Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with more than 780,000 deaths worldwide (as of 20 August 2020). To develop antiviral interventions quickly, drugs used for the treatment of unrelated diseases are currently being repurposed to treat COVID-19. Chloroquine is an anti-malaria drug that is used for the treatment of COVID-19 as it inhibits the spread of SARS-CoV-2 in the African green monkey kidney-derived cell line Vero1-3. Here we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells4, renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine. Moreover, we report that chloroquine does not block infection with SARS-CoV-2 in the TMPRSS2-expressing human lung cell line Calu-3. These results indicate that chloroquine targets a pathway for viral activation that is not active in lung cells and is unlikely to protect against the spread of SARS-CoV-2 in and between patients.

Higher-Dose Primaquine to Prevent Relapse of Plasmodium vivax Malaria.

BACKGROUND: In most of the Americas, the recommended treatment to prevent relapse of Plasmodium vivax malaria is primaquine at a total dose of 3.5 mg per kilogram of body weight, despite evidence of only moderate efficacy. METHODS: In this trial conducted in Brazil, we evaluated three primaquine regimens to prevent relapse of P. vivax malaria in children at least 5 years of age and in adults with microscopy-confirmed P. vivax monoinfection. All the patients received directly observed chloroquine for 3 days (total dose, 25 mg per kilogram). Group 1 received a total primaquine dose of 3.5 mg per kilogram (0.5 mg per kilogram per day) over 7 days with unobserved administration; group 2 received the same regimen as group 1 but with observed administration; and group 3 received a total primaquine dose of 7.0 mg per kilogram over 14 days (also 0.5 mg per kilogram per day) with observed administration. We monitored the patients for 168 days. RESULTS: We enrolled 63 patients in group 1, 96 in group 2, and 95 in group 3. The median age of the patients was 22.4 years (range, 5.4 to 79.8). By day 28, three P. vivax recurrences were observed: 2 in group 1 and 1 in group 2. By day 168, a total of 70 recurrences had occurred: 24 in group 1, 34 in group 2, and 12 in group 3. No serious adverse events were noted. On day 168, the percentage of patients without recurrence was 58% (95% confidence interval [CI], 44 to 70) in group 1, 59% (95% CI, 47 to 69) in group 2, and 86% (95% CI, 76 to 92) in group 3. Survival analysis showed a difference in the day 168 recurrence-free percentage of 27 percentage points (97.5% CI, 10 to 44; P<0.001) between group 1 and group 3 and a difference of 27 percentage points (97.5% CI, 12 to 42; P<0.001) between group 2 and group 3. CONCLUSIONS: The administration of primaquine at a total dose of 7.0 mg per kilogram had higher efficacy in preventing relapse of P. vivax malaria than a total dose of 3.5 mg per kilogram through day 168. (Supported by the U.S. Agency for International Development; ClinicalTrials.gov number, NCT03610399.).

PfCRT mutations conferring piperaquine resistance in falciparum malaria shape the kinetics of quinoline drug binding and transport.

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ.

Molecular Mechanisms of Drug Resistance in Plasmodium falciparum Malaria.

Understanding and controlling the spread of antimalarial resistance, particularly to artemisinin and its partner drugs, is a top priority. Plasmodium falciparum parasites resistant to chloroquine, amodiaquine, or piperaquine harbor mutations in the P. falciparum chloroquine resistance transporter (PfCRT), a transporter resident on the digestive vacuole membrane that in its variant forms can transport these weak-base 4-aminoquinoline drugs out of this acidic organelle, thus preventing these drugs from binding heme and inhibiting its detoxification. The structure of PfCRT, solved by cryogenic electron microscopy, shows mutations surrounding an electronegative central drug-binding cavity where they presumably interact with drugs and natural substrates to control transport. P. falciparum susceptibility to heme-binding antimalarials is also modulated by overexpression or mutations in the digestive vacuole membrane-bound ABC transporter PfMDR1 (P. falciparum multidrug resistance 1 transporter). Artemisinin resistance is primarily mediated by mutations in P. falciparum Kelch13 protein (K13), a protein involved in multiple intracellular processes including endocytosis of hemoglobin, which is required for parasite growth and artemisinin activation. Combating drug-resistant malaria urgently requires the development of new antimalarial drugs with novel modes of action.

Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT.

Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite's susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite's susceptibility to lumefantrine and mefloquine-a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite's digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine's access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.

Fluvastatin prevents lung metastasis in triple-negative breast cancer by triggering autophagy via the RhoB/PI3K/mTOR pathway.

Triple-negative breast cancer is more common among younger than older women and is associated with the poorest survival outcomes of all breast cancer types. Fluvastatin inhibits tumour progression and induces the autophagy of breast cancer cells; however, the role of autophagy in fluvastatin-induced inhibition of breast cancer metastasis is unknown. Therefore, this study aimed to determine this mechanism. The effect of fluvastatin on human hormone receptor-negative breast cancer cells was evaluated in vitro via migration and wound healing assays, western blotting, and morphological measurements, as well as in vivo using a mouse xenograft model. Chloroquine, a prophylactic medication used to prevent malaria in humans was used as an autophagy inhibitor. We found that fluvastatin administration effectively prevented the migration/invasion of triple-negative breast cancer cells, an effect that was largely dependent on the induction of autophagy. Administration of the autophagy inhibitor chloroquine prevented the fluvastatin-induced suppression of lung metastasis in the nude mouse model. Furthermore, fluvastatin increased Ras homolog family member B (RhoB) expression, and the autophagy and anti-metastatic activity induced by fluvastatin were predominantly dependent on the regulation of RhoB through the protein kinase B-mammalian target of rapamycin (Akt-mTOR) signaling pathway. These results suggest that fluvastatin inhibits the metastasis of triple-negative breast cancer cells by modulating autophagy via the up regulation of RhoB through the AKT-mTOR signaling pathway. Fluvastatin may be a promising therapeutic option for patients with triple-negative breast cancer.

Autophagy is required for mammary tumor recurrence by promoting dormant tumor cell survival following therapy.

BACKGROUND: Mortality from breast cancer is principally due to tumor recurrence. Recurrent breast cancers arise from the pool of residual tumor cells, termed minimal residual disease, that survive treatment and may exist in a dormant state for 20 years or more following treatment of the primary tumor. As recurrent breast cancer is typically incurable, understanding the mechanisms underlying dormant tumor cell survival is a critical priority in breast cancer research. The importance of this goal is further underscored by emerging evidence suggesting that targeting dormant residual tumor cells in early-stage breast cancer patients may be a means to prevent tumor recurrence and its associated mortality. In this regard, the role of autophagy in dormant tumor cell survival and recurrence remains unresolved, with conflicting reports of both pro-survival/recurrence-promoting and pro-death/recurrence-suppressing effects of autophagy inhibition in dormant tumor cells. Resolving this question has important clinical implications. METHODS: We used genetically engineered mouse models that faithfully recapitulate key features of human breast cancer progression, including minimal residual disease, tumor dormancy, and recurrence. We used genetic and pharmacological approaches to inhibit autophagy, including treatment with chloroquine, genetic knockdown of ATG5 or ATG7, or deletion of BECN and determined their effects on dormant tumor cell survival and recurrence. RESULTS: We demonstrate that the survival and recurrence of dormant mammary tumor cells following therapy is dependent upon autophagy. We find that autophagy is induced in vivo following HER2 downregulation and remains activated in dormant residual tumor cells. Using genetic and pharmacological approaches we show that inhibiting autophagy by chloroquine administration, ATG5 or ATG7 knockdown, or deletion of a single allele of the tumor suppressor Beclin 1 is sufficient to inhibit mammary tumor recurrence, and that autophagy inhibition results in the death of dormant mammary tumor cells in vivo. CONCLUSIONS: Our findings demonstrate a pro-tumorigenic role for autophagy in tumor dormancy and recurrence following therapy, reveal that dormant tumor cells are uniquely reliant upon autophagy for their survival, and indicate that targeting dormant residual tumor cells by inhibiting autophagy impairs tumor recurrence. These studies identify a pharmacological target for a cellular state that is resistant to commonly used anti-neoplastic agents and suggest autophagy inhibition as an approach to reduce dormant minimal residual disease in order to prevent lethal tumor recurrence.

