Composition of Anopheles species and bionomic characteristics over the peak malaria transmission season in Bandarban, Bangladesh.

BACKGROUND: Joint efforts by government and non-government organizations have helped to reduce malaria in Bangladesh and set the country on a clear path to eventual malaria elimination. However, achieving that goal would be challenging without a comprehensive understanding of vector bionomics. METHODS: Targeted capturing of Anopheles mosquitoes over a rainy season, utilizing specific sampling methods, including human landing catches (HLCs), CDC-light traps (CDC-LTs), and pyrethrum spray catches (PSCs) were aimed to characterize entomological drivers of transmission in four sites of Bandarban, Bangladesh. RESULTS: Molecular characterization of a subset of 4637 mosquitoes has demonstrated the presence of at least 17 species whose capture rates were representative of the rainy season. Species compositions and bionomic traits did not vary between sites with Anopheles maculatus having the highest landing rate by HLCs and Anopheles vagus having the highest capture rate with CDC-LTs. Interestingly, Anopheles species compositions and capture rates varied significantly (p < 0.05) for An. vagus, between HLCs and its often-used proxy-CDC-LTs- suggesting impacts on downstream analysis. CDC-LTs capture rates demonstrated differing compositions with indoor and outdoor biting rates. For example, Anopheles nigerrimus and Anopheles nivipes were more endophagic by HLCs and more exophagic by CDC-LTs. The use of a cow-baited CDC-LT also demonstrated significantly different results when compared to a human-baited CDC-LT considering the high degree of anthropophily in these species. The exception to both zoophily and indoor resting was An. vagus, which demonstrated both anthropophily and high resting rates indoors-pointing to this species being a possible primary vector at this site. CONCLUSION: A diverse Anopheles fauna in Bandarban has been confirmed through molecular methods, highlighting the potential impact of sampling techniques. Given the complexity of the local ecosystem, a better understanding of mosquito behaviour and ecology is required to achieve the goal of malaria elimination in Bangladesh.

Heterogeneity in prevalence of subclinical Plasmodium falciparum and Plasmodium vivax infections but no parasite genomic clustering in the Chittagong Hill Tracts, Bangladesh.

BACKGROUND: Malaria remains endemic in Bangladesh, with the majority of cases occurring in forested, mountainous region in the Chittagong Hill Tracts (CHT). This area is home to Bengali and diverse groups of indigenous people (Pahari) residing largely in mono-ethnic villages. METHODS: 1002 individuals of the 9 most prominent Pahari and the Bengali population were randomly selected and screened by RDT and qPCR. Parasites were genotyped by msp2 and deep sequencing of 5 amplicons (ama1-D3, cpmp, cpp, csp, and msp7) for Plasmodium falciparum (n = 20), and by microsatellite (MS) typing of ten loci and amplicon sequencing of msp1 for Plasmodium vivax (n = 21). Population structure was analysed using STRUCTURE software. Identity-by-state (IBS) was calculated as a measure of parasite relatedness and used to generate relatedness networks. RESULTS: The prevalence of P. falciparum and P. vivax infection was 0.7% by RDT (P. falciparum 6/1002; P. vivax 0/1002, mixed: 1/1002) and 4% by qPCR (P. falciparum 21/1002; P. vivax 16/1002, mixed: 5/1002). Infections were highly clustered, with 64% (27/42) of infections occurring in only two Pahari groups, the Khumi and Mro. Diversity was high; expected heterozygosity was 0.93 for P. falciparum and 0.81 for P. vivax. 85.7% (18/21) of P. vivax and 25% (5/20) of P. falciparum infections were polyclonal. No population structure was evident for either species, suggesting high transmission and gene flow among Pahari groups. CONCLUSIONS: High subclinical infection prevalence and genetic diversity mirror ongoing transmission. Control activities should be specifically directed to Pahari groups at greatest risk.

Human behaviour directs household-level exposure to malaria vectors in Bandarban, Bangladesh.

BACKGROUND: Bangladesh has reduced malaria incidence and mortality by over 75% between 2010 and 2020. Widespread long-lasting insecticidal net (LLIN) distribution and use is one of the measures responsible for this success. Recalcitrant malaria hotspots within the Chittagong Hill Tracts districts suggest important drivers of malaria risk may remain uncharacterized. METHODS: Towards understanding how household-level human behaviour impacts exposure to mosquitoes, parallel human landing catches and human behavioural observations were conducted in four households for 6 months (May-October) over the rainy season in the Bandarban District. Analysis quantifies spatiotemporal human behaviour-adjusted exposure to Anopheles with and without LLINs. RESULTS: This small-scale operational study demonstrates that human spatial and temporal presence along with LLIN use drives exposure to Anopheles. Though the four households had both outdoor and indoor exposure, especially in the evening (1800-2000 h) and early morning (0400-0500 h), data points to household-based heterogeneity in malaria exposure even with similar LLIN access. CONCLUSION: Incorporating human behaviour into exposure estimates can be used to understand the efficacy and limitations of local vector control strategies and identify gaps in protection, as well as where present intervention strategies may be optimized.

