Assessment of Plasmodium falciparum Artemisinin Resistance Independent of kelch13 Polymorphisms and with Escalating Malaria in Bangladesh.

Emerging resistance to artemisinin drugs threatens the elimination of malaria. Resistance is widespread in South East Asia (SEA) and Myanmar. Neighboring Bangladesh, where 90% of infections occur in the Chittagong Hill Tracts (CHTs), lacks recent assessment. We undertook a prospective study in the sole district-level hospital in Bandarban, a CHT district with low population densities but 60% of reported malaria cases. Thirty patients presented with malaria in 2018. An increase to 68 patients in 2019 correlated with the district-level rise in malaria, rainfall, humidity, and temperature. Twenty-four patients (7 in 2018 and 17 in 2019) with uncomplicated Plasmodium falciparum monoinfection were assessed for clearing parasites after starting artemisinin combination therapy (ACT). The median (range) time to clear half of the initial parasites was 5.6 (1.5 to 9.6) h, with 20% of patients showing a median of 8 h. There was no correlation between parasite clearance and initial parasitemia, blood cell counts, or mutations of P. falciparum gene Pfkelch13 (the molecular marker of artemisinin resistance [AR]). The in vitro ring-stage survival assay (RSA) revealed one (of four) culture-adapted strains with a quantifiable resistance of 2.01% ± 0.1% (mean ± standard error of the mean [SEM]). Regression analyses of in vivo and in vitro measurements of the four CHT strains and WHO-validated K13 resistance mutations yielded good correlation (R2 = 0.7; ρ = 0.9, P < 0.005), strengthening evaluation of emerging AR with small sample sizes, a challenge in many low/moderate-prevalence sites. There is an urgent need to deploy multiple, complementary approaches to understand the evolutionary dynamics of the emergence of P. falciparum resistant to artemisinin derivatives in countries where malaria is endemic. IMPORTANCE Malaria elimination is a Millennium Development Goal. Artemisinins, fast-acting antimalarial drugs, have played a key role in malaria elimination. Emergence of artemisinin resistance threatens the global elimination of malaria. Over the last decade, advanced clinical and laboratory methods have documented its spread throughout South East Asia and Myanmar. Neighboring Bangladesh lies in the historical path of dissemination of antimalarial resistance to the rest of the world, yet it has not been evaluated by combinations of leading methods, particularly in the highland Chittagong Hill Tracts adjacent to Myanmar which contain >90% of malaria in Bangladesh. We show the first establishment of capacity to assess clinical artemisinin resistance directly in patients in the hilltops and laboratory adaptation of Bangladeshi parasite strains from a remote, sparsely populated malaria frontier that is responsive to climate. Our study also provides a generalized model for comprehensive monitoring of drug resistance for countries where malaria is endemic.

Persistence of Markers of Chloroquine Resistance in Plasmodium falciparum in Bangladesh.

The control of malaria, in terms of drug resistance, remains a significant global challenge, with Bangladesh, a malaria-endemic country, being no exception. The aim of this study was to explore antimalarial resistance in Bangladesh by molecular analysis of Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance transporter 1 (pfmdr1) genetic markers of P. falciparum. Samples were obtained from uncomplicated malaria patients between 2009 and 2014 from six malaria-endemic districts. Based on parasite transmission intensity, the endemic districts were divided into high-transmission (Chittagong Hill Tracts [CHT]) and low-transmission (non-CHT) regions. Falciparum malaria-positive isolates were genotyped for K76T of the pfcrt gene, and N86Y and Y184F of the pfmdr1 gene: in total, 262 P. falciparum clinical isolates were analyzed. In CHT areas, the prevalence of polymorphisms was 70.6% for 76T, 14.4% for 86Y, and 7.8% for 184F. In non-CHT areas, 76T and 86Y mutations were found in 78.0% and 19.5% of the samples, respectively, whereas no 184F mutations were observed. We compared our data with previous similar molecular observations, which shows a significant decrease in pfcrt 76T mutation prevalence. No pfmdr1 amplification was observed in any of the samples suggesting an unaltered susceptibility to amino alcohol drugs such as mefloquine and lumefantrine. This study provides an updated assessment of the current status of pfcrt and pfmdr1 gene mutations in Bangladesh, and suggests there is persistent high prevalence of markers of resistance to aminoquinoline drugs.