Evaluation of hydroxychloroquine or chloroquine for the prevention of COVID-19 (COPCOV): A double-blind, randomised, placebo-controlled trial.

BACKGROUND: Hydroxychloroquine (HCQ) has proved ineffective in treating patients hospitalised with Coronavirus Disease 2019 (COVID-19), but uncertainty remains over its safety and efficacy in chemoprevention. Previous chemoprevention randomised controlled trials (RCTs) did not individually show benefit of HCQ against COVID-19 and, although meta-analysis did suggest clinical benefit, guidelines recommend against its use. METHODS AND FINDINGS: Healthy adult participants from the healthcare setting, and later from the community, were enrolled in 26 centres in 11 countries to a double-blind, placebo-controlled, randomised trial of COVID-19 chemoprevention. HCQ was evaluated in Europe and Africa, and chloroquine (CQ) was evaluated in Asia, (both base equivalent of 155 mg once daily). The primary endpoint was symptomatic COVID-19, confirmed by PCR or seroconversion during the 3-month follow-up period. The secondary and tertiary endpoints were: asymptomatic laboratory-confirmed Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection; severity of COVID-19 symptoms; all-cause PCR-confirmed symptomatic acute respiratory illness (including SARS-CoV-2 infection); participant reported number of workdays lost; genetic and baseline biochemical markers associated with symptomatic COVID-19, respiratory illness and disease severity (not reported here); and health economic analyses of HCQ and CQ prophylaxis on costs and quality of life measures (not reported here). The primary and safety analyses were conducted in the intention-to-treat (ITT) population. Recruitment of 40,000 (20,000 HCQ arm, 20,000 CQ arm) participants was planned but was not possible because of protracted delays resulting from controversies over efficacy and adverse events with HCQ use, vaccine rollout in some countries, and other factors. Between 29 April 2020 and 10 March 2022, 4,652 participants (46% females) were enrolled (HCQ/CQ n = 2,320; placebo n = 2,332). The median (IQR) age was 29 (23 to 39) years. SARS-CoV-2 infections (symptomatic and asymptomatic) occurred in 1,071 (23%) participants. For the primary endpoint the incidence of symptomatic COVID-19 was 240/2,320 in the HCQ/CQ versus 284/2,332 in the placebo arms (risk ratio (RR) 0.85 [95% confidence interval, 0.72 to 1.00; p = 0.05]). For the secondary and tertiary outcomes asymptomatic SARS-CoV-2 infections occurred in 11.5% of HCQ/CQ recipients and 12.0% of placebo recipients: RR: 0.96 (95% CI, 0.82 to 1.12; p = 0.6). There were no differences in the severity of symptoms between the groups and no severe illnesses. HCQ/CQ chemoprevention was associated with fewer PCR-confirmed all-cause respiratory infections (predominantly SARS-CoV-2): RR 0.61 (95% CI, 0.42 to 0.88; p = 0.009) and fewer days lost to work because of illness: 104 days per 1,000 participants over 90 days (95% CI, 12 to 199 days; p < 0.001). The prespecified meta-analysis of all published pre-exposure RCTs indicates that HCQ/CQ prophylaxis provided a moderate protective benefit against symptomatic COVID-19: RR 0.80 (95% CI, 0.71 to 0.91). Both drugs were well tolerated with no drug-related serious adverse events (SAEs). Study limitations include the smaller than planned study size, the relatively low number of PCR-confirmed infections, and the lower comparative accuracy of serology endpoints (in particular, the adapted dried blood spot method) compared to the PCR endpoint. The COPCOV trial was registered with ClinicalTrials.gov; number NCT04303507. INTERPRETATION: In this large placebo-controlled, double-blind randomised trial, HCQ and CQ were safe and well tolerated in COVID-19 chemoprevention, and there was evidence of moderate protective benefit in a meta-analysis including this trial and similar RCTs. TRIAL REGISTRATION: ClinicalTrials.gov NCT04303507; ISRCTN Registry ISRCTN10207947.