Glucose-6-phosphate dehydrogenase activity in individuals with and without malaria: Analysis of clinical trial, cross-sectional and case-control data from Bangladesh.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) activity is dependent upon G6PD genotype and age of the red blood cell (RBC) population, with younger RBCs having higher activity. Peripheral parasitemia with Plasmodium spp. induces hemolysis, replacing older RBCs with younger cells with higher G6PD activity. This study aimed to assess whether G6PD activity varies between individuals with and without malaria or a history of malaria. METHODS AND FINDINGS: Individuals living in the Chittagong Hill Tracts of Bangladesh were enrolled into 3 complementary studies: (i) a prospective, single-arm clinical efficacy trial of patients (n = 175) with uncomplicated malaria done between 2014 and 2015, (ii) a cross-sectional survey done between 2015 and 2016 (n = 999), and (iii) a matched case-control study of aparasitemic individuals with and without a history of malaria done in 2020 (n = 506). G6PD activity was compared between individuals with and without malaria diagnosed by microscopy, rapid diagnostic test (RDT), or polymerase chain reaction (PCR), and in aparasitemic participants with and without a history of malaria. In the cross-sectional survey and clinical trial, 15.5% (182/1,174) of participants had peripheral parasitemia detected by microscopy or RDT, 3.1% (36/1,174) were positive by PCR only, and 81.4% (956/1,174) were aparasitemic. Aparasitemic individuals had significantly lower G6PD activity (median 6.9 U/g Hb, IQR 5.2-8.6) than those with peripheral parasitemia detected by microscopy or RDT (7.9 U/g Hb, IQR 6.6-9.8, p < 0.001), but G6PD activity similar to those with parasitemia detected by PCR alone (submicroscopic parasitemia) (6.1 U/g Hb, IQR 4.8-8.6, p = 0.312). In total, 7.7% (14/182) of patients with malaria had G6PD activity < 70% compared to 25.0% (248/992) of participants with submicroscopic or no parasitemia (odds ratio [OR] 0.25, 95% CI 0.14-0.44, p < 0.001). In the case-control study, the median G6PD activity was 10.3 U/g Hb (IQR 8.8-12.2) in 253 patients with a history of malaria and 10.2 U/g Hb (IQR 8.7-11.8) in 253 individuals without a history of malaria (p = 0.323). The proportion of individuals with G6PD activity < 70% was 11.5% (29/253) in the cases and 15.4% (39/253) in the controls (OR 0.7, 95% CI 0.41-1.23, p = 0.192). Limitations of the study included the non-contemporaneous nature of the clinical trial and cross-sectional survey. CONCLUSIONS: Patients with acute malaria had significantly higher G6PD activity than individuals without malaria, and this could not be accounted for by a protective effect of G6PD deficiency. G6PD-deficient patients with malaria may have higher than expected G6PD enzyme activity and an attenuated risk of primaquine-induced hemolysis compared to the risk when not infected.

Barriers to routine G6PD testing prior to treatment with primaquine.

BACKGROUND: Primaquine is essential for the radical cure of vivax malaria, however its broad application is hindered by the risk of drug-induced haemolysis in individuals with glucose-6-phosphate-dehydrogenase (G6PD) deficiency. Rapid diagnostic tests capable of diagnosing G6PD deficiency are now available, but these are not used widely. METHODS: A series of qualitative interviews were conducted with policy makers and healthcare providers in four vivax-endemic countries. Routine G6PD testing is not part of current policy in Bangladesh, Cambodia or China, but it is in Malaysia. The interviews were analysed with regard to respondents perceptions of vivax malaria, -primaquine based treatment for malaria and the complexities of G6PD deficiency. RESULTS: Three barriers to the roll-out of routine G6PD testing were identified in all sites: (a) a perceived low risk of drug-induced haemolysis; (b) the perception that vivax malaria was benign and accordingly treatment with primaquine was not regarded as a priority; and, (c) the additional costs of introducing routine testing. In Malaysia, respondents considered the current test and treat algorithm suitable and the need for an alternative approach was only considered relevant in highly mobile and hard to reach populations. CONCLUSIONS: Greater efforts are needed to increase awareness of the benefits of the radical cure of Plasmodium vivax and this should be supported by economic analyses exploring the cost effectiveness of routine G6PD testing.

Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria.

Primaquine and tafenoquine are the only licensed drugs with activity against Plasmodium vivax hypnozoites but cause haemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Malaria also causes haemolysis, leading to the replacement of older erythrocytes with low G6PD activity by reticulocytes and young erythrocytes with higher activity. Aim of this study was to assess the impact of acute malaria on G6PD activity. Selected patients with uncomplicated malaria were recruited in Bangladesh (n = 87), Indonesia (n = 75), and Ethiopia (n = 173); G6PD activity was measured at the initial presentation with malaria and a median of 176 days later (range 140 to 998) in the absence of malaria. Among selected participants (deficient participants preferentially enrolled in Bangladesh but not at other sites) G6PD activity fell between malaria and follow up by 79.1% (95%CI: 40.4 to 117.8) in 6 participants classified as deficient (<30% activity), 43.7% (95%CI: 34.2 to 53.1) in 39 individuals with intermediate activity (30% to <70%), and by 4.5% (95%CI: 1.4 to 7.6) in 290 G6PD normal (≥70%) participants. In Bangladesh and Indonesia G6PD activity was significantly higher during acute malaria than when the same individuals were retested during follow up (40.9% (95%CI: 33.4-48.1) and 7.4% (95%CI: 0.2 to 14.6) respectively), whereas in Ethiopia G6PD activity was 3.6% (95%CI: -1.0 to -6.1) lower during acute malaria. The change in G6PD activity was apparent in patients presenting with either P. vivax or P. falciparum infection. Overall, 66.7% (4/6) severely deficient participants and 87.2% (34/39) with intermediate deficiency had normal activities when presenting with malaria. These findings suggest that G6PD activity rises significantly and at clinically relevant levels during acute malaria. Prospective case-control studies are warranted to confirm the degree to which the predicted population attributable risks of drug induced haemolysis is lower than would be predicted from cross sectional surveys.

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.

A five-day course of ivermectin for the treatment of COVID-19 may reduce the duration of illness.

Ivermectin, a US Food and Drug Administration-approved anti-parasitic agent, was found to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication in vitro. A randomized, double-blind, placebo-controlled trial was conducted to determine the rapidity of viral clearance and safety of ivermectin among adult SARS-CoV-2 patients. The trial included 72 hospitalized patients in Dhaka, Bangladesh, who were assigned to one of three groups: oral ivermectin alone (12 mg once daily for 5 days), oral ivermectin in combination with doxycycline (12 mg ivermectin single dose and 200 mg doxycycline on day 1, followed by 100 mg every 12 h for the next 4 days), and a placebo control group. Clinical symptoms of fever, cough, and sore throat were comparable among the three groups. Virological clearance was earlier in the 5-day ivermectin treatment arm when compared to the placebo group (9.7 days vs 12.7 days; p = 0.02), but this was not the case for the ivermectin + doxycycline arm (11.5 days; p = 0.27). There were no severe adverse drug events recorded in the study. A 5-day course of ivermectin was found to be safe and effective in treating adult patients with mild COVID-19. Larger trials will be needed to confirm these preliminary findings.

Precarity at the Margins of Malaria Control in the Chittagong Hill Tracts in Bangladesh: A Mixed-Methods Study.

Bangladesh has achieved significant progress towards malaria elimination, although health service delivery for malaria remains challenging in remote forested areas such as the Chittagong Hill Tracts (CHT). The aim of this study was to investigate perceptions of malaria and its treatment among the local population to inform contextualized strategies for rolling out radical cure for P. vivax in Bangladesh. The study comprised two sequential strands whereby the preliminary results of a qualitative strand informed the development of a structured survey questionnaire used in the quantitative strand. Results show that ethnic minority populations in the CHT live in precarious socio-economic conditions which increase their exposure to infectious diseases, and that febrile patients often self-treat, including home remedies and pharmaceuticals, before attending a healthcare facility. Perceived low quality of care and lack of communication between Bengali health providers and ethnic minority patients also affects access to public healthcare. Malaria is viewed as a condition that affects vulnerable people weakened by agricultural work and taking away blood is perceived to increase such vulnerability. Healthcare providers that initiate and sustain a dialogue about these issues with ethnic minority patients may foster the trust that is needed for local malaria elimination efforts.

Case Report: A Case of Primaquine-Induced Hemoglobinuria in Glucose-6-Phosphate Dehydrogenase Deficient Malaria Patient in Southeastern Bangladesh.

