Evolution of genetic markers for drug resistance after the introduction of dihydroartemisinin-piperaquine as first-line anti-malarial treatment for uncomplicated falciparum malaria in Indonesia.

BACKGROUND: Dihydroartemisinin-piperaquine has been Indonesia's first-line anti-malarial treatment since 2008. Annual therapeutic efficacy studies (TES) done in the last 12 years showed continued high treatment efficacy in uncomplicated Plasmodium falciparum malaria. Although these studies did not show evidence for artemisinin resistance, a slight increase in Late Treatment Failure was observed over time. It is highlight to explore the evolution of genetic markers for ACT partner drug resistance since adopting DHA-PPQ. METHODS: Dry blood spots were identified from a mass blood survey of uncomplicated falciparum malaria patients (N = 50) in Sumba from 2010 to 2018. Analysis of genotypic profile (N = 51) and a Therapeutic Efficacy Study (TES) from Papua (N = 142) from 2020 to 2021, 42-day follow-up. PCR correction using msp1, msp2, and glurp was used to distinguish recrudescence and reinfection. Parasite DNA from DBSs was used for genotyping molecular markers for antimalaria drug resistance, including in Pfk13, pfcrt, and pfmdr1, as well as gene copy number variation in pfpm2/3 and pfmdr1. RESULTS: The study revealed the absence of SNPs associated with ART resistance and several novel SNPs such as L396F, I526V, M579I and N537S (4.25%). In Sumba, the mutant haplotype SDD of pfmdr1 was found in one-third of the isolates, while only 8.9% in Papua. None of the pfcrt mutations linked to piperaquine resistance were observed, but 71% of isolates had pfcrt I356L. Amplification of the pfpm2/3 genes was in Sumba (17.02%) and Papua (13.7%), while pfmdr1 copy number prevalence was low (3.8%) in both areas. For the TES study, ten recurrences of infection were observed on days 28, 35, and 42. Late parasitological failure (LPF) was observed in 10/117 (8.5%) subjects by microscopy. PCR correction revealed that all nine cases were re-infections and one was confirmed as recrudescence. CONCLUSION: This study revealed that DHA-PPQ is still highly effective against P. falciparum. The genetic architecture of the parasite P. falciparum isolates during 2010-2021 revealed single copy of Pfpm2 and pfmdr1 were highly prevalent. The slight increase in DHA-PPQ LTF alerts researchers to start testing other ACTs as alternatives to DHA-PPQ for baseline data in order to get a chance of achieving malaria elimination wants by 2030.

Efficacy and safety of dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax malaria in Papua and Sumatra, Indonesia.

BACKGROUND: Dihydroartemisinin-piperaquine (DHA-PPQ) has been adopted as first-line therapy for uncomplicated falciparum malaria in Indonesia since 2010. The efficacy of DHA-PPQ was evaluated in 2 sentinel sites in Keerom District, Papua and Merangin District, Jambi, Sumatra from April 2017 to April 2018. METHODS: Clinical and parasitological parameters were monitored over a 42-day period following the World Health Organization standard in vivo protocol and subjects meeting the inclusion criteria were treated with DHA-PPQ once daily for 3 days, administered orally. RESULTS: In Papua, 6339 subjects were screened through active and passive cases detection. Of the 114 falciparum and 81 vivax cases enrolled, 102 falciparum and 80 vivax cases completed the 42 day follow up, and 12 falciparum and 1 vivax cases were either lost to follow up or withdrawn. Kaplan-Meier analysis of microscopy readings of 102 falciparum cases revealed 93.1% (95% CI 86.4-97.2) as Adequate Clinical and Parasitological Response (ACPR). No delay in parasite clearance nor severe adverse reaction was observed. Recurrent parasites of Plasmodium falciparum were detected in 7 cases and categorized as late treatment failures (LTF) at days 21, 35, and 42 and one of which was reinfected by Plasmodium vivax at day 42. Two cases were confirmed as recrudescent infection and 4 were re-infection. The PCR-corrected DHA-PPQ efficacy for P. falciparum was 97.9% (95% CI 92.7-99.7). Of the 80 cases of P. vivax that were followed up, 71 cases were completely cured and classified as ACPR (88.8%, 95% CI 79.7-94.7) and 9 cases showed recurrent infection at days 35 and 42, and classified as LTF. In Sumatra, of the 751 subjects screened, 35 vivax subjects enrolled, 34 completed the 42 day follow up. Thirty-three cases were completely cured and classified as ACPR (97.1%, 95% CI 84.7-99.9) and 1 recurrent infection was observed and classified as LTF. No delay in parasite clearance nor severe adverse reaction was observed. Analysis of the Pfk13 gene in P. falciparum cases from Papua revealed no mutations associated with artemisinin resistance in the 20 SNPs previously reported. Analysis of the Pfpm2 gene at day 0 and day of recurrence in recrudescent cases revealed the same single copy number, whereas 3 of the 4 re-infection cases carried 2-3 Pfpm2 gene copy numbers. CONCLUSION: Treatment of falciparum and vivax malaria cases with DHA-PPQ showed a high efficacy and safety.

Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in four malaria endemic states of India.

BACKGROUND: Malaria is a major public health problem in India and accounts for about 88% of malaria burden in South-East Asia. India alone accounted for 2% of total malaria cases globally. Anti-malarial drug resistance is one of the major problems for malaria control and elimination programme. Artemether-lumefantrine (AL) is the first-line treatment of uncomplicated Plasmodium falciparum in north eastern states of India since 2013 after confirming the resistance against sulfadoxine-pyrimethamine. In the present study, therapeutic efficacy of artemether-lumefantrine and k13 polymorphism was assessed in uncomplicated P. falciparum malaria. METHODS: This study was conducted at four community health centres located in Koraput district of Odisha, Bastar district of Chhattisgarh, Balaghat district of Madhya Pradesh and Gondia district of Maharashtra state. Patients with uncomplicated P. falciparum malaria were administered with fixed dose combination (6 doses) of artemether-lumefantrine for 3 days and clinical and parasitological response was recorded up to 28 days as per World Health Organization protocol. Nucleotide sequencing of msp1 and msp2 gene was performed to differentiate between recrudescence and reinfection. Amplification and sequencing of k13 propeller gene region covering codon 450-680 was also carried out to identify the polymorphism. RESULTS: A total 376 malaria patients who fulfilled the enrolment criteria as well as consented for the study were enrolled. Total 356 patients were followed up successfully up to 28 days. Overall, the adequate clinical and parasitological response was 98.9% and 99.4% with and without PCR correction respectively. No case of early treatment failure was observed. However, four cases (1.1%) of late parasitological failure were found from the Bastar district of Chhattisgarh. Genotyping of msp1 and msp2 confirmed 2 cases each of recrudescence and reinfection, respectively. Mutation analysis of k13 propeller gene showed one non-synonymous mutation Q613H in one isolate from Bastar. CONCLUSIONS: The study results showed that artemether-lumefantrine is highly effective in the treatment of uncomplicated P. falciparum malaria among all age groups. No functional mutation in k13 was found in the study area. The data from this study will be helpful in implementation of artemether-lumefantrine in case of treatment failure by artesunate plus sulfadoxine-pyrimethamine.

Clinical and molecular surveillance of artemisinin resistant falciparum malaria in Myanmar (2009-2013).

BACKGROUND: Emergence of artemisinin-resistant malaria in Southeast Asian countries threatens the global control of malaria. Although K13 kelch propeller has been assessed for artemisinin resistance molecular marker, most of the mutations need to be validated. In this study, artemisinin resistance was assessed by clinical and molecular analysis, including k13 and recently reported markers, pfarps10, pffd and pfmdr2. METHODS: A prospective cohort study in 1160 uncomplicated falciparum patients was conducted after treatment with artemisinin-based combination therapy (ACT), in 6 sentinel sites in Myanmar from 2009 to 2013. Therapeutic efficacy of ACT was assessed by longitudinal follow ups. Molecular markers analysis was done on all available day 0 samples. RESULTS: True recrudescence treatment failures cases and day 3 parasite positivity were detected at only the southern Myanmar sites. Day 3 positive and k13 mutants with higher prevalence of underlying genetic foci predisposing to become k13 mutant were detected only in southern Myanmar since 2009 and comparatively fewer mutations of pfarps10, pffd, and pfmdr2 were observed in western Myanmar. K13 mutations, V127M of pfarps10, D193Y of pffd, and T448I of pfmdr2 were significantly associated with day 3 positivity (OR: 6.48, 3.88, 2.88, and 2.52, respectively). CONCLUSIONS: Apart from k13, pfarps10, pffd and pfmdr2 are also useful for molecular surveillance of artemisinin resistance especially where k13 mutation has not been reported. Appropriate action to eliminate the resistant parasites and surveillance on artemisinin resistance should be strengthened in Myanmar. Trial registration This study was registered with ClinicalTrials.gov, identifier NCT02792816.

Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria from three highly malarious states in India.