Transmission-blocking activities of artesunate, chloroquine, and methylene blue on Plasmodium vivax gametocytes.

Plasmodium vivax is now the main cause of malaria outside Africa. The gametocytocidal effects of antimalarial drugs are important to reduce malaria transmissibility, particularly in low-transmission settings, but they are not well characterized for P. vivax. The transmission-blocking effects of chloroquine, artesunate, and methylene blue on P. vivax gametocytes were assessed. Blood specimens were collected from patients presenting with vivax malaria, incubated with or without the tested drugs, and then fed to mosquitos from a laboratory-adapted colony of Anopheles dirus (a major malaria vector in Southeast Asia). The effects on oocyst and sporozoite development were analyzed under a multi-level Bayesian model accounting for assay variability and the heterogeneity of mosquito Plasmodium infection. Artesunate and methylene blue, but not chloroquine, exhibited potent transmission-blocking effects. Gametocyte exposures to artesunate and methylene blue reduced the mean oocyst count 469-fold (95% CI: 345 to 650) and 1,438-fold (95% CI: 970 to 2,064), respectively. The corresponding estimates for the sporozoite stage were a 148-fold reduction (95% CI: 61 to 470) and a 536-fold reduction (95% CI: 246 to 1,311) in the mean counts, respectively. In contrast, high chloroquine exposures reduced the mean oocyst count only 1.40-fold (95% CI: 1.20 to 1.64) and the mean sporozoite count 1.34-fold (95% CI: 1.12 to 1.66). This suggests that patients with vivax malaria often remain infectious to anopheline mosquitos after treatment with chloroquine. Use of artemisinin combination therapies or immediate initiation of primaquine radical cure should reduce the transmissibility of P. vivax infections.

Evidence for the early emergence of piperaquine-resistant Plasmodium falciparum malaria and modeling strategies to mitigate resistance.

Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter's central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.

Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis.

The human malaria parasite Plasmodium falciparum is globally widespread, but its prevalence varies significantly between and even within countries. Most population genetic studies in P. falciparum focus on regions of high transmission where parasite populations are large and genetically diverse, such as sub-Saharan Africa. Understanding population dynamics in low transmission settings, however, is of particular importance as these are often where drug resistance first evolves. Here, we use the Pacific Coast of Colombia and Ecuador as a model for understanding the population structure and evolution of Plasmodium parasites in small populations harboring less genetic diversity. The combination of low transmission and a high proportion of monoclonal infections means there are few outcrossing events and clonal lineages persist for long periods of time. Yet despite this, the population is evolutionarily labile and has successfully adapted to changes in drug regime. Using newly sequenced whole genomes, we measure relatedness between 166 parasites, calculated as identity by descent (IBD), and find 17 distinct but highly related clonal lineages, six of which have persisted in the region for at least a decade. This inbred population structure is captured in more detail with IBD than with other common population structure analyses like PCA, ADMIXTURE, and distance-based trees. We additionally use patterns of intra-chromosomal IBD and an analysis of haplotypic variation to explore past selection events in the region. Two genes associated with chloroquine resistance, crt and aat1, show evidence of hard selective sweeps, while selection appears soft and/or incomplete at three other key resistance loci (dhps, mdr1, and dhfr). Overall, this work highlights the strength of IBD analyses for studying parasite population structure and resistance evolution in regions of low transmission, and emphasizes that drug resistance can evolve and spread in small populations, as will occur in any region nearing malaria elimination.

