Polymorphisms in Pvkelch12 and gene amplification of Pvplasmepsin4 in Plasmodium vivax from Thailand, Lao PDR and Cambodia.

BACKGROUND: Mutations in Pfkelch13 and Pfplasmepsin2/3 gene amplification are well-established markers for artemisinin and piperaquine resistance in Plasmodium falciparum, a widespread problem in the Greater Mekong Subregion (GMS). The Plasmodium vivax parasite population has experienced varying drug pressure dependent on local drug policies. We investigated the correlation between drug pressure from artemisinins and piperaquine and mutations in the P. vivax orthologous genes Pvkelch12 and Pvplasmepsin4 (Pvpm4), as candidate resistance markers. METHODS: Blood samples from 734 P. vivax patients were obtained from Thailand (n = 399), Lao PDR (n = 296) and Cambodia (n = 39) between 2007 and 2017. Pvkelch12 and Pvpm4 was amplified and sequenced to assess gene mutations. To assess PvPM4 gene amplification, a Taqman® Real-Time PCR method was developed and validated. Selection of non-synonymous mutations was assessed by its ratio with synonymous mutations (Ka/Ks ratios). Mutation rates were compared to the estimated local drug pressure. RESULTS: Polymorphisms in Pvkelch12 were rare. Pvkelch12 mutations V552I, K151Q and M124I were observed in 1.0% (7/734) of P. vivax samples. V552I was the most common mutation with a frequency of 0.7% (5/734), most of which (4/5) observed in Ubon Ratchathani, Thailand. Polymorphisms in Pvpm4 were more common, with a frequency of 40.3% (123/305) in 305 samples from Thailand, Lao PDR and Cambodia, but this was not related to the estimated piperaquine drug pressure in these areas (Pearson's χ2 test, p = 0.50). Pvpm4 mutation V165I was most frequent in Tak, Thailand (40.2%, 43/107) followed by Pailin, Cambodia (43.5%, 37/85), Champasak, Lao PDR (40.4%, 23/57) and Ubon Ratchathani, Thailand (35.7%, 20/56). Pvpm4 amplification was not observed in 141 samples from Thailand and Cambodia. For both Pvkelch12 and Pvpm4, in all areas and at all time points, the Ka/Ks values were < 1, suggesting no purifying selection. CONCLUSIONS: A novel real-time PCR-based method to assess P. vivax Pvpm4 gene amplification was developed. Drug pressure with artemisinins and piperaquine in the GMS was not clearly related to signatures of selection for mutations in the P. vivax orthologous resistance genes Pvkelch12 and Pvpm4 in areas under investigation. Current resistance of P. vivax to these drugs is unlikely and additional observations including analysis of associated clinical data from these regions could further clarify current findings.

Induction of Multidrug Tolerance in Plasmodium falciparum by Extended Artemisinin Pressure.

Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia threatens global malaria control strategies. Whether delayed parasite clearance, which exposes larger parasite numbers to artemisinins for longer times, selects higher-grade resistance remains unexplored. We investigated whether long-lasting artemisinin pressure selects a novel multidrug-tolerance profile. Although 50% inhibitory concentrations for 10 antimalarial drugs tested were unchanged, drug-tolerant parasites showed higher recrudescence rates for endoperoxides, quinolones, and an antifolate, including partner drugs of recommended combination therapies, but remained susceptible to atovaquone. Moreover, the age range of intraerythrocytic stages able to resist artemisinin was extended to older ring forms and trophozoites. Multidrug tolerance results from drug-induced quiescence, which enables parasites to survive exposure to unrelated antimalarial drugs that inhibit a variety of metabolic pathways. This novel resistance pattern should be urgently monitored in the field because this pattern is not detected by current assays and represents a major threat to antimalarial drug policy.

Prevalence and predictors of residual antibiotics in children's blood in community settings in Tanzania.

OBJECTIVES: Children account for a significant proportion of antibiotic consumption in low- and middle-income countries, with overuse occurring in formal and informal health sectors. This study assessed the prevalence and predictors of residual antibiotics in the blood of children in the Mbeya and Morogoro regions of Tanzania. METHODS: The cross-sectional community-based survey used two-stage cluster sampling to include children aged under 15 years. For each child, information on recent illness, healthcare-seeking behaviour, and use of antibiotics, as well as a dried blood spot sample, were collected. The samples underwent tandem mass spectrometry analysis to quantify the concentrations of 15 common antibiotics. Associations between survey variables and the presence of residual antibiotics were assessed using mixed-effects logistic regression. RESULTS: In total, 1742 children were surveyed, and 1699 analysed. The overall prevalence of residual antibiotics in the blood samples was 17.4% (296/1699), the highest among children under the age of 5 years. The most frequently detected antibiotics were trimethoprim (144/1699; 8.5%), sulfamethoxazole (102/1699; 6.0%), metronidazole (61/1699; 3.6%), and amoxicillin (43/1699; 2.5%). The strongest predictors of residual antibiotics in the blood were observed presence of antibiotics at home (adjusted odds ratio [aOR] = 2.9; 95% CI, 2.0-4.1) and reported consumption of antibiotics in the last 2 weeks (aOR = 2.5; 95% CI, 1.6-3.9). However, half (145/296) of the children who had residual antibiotics in their blood, some with multiple antibiotics, had no reported history of illness or antibiotic consumption in the last 2 weeks, and antibiotics were not found at home. DISCUSSION: This study demonstrated a high prevalence of antibiotic exposure among children in Tanzanian communities, albeit likely underestimated, especially for compounds with short half-lives. A significant proportion of antibiotic exposure was unexplained and may have been due to unreported self-medication or environmental pathways. Incorporating biomonitoring into surveillance strategies can help better understand exposure patterns and design antibiotic stewardship interventions.

