Proteins/genetics , Quinolines/therapeutic use , Animals , Antimalarials/therapeutic use , Cambodia/epidemiology , Drug Resistance/drug effects , Malaria, Falciparum/epidemiology , Mefloquine/therapeutic use , Mutation/drug effects , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Prevalence , Real-Time Polymerase Chain ReactionABSTRACT
The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.
Subject(s)
Antiviral Agents/therapeutic use , COVID-19 Drug Treatment , SARS-CoV-2/drug effects , Antiviral Agents/pharmacology , COVID-19/virology , Drug Development , Humans , National Institutes of Health (U.S.) , Peptide Hydrolases/metabolism , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , United States , Virus Replication/drug effectsABSTRACT
BACKGROUND: In Southeast Asia, people are often coinfected with different species of malaria (Plasmodium falciparum [Pf] and Plasmodium vivax [Pv]) as well as with multiple clones of the same species. Whether particular species or clones within mixed infections are more readily transmitted to mosquitoes remains unknown. METHODS: Laboratory-reared Anopheles dirus were fed on blood from 119 Pf-infected Cambodian adults, with 5950 dissected to evaluate for transmitted infection. Among 12 persons who infected mosquitoes, polymerase chain reaction and amplicon deep sequencing were used to track species and clone-specific transmission to mosquitoes. RESULTS: Seven of 12 persons that infected mosquitoes harbored mixed Pf/Pv infection. Among these 7 persons, all transmitted Pv with 2 transmitting both Pf and Pv, leading to Pf/Pv coinfection in 21% of infected mosquitoes. Up to 4 clones of each species were detected within persons. Shifts in clone frequency were detected during transmission. However, in general, all parasite clones in humans were transmitted to mosquitoes, with individual mosquitoes frequently carrying multiple transmitted clones. CONCLUSIONS: Malaria diversity in human hosts was maintained in the parasite populations recovered from mosquitoes fed on their blood. However, in persons with mixed Pf/Pv malaria, Pv appears to be transmitted more readily, in association with more prevalent patent gametocytemia.
Subject(s)
Anopheles/parasitology , Malaria, Falciparum/transmission , Malaria, Vivax/transmission , Mosquito Vectors/parasitology , Plasmodium falciparum/genetics , Plasmodium vivax/genetics , Adult , Animals , Cohort Studies , Female , Haplotypes , High-Throughput Nucleotide Sequencing , Humans , Malaria, Falciparum/parasitology , Malaria, Vivax/parasitology , Plasmodium falciparum/isolation & purification , Plasmodium vivax/isolation & purification , Polymerase Chain ReactionABSTRACT
Cambodia, in which both Plasmodium vivax and Plasmodium falciparum are endemic, has been the focus of numerous malaria-control interventions, resulting in a marked decline in overall malaria incidence. Despite this decline, the number of P vivax cases has actually increased. To understand better the factors underlying this resilience, we compared the genetic responses of the two species to recent selective pressures. We sequenced and studied the genomes of 70 P vivax and 80 P falciparum isolates collected between 2009 and 2013. We found that although P falciparum has undergone population fracturing, the coendemic P vivax population has grown undisrupted, resulting in a larger effective population size, no discernable population structure, and frequent multiclonal infections. Signatures of selection suggest recent, species-specific evolutionary differences. Particularly, in contrast to P falciparum, P vivax transcription factors, chromatin modifiers, and histone deacetylases have undergone strong directional selection, including a particularly strong selective sweep at an AP2 transcription factor. Together, our findings point to different population-level adaptive mechanisms used by P vivax and P falciparum parasites. Although population substructuring in P falciparum has resulted in clonal outgrowths of resistant parasites, P vivax may use a nuanced transcriptional regulatory approach to population maintenance, enabling it to preserve a larger, more diverse population better suited to facing selective threats. We conclude that transcriptional control may underlie P vivax's resilience to malaria control measures. Novel strategies to target such processes are likely required to eradicate P vivax and achieve malaria elimination.