Investigation of antimalarial activity and cytotoxicity profiling of a Bangladeshi plant Syzygium cymosum.

INTRODUCTION: The persistent increase of resistance to existing antimalarials underscores the needs for new drugs. Historically, most of the successful antimalarial are derived from plants. The leaves of the S. cymosum is one of the plant materials used by traditional healers in malaria-endemic areas in Bangladesh for treatment of malaria. Here, we investigated the crude extract and its fractions against chloroquine (CQ)-sensitive 3D7, CQ-resistant Dd2, and artemisinin (ART)-resistant IPC 4912 Mondulkiri strains of Plasmodium falciparum. METHODOLOGY: The antimalarial activities were tested using HRP II based in-vitro antimalarial drug sensitivity ELISA described by WWARN and half inhibitory concentrations (IC50) were calculated by non-linear regression analysis using GraphaPad Prism. The cytotoxicity of the crude methanolic extract was assessed using the MTT assay on Vero cell line. RESULTS: The methanolic crude extract revealed promising activity against 3D7 (IC50 6.28 µg/mL), Dd2 (IC50 13.42 µg/mL), and moderate activity against IPC 4912 Mondulkiri (IC50 17.47 µg/mL). Among the fractionated portions, the chloroform fraction revealed highest activity against IPC 4912 Mondulkiri (IC50 1.65 µg/mL) followed by Dd2 (1.73 µg/mL) and 3D7 (2.39 µg/mL). The crude methanolic extract also demonstrated good selectivity with the selectivity indices of > 15.92, > 7.45, and > 6.91 against 3D7, Dd2, and IPC 4912, respectively when tested against Vero cell line. CONCLUSIONS: This is the first report on S. cymosum for its putative antimalarial activity, and is imperative to go for further phytochemical analyses in order to investigate possible novel antimalarial drug compound(s).

Molecular analysis demonstrates high prevalence of chloroquine resistance but no evidence of artemisinin resistance in Plasmodium falciparum in the Chittagong Hill Tracts of Bangladesh.

BACKGROUND: Artemisinin resistance is present in the Greater Mekong region and poses a significant threat for current anti-malarial treatment guidelines in Bangladesh. The aim of this molecular study was to assess the current status of drug resistance in the Chittagong Hill Tracts of Bangladesh near the Myanmar border. METHODS: Samples were obtained from patients enrolled into a Clinical Trial (NCT02389374) conducted in Alikadam, Bandarban between August 2014 and January 2015. Plasmodium falciparum infections were confirmed by PCR and all P. falciparum positive isolates genotyped for the pfcrt K76T and pfmdr1 N86Y markers. The propeller region of the kelch 13 (k13) gene was sequenced from isolates from patients with delayed parasite clearance. RESULTS: In total, 130 P. falciparum isolates were available for analysis. The pfcrt mutation K76T, associated with chloroquine resistance was found in 81.5% (106/130) of cases and the pfmdr1 mutation N86Y in 13.9% (18/130) cases. No single nucleotide polymorphisms were observed in the k13 propeller region. CONCLUSION: This study provides molecular evidence for the ongoing presence of chloroquine resistant P. falciparum in Bangladesh, but no evidence of mutations in the k13 propeller domain associated with artemisinin resistance. Monitoring for artemisinin susceptibility in Bangladesh is needed to ensure early detection and containment emerging anti-malarial resistance.

Case Report: A Case of Plasmodium falciparum hrp2 and hrp3 Gene Mutation in Bangladesh.

Several species of Plasmodium are responsible for causing malaria in humans. Proper diagnoses are crucial to case management, because severity and treatment varies between species. Diagnoses can be made using rapid diagnostic tests (RDTs), which detect Plasmodium proteins. Plasmodium falciparum causes the most virulent cases of malaria, and P. falciparum histidine-rich protein 2 (PfHRP2) is a common target of falciparum malaria RDTs. Here we report a case in which a falciparum malaria patient in Bangladesh tested negative on PfHRP2-based RDTs. The negative results can be attributed to a deletion of part of the pfhrp2 gene and frameshift mutations in both pfhrp2 and pfhrp3 gene. This finding may have implications for malaria diagnostics and case management in Bangladesh and other regions of South Asia.