Chloroquine has shown high therapeutic efficacy against uncomplicated Plasmodium vivax malaria in southern Ethiopia: seven decades after its introduction.

BACKGROUND: Plasmodium vivax malaria is a leading cause of morbidity in Ethiopia. The first-line treatment for P. vivax is chloroquine (CQ) and primaquine (PQ), but there have been local reports of CQ resistance. A clinical study was conducted to determine the efficacy of CQ for the treatment of P. vivax malaria in southern Ethiopia. METHODS: In 2021, patients with P. vivax mono-infection and uncomplicated malaria were enrolled and treated with 25 mg/kg CQ for 3 consecutive days. Patients were followed for 28 days according to WHO guidelines. The data were analysed using per-protocol (PP) and Kaplan‒Meier (K‒M) analyses to estimate the risk of recurrent P. vivax parasitaemia on day 28. RESULTS: A total of 88 patients were enrolled, 78 (88.6%) of whom completed the 28 days of follow-up. Overall, 76 (97.4%) patients had adequate clinical and parasitological responses, and two patients had late parasitological failures. The initial therapeutic response was rapid, with 100% clearance of asexual parasitaemia within 48 h. CONCLUSION: Despite previous reports of declining chloroquine efficacy against P. vivax, CQ retains high therapeutic efficacy in southern Ethiopia, supporting the current national treatment guidelines. Ongoing clinical monitoring of CQ efficacy supported by advanced molecular methods is warranted to inform national surveillance and ensure optimal treatment guidelines.

Intervention portfolios analysis of Plasmodium vivax control in central China.

BACKGROUND: The effects of a diverse spectrum of malaria interventions were evaluated through a deterministic Plasmodium vivax transmission model. This approach aimed to provide theoretical evidence of the performance of these interventions once implemented for achieving malaria elimination. METHODS: An integrated intervention portfolio, including mass drug administration, insecticide treatment, and untreated bed nets, was analyzed through modeling. Additionally, data-driven calibration was implemented to infer coverages that effectively reproduced historical malaria patterns in China from 1971 to 1983. RESULTS: MDA utilizing primaquine emerged as the most effective single intervention, achieving a 70% reduction in malaria incidence when implemented at full coverage. Furthermore, a strategic combination of MDA with primaquine, chloroquine, untreated bed nets, and seasonal insecticide treatments effectively eradicated malaria, attaining elimination at a coverage level of 70%. It was conclusively demonstrated that an integrated approach combining MDA and vector control measures is essential for the successful elimination of malaria. CONCLUSION: High coverage of mass drug administration with primaquine and chloroquine before transmission was the key driver of the malaria decline in China from 1971 to 1983. The best-fit intervention coverage combinations derived from calibration are provided as a reference for malaria control in other countries.

Efficacy and safety of chloroquine plus primaquine for the treatment of Plasmodium vivax malaria in Hamusit site, Northwestern Ethiopia.