Subject(s)
Malaria, Vivax/prevention & control , Malaria, Vivax/parasitology , Plasmodium vivax/genetics , Cambodia/epidemiology , DNA Copy Number Variations , DNA, Protozoan/genetics , Drug Resistance/genetics , Endemic Diseases/prevention & control , Genetic Variation , Genome, Protozoan , Haplotypes , Humans , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Malaria, Falciparum/prevention & control , Malaria, Vivax/epidemiology , Plasmodium falciparum/genetics , Polymorphism, Single Nucleotide , Selection, Genetic , Species Specificity , Transcription, GeneticABSTRACT
Background: Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine. Methods: Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset. Results: Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity. Conclusions: Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.
Subject(s)
Drug Resistance/genetics , Membrane Transport Proteins/genetics , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Quinolines/pharmacology , Artemisinins/pharmacology , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Cambodia , DNA Copy Number Variations , DNA, Protozoan/genetics , Genetic Loci , Genome-Wide Association Study , Genotyping Techniques , Humans , Inhibitory Concentration 50 , Linkage Disequilibrium , Membrane Transport Proteins/metabolism , Mutation , Plasmodium falciparum/drug effects , Polymorphism, Single Nucleotide , Proportional Hazards Models , Protozoan Proteins/metabolism , Sensitivity and Specificity , Treatment FailureABSTRACT
Despite the rising rates of resistance to dihydroartemisinin-piperaquine (DP), DP remains a first-line therapy for uncomplicated malaria in many parts of Cambodia. While DP is generally well tolerated as a 3-day DP (3DP) regimen, compressed 2-day DP (2DP) regimens were associated with treatment-limiting cardiac repolarization effects in a recent clinical trial. To better estimate the risks of piperaquine on QT interval prolongation, we pooled data from three randomized clinical trials conducted between 2010 and 2014 in northern Cambodia. A population pharmacokinetic model was developed to compare exposure-response relationships between the 2DP and 3DP regimens while accounting for differences in regimen and sample collection times between studies. A 2-compartment model with first-order absorption and elimination without covariates best fit the data. The linear slope-intercept model predicted a 0.05-ms QT prolongation per ng/ml of piperaquine (5 ms per 100 ng/ml) in this largely male population. Though the plasma half-life was similar in both regimens, peak and total piperaquine exposures were higher in those treated with the 2DP regimen. Furthermore, the correlation between the plasma piperaquine concentration and the QT interval prolongation was stronger in the population receiving the 2DP regimen. Neither the time since the previous meal nor the baseline serum magnesium or potassium levels had additive effects on QT interval prolongation. As electrocardiographic monitoring is often nonexistent in areas where malaria is endemic, 2DP regimens should be avoided and the 3DP regimen should be carefully considered in settings where viable alternative therapies exist. When DP is employed, the risk of cardiotoxicity can be mitigated by combining a 3-day regimen, enforcing a 3-h fast before and after administration, and avoiding the concomitant use of QT interval-prolonging medications. (This study used data from three clinical trials that are registered at ClinicalTrials.gov under identifiers NCT01280162, NCT01624337, and NCT01849640.).
Subject(s)
Antimalarials/adverse effects , Arrhythmias, Cardiac/chemically induced , Artemisinins/pharmacokinetics , Malaria, Falciparum/drug therapy , Myocardial Contraction/drug effects , Quinolines/pharmacokinetics , Antimalarials/therapeutic use , Artemisinins/adverse effects , Artemisinins/therapeutic use , Cambodia , Cardiotoxicity , Drug Therapy, Combination , Female , Humans , Malaria, Falciparum/parasitology , Male , Myocardial Contraction/physiology , Plasmodium falciparum/drug effects , Quinolines/blood , Quinolines/therapeutic useABSTRACT
BACKGROUND: While intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013-2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates. RESULTS: Ex vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013-4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification. CONCLUSION: The findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method.