We herein report the first case of Mediterranean glucose-6-phosphate dehydrogenase (G6PD) variant from Bangladesh. A boy had been admitted to hospital and was diagnosed with uncomplicated Plasmodium vivax infection and treated with 30 mg/kg body weight (BW) chloroquine for 3 days and 4.8 mg/kg BW primaquine (PQ) to be taken over 14 days. The boy was discharged but represented 4 days later with severe hemoglobinuria and fatigue. Hemoglobin was measured at 6.0 g/dL and serum bilirubin was at 5.6 mg/dL, although malaria microscopy was negative. The boy had taken the 4-fold recommended daily dose of PQ and was treated with two fresh blood transfusions. Subsequent molecular analysis showed the boy to have the Mediterranean G6PD variant and a G6PD activity of 0.93 U/gHb.

Wide range of G6PD activities found among ethnic groups of the Chittagong Hill Tracts, Bangladesh.

The proportion of Plasmodium vivax malaria among all malarias is increasing worldwide. Treatment with 8-aminoquinolines remain the only radical cure. However, 8-aminoquinolines can cause severe hemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient patients. The population of the multi-ethnic Chittagong Hill Tracts (CHT) carry the highest malaria burden within Bangladesh. As in many countries the national treatment guidelines recommend 8-aminoquinoline based radical cure without routine G6PD deficiency (G6PDd) testing to guide treatment. Aim of this study was to determine the need for routine testing within a multi-ethnic population by assessing the prevalence of G6PDd among the local population. Participants from 11 ethnicities were randomly selected and malaria status was assessed by microscopy, rapid diagnostic test (RDT) and polymerase chain reaction (PCR). G6PD status was determined by spectrophotometry and G6PD genotyping. The adjusted male median (AMM) was defined as 100% G6PD activity, participants were categorized as G6PD deficient (<30% activity), G6PD intermediate (30% to 70% activity) or G6PD normal (>70% activity). Median G6PD activities between ethnicities were compared and the association between G6PD activity and malaria status was assessed. 1002 participants were enrolled and tested for malaria. G6PD activity was measured by spectrophotometry in 999 participants and host G6PD genotyping undertaken in 323 participants. Seven participants (0.7%) had peripheral parasitaemia detected by microscopy or RDT and 42 by PCR (4.2%). Among 106 participants (32.8%) with confirmed genotype, 99 (93.4%) had the Mahidol variant. The AMM was 7.03U/gHb with 90 (9.0%) G6PD deficient participants and 133 (13.3%) with intermediate G6PD activity. Median G6PD activity differed significantly between ethnicities (p<0.001), proportions of G6PD deficient individuals ranged from 2% to 26% but did not differ between participants with and without malaria. The high G6PDd prevalence and significant variation between ethnicities suggest routine G6PDd testing to guide 8-aminoquinoline based radical in the CHT and comparable settings.

Field evaluation of quantitative point of care diagnostics to measure glucose-6-phosphate dehydrogenase activity.

BACKGROUND: Glucose-6-Phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy worldwide, no reliable bedside diagnostic tests to quantify G6PD activity exist. This study evaluated two novel quantitative G6PD diagnostics. METHODS: Participants with known G6PD activity were enrolled in Bangladesh. G6PD activity was measured by spectrophotometry, Biosensor (BS; AccessBio/CareStart, USA) and STANDARD G6PD (SG; SDBiosensor, ROK). G6PD activity was measured repeatedly in a subset of samples stored at room temperature and 4°C. RESULTS: 158 participants were enrolled, 152 samples tested by BS, 108 samples by SG and 102 samples were tested by all three methods. In comparison to spectrophotometry BS had sensitivity and specificity of 72% (95%CI: 53-86) and 100% (95%CI: 97-100) at 30% cut off respectively, while SG had a sensitivity of 100% (95%CI: 88-100) and specificity of 97% (95%CI: 91-99) at the same cut off. The sensitivity and specificity at 70% cut off activity were 71% (95%CI: 59-82) and 98% (95%CI, 92-100) respectively for BS and 89% (95%CI: 77-96) and 93% (95%CI: 83-98) respectively for SG. When an optimal cut-off was applied the sensitivity of the BS at 70 cut off rose to 91% [95%CI: 80-96] and specificity to 82% [95%CI: 83-89]; a diagnostic accuracy comparable to that of the SG (p = 0.879). G6PD activity dropped significantly (-0.31U/gHb, 95%CI: -0.61 to -0.01, p = 0.022) within 24 hours in samples stored at room temperature, but did not fall below 90% of baseline activity until day 13 (-0.87U/gHb, 95%CI: (-1.11 to -0.62), p<0.001). CONCLUSION: BS and SG are the first quantitative diagnostics to measure G6PD activity reliably at the bedside and represent suitable alternatives to spectrophotometry in resource poor settings. If samples are stored at 4°C, G6PD activity can be measured reliably for at least 7 days after sample collection.