BACKGROUND: Plasmodium vivax malaria is still an important public health problem in Ethiopia. Unlike Plasmodium falciparum, P. vivax has a dormant liver stage (hypnozoite) that can be a risk of recurrent vivax malaria unless treated by radical cure with primaquine. Drug resistance to chloroquine is threatening malaria control and elimination efforts. This study assessed the therapeutic efficacy and safety of chloroquine plus 14 days of primaquine on P. vivax infection based on parasitological, clinical, and haematological parameters. METHODS: A single-arm in vivo prospective therapeutic efficacy study was conducted to assess the clinical and parasitological response to the first-line treatment of P. vivax in Ethiopia, chloroquine plus 14 days low dose of (0.25 mg/kg/day) primaquine between December 2022 and March 2023 at Hamusit Health Centre using the standard World Health Organization (WHO) protocol. A total of 100 study participants with P. vivax mono-infection who were over 6 months old were enrolled and monitored for adequate clinical and parasitological responses for 42 days. The WHO double-entry Excel sheet and SPSS v.25 software were used for Kaplan-Meier survival analysis, and a paired t-test was used for analysis of haemoglobin improvements between follow up days. RESULTS: A total of 100 patients were enrolled among those, 96% cases were rural residents, 93% had previous malaria exposure, and predominant age group was 5-15 years (61%). 92.6% (95% CI 85.1-96.4%) of enrolled patients were adequate clinical and parasitological response, and 7.4% (95% CI 3.6-14.9%) recurrences were observed among treated patients. The fever and parasite clearance rate on day 3 were 98% and 94%, respectively. The baseline haemoglobin levels improved significantly compared to those days 14 and 42 (p < 0.001). No serious adverse event was observed during the study period. CONCLUSIONS: In this study, co-administration of chloroquine with primaquine was efficacious and well-tolerated with fast resolution of fever and high parasites clearance rate. However, the 7.4% failure is reported is alarming that warrant further monitoring of the therapeutic efficacy study of P. vivax.

In vivo efficacy of chloroquine plus primaquine combination therapy against uncomplicated Plasmodium vivax malaria in Limu Kossa District, Jimma Zone, Southwest Ethiopia.

BACKGROUND: Plasmodium vivax is the second most common malaria parasite in Ethiopia. It has been treated with chloroquine (CQ) for the past seven decades. However, the emergence of CQ-resistant strains in the nation urged the Federal Ministry of Health of Ethiopia to review its national malaria treatment guideline in 2018. In the revised guideline, the first-line treatment for uncomplicated P. vivax infection is a combination of CQ and primaquine (PQ). Thus, the present study was designed to evaluate the in vivo efficacy of CQ and PQ combination therapy against clinical P. vivax mono-infection in one of the malaria-endemic areas of Ethiopia. METHODS: An open-label prospective clinical trial was conducted in the Limmu Kossa District, Jimma zone, Southwest Ethiopia, from September 2023 to March 2024. A total of 108 patients were recruited for the study. All participants received treatment with CQ at a dosage of 25 mg/kg over three days, followed by PQ at 0.25 mg/kg for 14 consecutive days. Patients were monitored for 42 days for any signs of treatment failure and malaria clinical symptoms, as per the World Health Organization (WHO) guidelines for anti-malarial drug evaluation. Additionally, haemoglobin (Hb) levels, body temperature, any adverse events, and signs of haemolysis were assessed. Data was analysed using R-software (version 4.0.0) and a significant level was considered at p < 0.05. RESULTS: The median age of the patients was 23 years, ranging from 2.5 to 62 years. Of the 108 patients initially recruited, 100 completed the 42-day follow-up period. The combination therapy of CQ and PQ for uncomplicated clinical P. vivax malaria demonstrated excellent therapeutic efficacy, with a 100% cure rate observed at both day 28 and day 42. Additionally, the recommended low dose of PQ (0.25 mg/kg) was well-tolerated, with no signs of. Additionally, most common malaria symptoms were disappeared early in the follow-up period. CONCLUSION: The combination of CQ plus PQ has exhibited excellent efficacy against uncomplicated P. vivax malaria mono-infections. To preserve this efficacy, it is critical to ensure patients adhere to the full course of PQ treatment, despite its extended duration. Therefore, health authorities should put emphasis on the boosting of the public on the importance of finishing the prescribed medication regimen.

Kinetics of glucose-6-phosphate dehydrogenase (G6PD) activity during Plasmodium vivax infection: implications for early radical malaria treatment.