Subject(s)
Antimalarials/pharmacology , Drug Resistance , Enzyme-Linked Immunosorbent Assay/methods , Microscopy/methods , Plasmodium vivax/drug effects , Cambodia , DNA Copy Number Variations , L-Lactate Dehydrogenase/genetics , L-Lactate Dehydrogenase/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Mutation , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Schizonts/growth & developmentABSTRACT
Although gametocytes are essential for malaria transmission, in Africa many falciparum-infected persons without smear-detectable gametocytes still infect mosquitoes. To see whether the same is true in Southeast Asia, we determined the infectiousness of 119 falciparum-infected Cambodian adults to Anopheles dirus mosquitoes by membrane feeding. Just 5.9% of subjects infected mosquitoes. The 8.4% of patients with smear-detectable gametocytes were >20 times more likely to infect mosquitoes than those without and were the source of 96% of all mosquito infections. In low-transmission settings, targeting transmission-blocking interventions to those with microscopic gametocytemia may have an outsized effect on malaria control and elimination.
Subject(s)
Anopheles/parasitology , Insect Vectors/parasitology , Malaria, Falciparum/parasitology , Malaria, Falciparum/transmission , Parasitemia/parasitology , Parasitemia/transmission , Plasmodium falciparum/pathogenicity , Adolescent , Adult , Aged , Animals , Female , Humans , Middle Aged , Parasite Load , Young AdultABSTRACT
BACKGROUND: Knowledge of the population genetics and transmission dynamics of Plasmodium vivax is crucial in predicting the emergence of drug resistance, relapse pattern and novel parasite phenotypes, all of which are relevant to the control of vivax infections. The aim of this study was to analyse changes in the genetic diversity of P. vivax genes from field isolates collected at different times along the Thai-Myanmar border. METHODS: Two hundred and fifty-four P. vivax isolates collected during two periods 10 years apart along the Thai-Myanmar border were analysed. The parasites were genotyped by nested-PCR and PCR-RFLP targeting selected polymorphic loci of Pvmsp1, Pvmsp3α and Pvcsp genes. RESULTS: The total number of distinguishable allelic variants observed for Pvcsp, Pvmsp1, and Pvmsp3α was 17, 7 and 3, respectively. High genetic diversity was observed for Pvcsp (H E = 0.846) and Pvmsp1 (H E = 0.709). Of the 254 isolates, 4.3 and 14.6 % harboured mixed Pvmsp1 and Pvcsp genotypes with a mean multiplicity of infection (MOI) of 1.06 and 1.15, respectively. The overall frequency of multiple genotypes was 16.9 %. When the frequencies of allelic variants of each gene during the two distinct periods were analysed, significant differences were noted for Pvmsp1 (P = 0.018) and the Pvcsp (P = 0.033) allelic variants. CONCLUSION: Despite the low malaria transmission levels in Thailand, P. vivax population exhibit a relatively high degree of genetic diversity along the Thai-Myanmar border of Thailand, in particular for Pvmsp1 and Pvcsp, with indication of geographic and temporal variation in frequencies for some variants. These results are of relevance to monitoring the emergence of drug resistance and to the elaboration of measures to control vivax malaria.
Subject(s)
Plasmodium vivax/genetics , Protozoan Proteins/genetics , Adolescent , Adult , Female , Genetic Variation/genetics , Genotype , Humans , Malaria, Vivax/parasitology , Male , Middle Aged , Plasmodium vivax/classification , Polymerase Chain Reaction , Thailand , Young AdultABSTRACT
BACKGROUND: The recent dramatic decline in dihydroartemisinin-piperaquine (DHA-PPQ) efficacy in northwestern Cambodia has raised concerns about the rapid spread of piperaquine resistance just as DHA-PPQ is being introduced as first-line therapy in neighbouring countries. METHODS: Ex vivo parasite susceptibilities were tracked to determine the rate of progression of DHA, PPQ and mefloquine (MQ) resistance from sentinel sites on the Thai-Cambodian and Thai-Myanmar borders from 2010 to 2015. Immediate ex vivo (IEV) histidine-rich protein 2 (HRP-2) assays were used on fresh patient Plasmodium falciparum isolates to determine drug susceptibility profiles. RESULTS: IEV HRP-2 assays detected the precipitous emergence of PPQ resistance in Cambodia beginning in 2013 when 40 % of isolates had an IC90 greater than the upper limit of prior years, and this rate doubled to 80 % by 2015. In contrast, Thai-Myanmar isolates from 2013 to 14 remained PPQ-sensitive, while northeastern Thai isolates appeared to have an intermediate resistance profile. The opposite trend was observed for MQ where Cambodian isolates appeared to have a modest increase in overall sensitivity during the same period, with IC50 declining to median levels comparable to those found in Thailand. A significant association between increased PPQ IC50 and IC90 among Cambodian isolates with DHA-PPQ treatment failure was observed. Nearly all Cambodian and Thai isolates were deemed artemisinin resistant with a >1 % survival rate for DHA in the ring-stage assay (RSA), though there was no correlation among isolates to indicate cross-resistance between PPQ and artemisinins. CONCLUSIONS: Clinical DHA-PPQ failures appear to be associated with declines in the long-acting partner drug PPQ, though sensitivity appears to remain largely intact for now in western Thailand. Rapid progression of PPQ resistance associated with DHA-PPQ treatment failures in northern Cambodia limits drugs of choice in this region, and urgently requires alternative therapy. The temporary re-introduction of artesunate AS-MQ is the current response to PPQ resistance in this area, due to inverse MQ and PPQ resistance patterns. This will require careful monitoring for re-emergence of MQ resistance, and possible simultaneous resistance to all three drugs (AS, MQ and PPQ).
