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.

Trends and clinico-epidemiological features of human rabies cases in Bangladesh 2006-2018.

Vaccinating dogs against rabies is an effective means of reducing human rabies. We subjected 1327 clinically diagnosed human rabies death and mass dog vaccination (MDV) data during 2006-2018 to quantify the impacts of MDV on human rabies incidence in Bangladesh and a subset of rabies death data (422) for clinico-epidemiological analysis. A positive and increasing trend of MDV (p = 0.01 and tau = 0.71) and a negative and declining trend (p < 0.001 and tau = -0.88) of human rabies cases (Correlation coefficient: -0.82) have been observed. Among 422 deaths, the majority (78%) of the victims sought treatment from traditional healers, and 12% received post-exposure prophylaxis (PEP). The mean incubation period of rabies in cases with exposure sites on the head & neck (35 days) was shorter than the upper limb (mean = 64 days, p = 0.02) and lower limb (mean = 89 days, p < 0.01). MDV has been found to be effective for reducing human rabies cases in Bangladesh. Creating awareness among the animal bite victims to stop reliance on traditional healers rather seeking PEP, addressing the role of traditional healers through awareness education programme with respect to the treatment of dog bites, ensuring availability of PEP, and continuing to scale up MDV may help to prevent human rabies deaths.

Ordered accumulation of mutations conferring resistance to sulfadoxine-pyrimethamine in the Plasmodium falciparum parasite.

BACKGROUND: Monitoring the prevalence of drug resistant Plasmodium falciparum is essential for effective malaria control. Resistance to pyrimethamine and sulfadoxine increases as mutations accumulate in the parasite genes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps), respectively. Although parasites are exposed to these antifolate drugs simultaneously, it remains virtually unknown whether dhfr and dhps mutations accumulate along interrelated paths. METHODS: We investigated the order of step-wise accumulation in dhfr and dhps by cumulative analyses using binomial tests in 575 P. falciparum isolates obtained from 7 countries in Asia and Melanesia. RESULTS: An initial step in the accumulation of mutations preferentially occurred in dhfr (2 mutations), followed by 1 mutation in dhps. In a subsequent step, mutations were estimated separately for 5 dhfr/dhps-resistant lineages identified using 12 microsatellites flanking dhfr and dhps. Among these lineages, we found 3 major mutational paths, each of which follows a unique stepwise trajectory to produce the most highly resistant form with 4 mutations in dhfr and 3 in dhps. CONCLUSIONS: The ordered accumulation of mutations in dhfr and dhps elucidated here will assist in predicting the status and progression of antifolate resistance in malaria-endemic regions where antifolate drugs are used for intermittent preventive treatment.

A survey of the dog population in rural Bangladesh.

Globally, Bangladesh ranks third in the number of human deaths from rabies. Although dogs are the principal known transmitters of rabies and knowledge of dog populations is essential for effective national control and proper planning, dog control programs are scarce in Bangladesh. Our objective was to count dogs in a rural area to understand the dog population of the country. For this purpose we selected six unions of Raipura upazila in Narsingdi district. Dog counting was done by direct observation following accepted guidelines. We determined the mean density of the dog population in Bangladesh to be 14 dog/km(2) (95% CI 3.7, 24) and the human:dog ratio to be 120 (95% CI 55, 184). Our paper contribute to the literature which shows great variation in the human:dog ratio across regions of the developing world. The human:dog ratio depends on the area's human (as well as dog) population, whereas dog density per unit area indicates the true number of dogs. We propose that extrapolating from the human:dog ratios of other regions not be relied upon for estimating dog populations, unless the ratios can be supplemented by actual counts of dogs within the target area.

Spontaneous mutations in the Plasmodium falciparum sarcoplasmic/ endoplasmic reticulum Ca2+-ATPase (PfATP6) gene among geographically widespread parasite populations unexposed to artemisinin-based combination therapies.

Recent reports on the decline of the efficacy of artemisinin-based combination therapies (ACTs) indicate a serious threat to malaria control. The endoplasmic/sarcoplasmic reticulum Ca(2+)-ATPase ortholog of Plasmodium falciparum (PfSERCA) has been suggested to be the target of artemisinin and its derivatives. It is assumed that continuous artemisinin pressure will affect polymorphism of the PfSERCA gene (serca) if the protein is the target. Here, we investigated the polymorphism of serca in parasite populations unexposed to ACTs to obtain baseline information for the study of potential artemisinin-driven selection of resistant parasites. Analysis of 656 full-length sequences from 13 parasite populations in Africa, Asia, Oceania, and South America revealed 64 single nucleotide polymorphisms (SNPs), of which 43 were newly identified and 38 resulted in amino acid substitutions. No isolates showed L263E and S769N substitutions, which were reportedly associated with artemisinin resistance. Among the four continents, the number of SNPs was highest in Africa. In Africa, Asia, and Oceania, common SNPs, or those with a minor allele frequency of ≥0.05, were less prevalent, with most SNPs noted to be continent specific, whereas in South America, common SNPs were highly prevalent and often shared with those in Africa. Of 50 amino acid haplotypes observed, only one haplotype (3D7 sequence) was seen in all four continents (64%). Forty-eight haplotypes had frequencies of less than 5%, and 40 haplotypes were continent specific. The geographical difference in the diversity and distribution of serca SNPs and haplotypes lays the groundwork for assessing whether some artemisinin resistance-associated mutations and haplotypes are selected by ACTs.