BACKGROUND: Anti-malarial drug resistance continues to be a leading threat to malaria control efforts and calls for continued monitoring of waning efficacy of artemisinin-based combination therapy (ACT). Artesunate + sulfadoxine/pyrimethamine (AS + SP) is used for the treatment of uncomplicated Plasmodium falciparum malaria in India. However, resistance against AS + SP is emerged in northeastern states. Therefore, artemether-lumefantrine (AL) is the recommended first line treatment for falciparum malaria in north eastern states. This study investigates the therapeutic efficacy and safety of AL for the treatment of uncomplicated falciparum malaria in three malaria-endemic states in India. The data generated through this study will benefit the immediate implementation of second-line ACT as and when required. METHODS: This was a one-arm prospective evaluation of clinical and parasitological responses for uncomplicated falciparum malaria using WHO protocol. Patients diagnosed with uncomplicated mono P. falciparum infection were administered six-dose regimen of AL over 3 days and subsequent follow-up was carried out up to 28 days. Molecular markers msp-1 and msp-2 were used to differentiate recrudescence and re-infection and K13 propeller gene was amplified and sequenced covering the codon 450-680. RESULTS: A total of 402 eligible patients were enrolled in the study from all four sites. Overall, adequate clinical and parasitological response (ACPR) was 98 % without PCR correction and 99 % with PCR correction. At three study sites, ACPR rates were 100 %, while at Bastar, cure rate was 92.5 % on day 28. No early treatment failure was found. The PCR-corrected endpoint finding confirmed that one late clinical failure (LCF) and two late parasitological failures (LPF) were recrudescences. The PCR corrected cure rate was 96.5 %. The mean fever clearance time was 27.2 h ± 8.2 (24-48 h) and the mean parasite clearance time was 30.1 h ± 11.0 (24-72 h). Additionally, no adverse event was recorded. Analysis of total 186 samples revealed a mutation in the k13 gene along with non-synonymous mutation at codon M579T in three (1.6 %) samples. CONCLUSION: AL is an efficacious drug for the treatment of uncomplicated falciparum malaria. However, regular monitoring of AL is required in view of malaria elimination initiatives, which will be largely dependent on therapeutic interventions, regular surveillance and targeted vector control.

Pyronaridine-artesunate granules versus artemether-lumefantrine crushed tablets in children with Plasmodium falciparum malaria: a randomized controlled trial.

BACKGROUND: Children are most vulnerable to malaria. A pyronaridine-artesunate pediatric granule formulation is being developed for the treatment of uncomplicated Plasmodium falciparum malaria. METHODS: This phase III, multi-center, comparative, open-label, parallel-group, controlled clinical trial included patients aged ≤12 years, bodyweight ≥5 to <25 kg, with a reported history of fever at inclusion or in the previous 24 h and microscopically-confirmed uncomplicated P. falciparum malaria. Patients were randomized (2:1) to pyronaridine-artesunate granules (60/20 mg) once daily or artemether-lumefantrine crushed tablets (20/120 mg) twice daily, both dosed by bodyweight, orally (liquid suspension) for three days. RESULTS: Of 535 patients randomized, 355 received pyronaridine-artesunate and 180 received artemether-lumefantrine. Day-28 adequate clinical and parasitological response (ACPR), corrected for re-infection using polymerase chain reaction (PCR) genotyping (per-protocol population) was 97.1% (329/339; 95% CI 94.6, 98.6) for pyronaridine-artesunate; 98.8% (165/167; 95% CI 95.7, 99.9) for artemether-lumefantrine. The primary endpoint was achieved: pyronaridine-artesunate PCR-corrected day-28 ACPR was statistically significantly >90% (P < .0001). Pyronaridine-artesunate was non-inferior to artemether-lumefantrine: treatment difference -1.8% (95% CI -4.3 to 1.6). The incidence of drug-related adverse events was 37.2% (132/355) with pyronaridine-artesunate, 44.4% (80/180) with artemether-lumefantrine. Clinical biochemistry results showed similar mean changes versus baseline in the two treatment groups. From day 3 until study completion, one patient in each treatment group had peak alanine aminotransferase (ALT) >3 times the upper limit of normal (ULN) and peak total bilirubin >2xULN (i.e. within the Hy's law definition). CONCLUSIONS: The pyronaridine-artesunate pediatric granule formulation was efficacious and was non-inferior to artemether-lumefantrine. The adverse event profile was similar for the two comparators. Pyronaridine-artesunate should be considered for inclusion in paediatric malaria treatment programmes. TRIAL REGISTRATION: ClinicalTrials.gov: identifier NCT00541385.