Subject(s)
Antimalarials/pharmacology , Drug Resistance , Plasmodium falciparum/drug effects , Quinolines/pharmacology , Antigens, Protozoan/analysis , Artemisinins/pharmacology , Cambodia , Humans , Inhibitory Concentration 50 , Mefloquine/pharmacology , Parasitic Sensitivity Tests , Plasmodium falciparum/isolation & purification , Protozoan Proteins/analysis , ThailandABSTRACT
Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology.
Subject(s)
DNA, Protozoan/genetics , Malaria, Vivax/parasitology , Plasmodium vivax/genetics , Cambodia/epidemiology , Gene Expression Regulation , Genetic Variation , Haplotypes , Humans , Malaria, Vivax/epidemiology , Merozoite Surface Protein 1/genetics , Merozoite Surface Protein 1/metabolism , Microsatellite Repeats/genetics , Phylogeny , RecurrenceABSTRACT
Our recent report of dihydroartemisinin-piperaquine failure to treat Plasmodium falciparum infections in Cambodia adds new urgency to the search for alternative treatments. Despite dihydroartemisinin-piperaquine failure, and higher piperaquine 50% inhibitory concentrations (IC50s) following reanalysis than those previously reported, P. falciparum remained sensitive to atovaquone (ATQ) in vitro. There were no point mutations in the P. falciparum cytochrome b ATQ resistance gene. Mefloquine, artemisinin, chloroquine, and quinine IC50s remained comparable to those from other recent reports. Atovaquone-proguanil may be a useful stopgap but remains susceptible to developing resistance when used as blood-stage therapy.
Subject(s)
Antimalarials/therapeutic use , Atovaquone/therapeutic use , Drug Resistance, Multiple/genetics , Malaria, Falciparum/drug therapy , Plasmodium falciparum/drug effects , Proguanil/therapeutic use , Artemisinins/therapeutic use , Base Sequence , Cambodia , DNA, Protozoan/genetics , Drug Combinations , Humans , Malaria, Falciparum/parasitology , Parasitic Sensitivity Tests , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Quinolines/therapeutic use , Sequence Analysis, DNA , ThailandABSTRACT
Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.