High prevalence of sulfadoxine/pyrimethamine resistance alleles in Plasmodium falciparum parasites from Bangladesh.

In Bangladesh, despite the official introduction of artemisinin combination therapy in 2004, chloroquine+sulfadoxine/pyrimethamine has been used for the treatment of uncomplicated malaria. To assess the distribution of pfcrt, pfmdr1, dhfr, and dhps genotypes in Plasmodium falciparum, we conducted hospital- and community-based surveys in Bandarban, Bangladesh (near the border with Myanmar) in 2007 and 2008. Using nested PCR followed by digestion, 139 P. falciparum isolates were genotyped. We found fixation of a mutation at position 76 in pfcrt and low prevalence of a mutation at position 86 in pfmdr1. In dhfr, the highest pyrimethamine resistant genotype quadruple mutant was found in 19% of isolates, which is significantly higher prevalence than reported in a previous study in Khagrachari (1%) in 2002. Microsatellite haplotypes flanking dhfr of the quadruple mutants in Bangladesh were identical or very similar to those found in Thailand and Cambodia, indicating a common origin for the mutant in these countries. These observations suggest that the higher prevalence of the dhfr quadruple mutant in Bandarban is because of parasite migration from Myanmar. However, continuous use of sulfadoxine/pyrimethamine would have also played a role through selection for the dhfr quadruple mutant. These results indicate an urgent need to collect molecular epidemiological information regarding dhfr and dhps genes, and a review of current sulfadoxine/pyrimethamine usage with the aim of avoiding the widespread distribution of high levels of resistant parasites in Bangladesh.

[In vitro interaction studies of azithromycin and dihydroartemisinin in Plasmodium falciparum isolates from Bangladesh]. / In vitro Interaktionsstudien mit Azithromycin und Dihydroartemisinin in Plasmodium falciparum Isolaten aus Bangladesh.

In recent clinical trials acithromycin in combination with artemisinin derivatives proved to be a promising combination therapy with indifferent to synergistic interaction. The aim of the present study was the assessment of optimal combination ratios for dihydroartemisinin and azithromycin for the treatment of uncomplicated falciparum malaria. The study was conducted in Bandarban, in Southeastern Bangladesh. Plasmodium falciparum isolates collected as part of a clinical trial were cultured for 72 hours. Samples were analyzed using the HRP2 drug sensitivity assay in fixed combinations and checkerboard assays. An indifferent mode of interaction was found for the 1:500 combination of dihydroartemisinine and azithromycin. The sum fractional inhibitory concentrations (SFICs) at IC95 ranged from 0.89 to 1.16 for combination ratios of 1:500 and 1:5000, respectively. A trend towards lower SFICs was observed with rising inhibitory concentrations (i.e. at IC90 and IC95). Correlation analysis suggests a different mode of action for azithromycin as compared to traditional antimalarials.

In vitro interaction of dihydroartemisin and lumefantrine in clinical field isolates from Bangladesh.

The combination of artemether and lumefantrine was introduced in 2005 as the official first line therapy for uncomplicated falciparum malaria in Bangladesh. Fresh P. falciparum samples from patients with acute uncomplicated falciparum malaria who presented to the field site at the Bandarban Sadar Hospital in Bangladesh were tested in checkerboard in vitro drug sensitivity assays to assess the interaction between dihydroartemisinin (DHA) and lumefantrine (LUM). Clearly synergistic interactions with an overall mean FIC(50) of 0.52 and individual mean FICs between 0.26 and 0.85 were found. Lowest FICs were 0.41, 0.18, 0.22, 0.15 and 0.11 at different combination ratios. The optimal combination ratio of the drug combination indicated by the lowest mean FIC average was found to be 1:150 DHA:LUM. Although activity correlations between DHA and lumefantrine were significant, indicating possible cross sensitivity patterns, our data confirm that artemether-lumefantrine is a highly synergistic drug combination.