BACKGROUND: Plasmodium vivax relapses due to dormant liver hypnozoites can be prevented with primaquine. However, the dose must be adjusted in individuals with glucose-6-phosphate-dehydrogenase (G6PD) deficiency. In French Guiana, assessment of G6PD activity is typically delayed until day (D)14 to avoid the risk if misclassification. This study assessed the kinetics of G6PD activity throughout P. vivax infection to inform the timing of treatment. METHODS: For this retrospective monocentric study, data on G6PD activity between D1 and D28 after treatment initiation with chloroquine or artemisinin-based combination therapy were collected for patients followed at Cayenne Hospital, French Guiana, between January 2018 and December 2020. Patients were divided into three groups based on the number of available G6PD activity assessments: (i) at least two measurements during the P. vivax malaria infection; (ii) two measurements: one during the current infection and one previously; (iii) only one measurement during the malaria infection. RESULTS: In total, 210 patients were included (80, 20 and 110 in groups 1, 2 and 3, respectively). Data from group 1 showed that G6PD activity remained stable in each patient over time (D1, D3, D7, D14, D21, D28). None of the patients with normal G6PD activity during the initial phase (D1-D3) of the malaria episode (n = 44) was categorized as G6PD-deficient at D14. Patients with G6PD activity < 80% at D1 or D3 showed normal activity at D14. Sex and reticulocyte count were statistically associated with G6PD activity variation. In the whole sample (n = 210), no patient had severe G6PD deficiency (< 10%) and only three between 10 and 30%, giving a G6PD deficiency prevalence of 1.4%. Among the 100 patients from group 1 and 2, 30 patients (26.5%) were lost to follow-up before primaquine initiation. CONCLUSIONS: In patients treated for P. vivax infection, G6PD activity did not vary over time. Therefore, G6PD activity on D1 instead of D14 could be used for primaquine dose-adjustment. This could allow earlier radical treatment with primaquine, that could have a public health impact by decreasing early recurrences and patients lost to follow-up before primaquine initiation. This hypothesis needs to be confirmed in larger prospective studies.

Optimal balance of benefit versus risk for tafenoquine in the treatment of Plasmodium vivax malaria.

A single 300 mg dose of tafenoquine (an 8-aminoquinoline), in combination with a standard 3-day course of chloroquine, is approved in several countries for the radical cure (prevention of relapse) of Plasmodium vivax malaria in patients aged ≥ 16 years. Despite this, questions have arisen on the optimal dose of tafenoquine. Before the availability of tafenoquine, a 3-day course of chloroquine in combination with the 8-aminoquinoline primaquine was the only effective radical cure for vivax malaria. The World Health Organization (WHO)-recommended standard regimen is 14 days of primaquine 0.25 mg/kg/day or 7 days of primaquine 0.5 mg/kg/day in most regions, or 14 days of primaquine 0.5 mg/kg/day in East Asia and Oceania, however the long treatment courses of 7 or 14 days may result in poor adherence and, therefore, low treatment efficacy. A single dose of tafenoquine 300 mg in combination with a 3-day course of chloroquine is an important advancement for the radical cure of vivax malaria in patients without glucose-6-phosphate dehydrogenase (G6PD) deficiency, as the use of a single-dose treatment will improve adherence. Selection of a single 300 mg dose of tafenoquine for the radical cure of P. vivax malaria was based on collective efficacy and safety data from 33 studies involving more than 4000 trial participants who received tafenoquine, including over 800 subjects who received the 300 mg single dose. The safety profile of single-dose tafenoquine 300 mg is similar to that of standard-dosage primaquine 0.25 mg/kg/day for 14 days. Both primaquine and tafenoquine can cause acute haemolytic anaemia in individuals with G6PD deficiency; severe haemolysis can lead to anaemia, kidney damage, and, in some cases, death. Therefore, relapse prevention using an 8-aminoquinoline must be balanced with the need to avoid clinical haemolysis associated with G6PD deficiency. To minimize this risk, the WHO recommends G6PD testing for all individuals before the administration of curative doses of 8-aminoquinolines. In this article, the authors review key efficacy and safety data from the pivotal trials of tafenoquine and argue that the currently approved dose represents a favourable benefit-risk profile.

Magnolol induces cytotoxic autophagy in glioma by inhibiting PI3K/AKT/mTOR signaling.