Subject(s)
Antimalarials/therapeutic use , Drug Resistance/drug effects , Malaria, Falciparum/drug therapy , Plasmodium falciparum/drug effects , Quinolines/therapeutic use , Adolescent , Adult , Aged , Artemisinins/therapeutic use , Cambodia , Chloroquine/therapeutic use , Female , Humans , Inhibitory Concentration 50 , Malaria, Falciparum/microbiology , Male , Mefloquine/therapeutic use , Membrane Transport Proteins/metabolism , Middle Aged , Multidrug Resistance-Associated Proteins/metabolism , Parasitic Sensitivity Tests/methods , Plasmodium falciparum/isolation & purification , Plasmodium falciparum/metabolism , Young AdultABSTRACT
BACKGROUND: There is currently no standardized approach for assessing in vitro anti-malarial drug susceptibility. Potential alterations in drug susceptibility results between fresh immediate ex vivo (IEV) and cryopreserved culture-adapted (CCA) Plasmodium falciparum isolates, as well as changes in parasite genotype during culture adaptation were investigated. METHODS: The 50 % inhibitory concentration (IC50) of 12 P. falciparum isolates from Cambodia against a panel of commonly used drugs were compared using both IEV and CCA. Results were compared using both histidine-rich protein-2 ELISA (HRP-2) and SYBR-Green I fluorescence methods. Molecular genotyping and amplicon deep sequencing were also used to compare multiplicity of infection and genetic polymophisms in fresh versus culture-adapted isolates. RESULTS: IC50 for culture-adapted specimens were significantly lower compared to the original fresh isolates for both HRP-2 and SYBR-Green I assays, with greater than a 50 % decline for the majority of drug-assay combinations. There were correlations between IC50s from IEV and CCA for most drugs assays. Infections were nearly all monoclonal, with little or no change in merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP) or apical membrane antigen 1 (AMA1) polymorphisms, nor differences in P. falciparum multidrug resistance 1 gene (PfMDR1) copy number or single nucleotide polymorphisms following culture adaptation. CONCLUSIONS: The overall IC50 reduction combined with the correlation between fresh isolates and culture-adapted drug susceptibility assays suggests the utility of both approaches, as long as there is consistency of method, and remaining mindful of possible attenuation of resistance phenotype occurring in culture. Further study should be done in higher transmission settings where polyclonal infections are prevalent.
Subject(s)
Antimalarials/pharmacology , Drug Resistance , Parasitic Sensitivity Tests/methods , Plasmodium falciparum/drug effects , Adolescent , Adult , Cambodia , DNA, Protozoan/genetics , Genetic Variation , Genotype , Humans , Inhibitory Concentration 50 , Malaria, Falciparum/parasitology , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Plasmodium falciparum/isolation & purification , Young AdultABSTRACT
Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC50s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC50 susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC50s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.
Subject(s)
Antimalarials/pharmacology , Artemisinins/pharmacology , Heterocyclic Compounds, 1-Ring/pharmacology , Peroxides/pharmacology , Plasmodium falciparum/drug effects , Spiro Compounds/pharmacology , Artesunate , Cambodia , Chloroquine/pharmacology , Parasitic Sensitivity TestsABSTRACT
Primaquine (PQ) remains the sole available drug to prevent relapse of Plasmodium vivax malaria more than 60 years after licensure. While this drug was administered as a racemic mixture, prior studies suggested a pharmacodynamic advantage based on differential antirelapse activity and/or toxicities of its enantiomers. Oral primaquine enantiomers prepared using a novel, easily scalable method were given for 7 days to healthy rhesus macaques in a dose-rising fashion to evaluate their effects on the blood, liver, and kidneys. The enantiomers were then administered to Plasmodium cynomolgi-infected rhesus macaques at doses of 1.3 and 0.6 mg/kg of body weight/day in combination with chloroquine. The (-)-PQ enantiomer had higher clearance and apparent volume of distribution than did (+)-PQ and was more extensively converted to the carboxy metabolite. There is evidence for differential oxidative stress with a concentration-dependent rise in methemoglobin (MetHgb) with increasing doses of (+)-PQ greater than that seen for (-)-PQ. There was a marked, reversible hepatotoxicity in 2 of 3 animals dosed with (-)-PQ at 4.5 mg/kg. (-)-PQ in combination with chloroquine was successful in preventing P. cynomolgi disease relapse at doses of 0.6 and 1.3 mg/kg/day, while 1 of 2 animals receiving (+)-PQ at 0.6 mg/kg/day relapsed. While (-)-PQ was also associated with hepatotoxicity at higher doses as seen previously, this has not been identified as a clinical concern in humans during >60 years of use. Limited evidence for increased MetHgb generation with the (+) form in the rhesus macaque model suggests that it may be possible to improve the therapeutic window for hematologic toxicity in the clinic by separating primaquine into its enantiomers.