Drug selection pressure and fitness cost for artemether-resistant Plasmodium berghei ANKA parasites in vivo.

BACKGROUND: The clinical use of artemisinin-based combination therapies is threatened by increasing failure rates due to the emergence and spread of multiple drug resistance genes in most human Plasmodium strains. The aim of this study was to generate artemether-resistant (AMR) parasites from Plasmodium berghei ANKA (AMS), and determine their fitness cost. METHODS: Artemether resistance was generated by increasing drug pressure doses gradually for 9 months. Effective doses (ED50 and ED90) were determined using the 4-day suppressive test, and the indices of resistance (I) at 50% and 90% (I50 and I90) were determined using the ratio of either ED50 or ED90 of AMR to AMS, respectively. The stability of the AMR parasites was evaluated by: five drug-free passages (5DFPs), 3 months of cryopreservation (CP), and drug-free serial passages (DFSPs) for 4 months. Analysis of variance was used to compare differences in growth rates between AMR and AMS with 95% confidence intervals. RESULTS: ED50 and ED90 of AMS were 0.61 and 3.43 mg/kg/day respectively. I50 and I90 after 20 cycles of artemether selection pressure were 19.67 and 21.45, respectively; 5DFP values were 39.16 and 15.27, respectively; 3-month CP values were 29.36 and 10.79, respectively; and DFSP values were 31.34 and 12.29, respectively. The mean parasitaemia value of AMR (24.70% ± 3.60) relative to AMS (37.66% ± 3.68) at Day 7 post infection after DFSPs revealed a fitness cost of 34.41%. CONCLUSION: A moderately stable AMRP. berghei line was generated. Known and unknown mutations may be involved in modulating artemether resistance, and therefore molecular investigations are recommended.

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.

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.

Drug resistance and in vitro susceptibility of Plasmodium falciparum in Thailand during 1988-2003.

The aim of the present study was to investigate antimalarial drug pressure resulting from the clinical use of different antimalarials in Thailand. The phenotypic diversity of the susceptibility profiles of antimalarials, i.e., chloroquine (CQ), quinine (QN), mefloquine (MQ), and artesunate (ARS) in Plasmodium falciparum isolates collected during the period from 1988 to 2003 were studied. P. falciparum isolates from infected patients were collected from the Thai-Cambodian border area at different time periods (1988-1989, 1991-1992, and 2003), during which 3 different patterns of drug use had been implemented: MQ + sulphadoxine (S) + pyrimethamine (P), MQ alone and MQ + ARS, respectively. The in vitro drug susceptibilities were investigated using a method based on the incorporation of [(3)H] hypoxanthine. A total of 50 isolates were tested for susceptibilities to CQ, QN, MQ, and ARS. Of these isolates, 19, 16, and 15 were adapted during the periods 1988-1989, 1991-1993, and 2003, respectively. P. falciparum isolates collected during the 3 periods were resistant to CQ. Sensitivities to MQ declined from 1988 to 2003. In contrast, the parasite was sensitive to QN, and similar sensitivity profile patterns were observed during the 3 time periods. There was a significantly positive but weak correlation between the IC(50) values of CQ and QN, as well as between the IC(50) values of QN and MQ. Drug pressure has impact on sensitivity of P. falciparum to MQ. A combination therapy of MQ and ARS is being applied to reduce the parasite resistance, and also increasing the efficacy of the drug.

Drug Resistance and in Vitro Susceptibility of Plasmodium falciparum in Thailand during 1988-2003

The aim of the present study was to investigate antimalarial drug pressure resulting from the clinical use of different antimalarials in Thailand. The phenotypic diversity of the susceptibility profiles of antimalarials, i.e., chloroquine (CQ), quinine (QN), mefloquine (MQ), and artesunate (ARS) in Plasmodium falciparum isolates collected during the period from 1988 to 2003 were studied. P. falciparum isolates from infected patients were collected from the Thai-Cambodian border area at different time periods (1988-1989, 1991-1992, and 2003), during which 3 different patterns of drug use had been implemented: MQ + sulphadoxine (S) + pyrimethamine (P), MQ alone and MQ + ARS, respectively. The in vitro drug susceptibilities were investigated using a method based on the incorporation of [3H] hypoxanthine. A total of 50 isolates were tested for susceptibilities to CQ, QN, MQ, and ARS. Of these isolates, 19, 16, and 15 were adapted during the periods 1988-1989, 1991-1993, and 2003, respectively. P. falciparum isolates collected during the 3 periods were resistant to CQ. Sensitivities to MQ declined from 1988 to 2003. In contrast, the parasite was sensitive to QN, and similar sensitivity profile patterns were observed during the 3 time periods. There was a significantly positive but weak correlation between the IC50 values of CQ and QN, as well as between the IC50 values of QN and MQ. Drug pressure has impact on sensitivity of P. falciparum to MQ. A combination therapy of MQ and ARS is being applied to reduce the parasite resistance, and also increasing the efficacy of the drug.