Glioma is difficult-to-treat because of its infiltrative nature and the presence of the blood-brain barrier. Temozolomide is the only FDA-approved drug for its management. Therefore, finding a novel chemotherapeutic agent for glioma is of utmost importance. Magnolol, a neolignan, has been known for its apoptotic role in glioma. In this work, we have explored a novel anti-glioma mechanism of Magnolol associated with its role in autophagy modulation. We found increased expression levels of Beclin-1, Atg5-Atg12, and LC3-II and lower p62 expression in Magnolol-treated glioma cells. PI3K/AKT/mTOR pathway proteins were also downregulated in Magnolol-treated glioma cells. Next, we treated the glioma cells with Insulin, a stimulator of PI3K/AKT/mTOR signaling, to confirm that Magnolol induced autophagy by inhibiting this pathway. Insulin reversed the effect on Magnolol-mediated autophagy induction. We also established the same in in vivo glioma model where Magnolol showed an anti-glioma effect by inducing autophagy. To confirm the cytotoxic effect of Magnolol-induced autophagy, we used Chloroquine, a late-stage autophagy inhibitor. Chloroquine efficiently reversed the anti-glioma effects of Magnolol both in vitro and in vivo. Our study revealed the cytotoxic effect of Magnolol-induced autophagy in glioma, which was not previously reported. Additionally, Magnolol showed no toxicity in non-cancerous cell lines as well as rat organs. Thus, we concluded that Magnolol is an excellent candidate for developing new therapeutic strategies for glioma management.

Efficacy of antimalarials in oral lichen planus: A systematic review.

OBJECTIVE: To evaluate whether hydroxychloroquine (HCQ) or chloroquine (CQ) are effective for the treatment of oral lichen planus (OLP). MATERIALS AND METHODS: A literature search was conducted in four databases. Clinical studies investigating the effect of HCQ/CQ in patients with OLP were included. RESULTS: Eleven studies were included. Four were RCTs and seven quasi-experimental studies. The studies included 390 patients diagnosed with OLP, of which 326 and 7 received HCQ and CQ, respectively. 46 patients received topical dexamethasone, 5 placebo and 6 griseofulvin as controls. Five studies assessed pain, and all of them obtained pain reduction with the use of HCQ. Six studies reported objective clinical improvement of OLP with the use of HCQ. Five studies that used a subjective scale obtained that 24%-100% of the patients achieved a complete/almost complete improvement of OLP lesions and its symptomatology. The most frequent side effects were vision problems, gastric discomfort, rash, nauseas, headaches, skin pigmentation, and elevated kidney function. 17 patients had to withdraw from the studies. CONCLUSIONS: Current evidence is scarce to confirm HCQ as a therapeutic option for OLP. More RCTs are needed to compare its efficacy with topical corticosteroids and to evaluate whether HCQ reduces relapses of OLP.

The effects of hydroxychloroquine and its promising use in refractory obstetric antiphospholipid syndrome.

Hydroxychloroquine (HCQ) is obtained by hydroxylation of chloroquine (CQ) and the first indication was malaria. Nowadays, HCQ is commonly used in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) with favorable results. Antiphospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity and persistent positivity of antiphospholipid antibodies. Around 20-30% of pregnant women with APS develop adverse pregnancy outcomes despite conventional treatment with aspirin and heparin, called refractory obstetric APS. Interestingly, HCQ has shown positive effects on top of the standard of care in some refractory obstetric APS patients. HCQ mechanisms of action in APS comprise its ability to bind sialic acid present in cell membranes, its capacity to block the binding of antiphospholipid antibodies to the cell and the induced increase of pH in extracellular and intracellular compartments. However, the precise mechanisms of HCQ in the specific situation of refractory APS still need to be fully clarified. Therefore, this review summarizes the known modulating effects of HCQ and CQ, their side effects and use in APS and different pathologies to understand the benefit effects and the mechanism of action of HCQ in refractory obstetric APS.