A systematic review: is Anopheles vagus a species complex?

BACKGROUND: Anopheles vagus (subgenus Cellia) has been identified as a vector for malaria, filariasis, and Japanese encephalitis in Asia. Sporozoites of Plasmodium falciparum and Plasmodium vivax have been found in this zoophilic mosquito in Asia and Indonesia. This study systematically reviews publications regarding An. vagus species, variation, bio-ecology, and malaria transmission in various localities in Asia, especially Indonesia, to determine whether the current data support An. vagus as a species complex. METHODS: The databases Pubmed, Scopus, Europe PMC, and Proquest were searched to identify information regarding the morphology, karyotypes, polytene chromosome, cross-mating, ecology, and molecular identification of An. vagus was then evaluated to determine whether there were possible species complexes. RESULTS: Of the 1326 articles identified, 15 studies were considered for synthesis. The Anopheles spp. samples for this study came from Asia. Eleven studies used morphology to identify An. vagus, with singular studies using each of karyotype identification, chromosomal polytene identification, and cross-breeding experiments. Ten studies used molecular techniques to identify Anopheles spp., including An. vagus. Most studies discovered morphological variations of An. vagus either in the same or different areas and ecological settings. In this review, the members of An. vagus sensu lato grouped based on morphology (An. vagus, An. vagus vagus, An. vagus limosus, and An. limosus), karyotyping (form A and B), and molecular (An. vagus genotype A and B, An. vagus AN4 and AN5). Genetic analysis revealed a high conservation of the ITS2 fragment among members except for the An. vagus genotype B, which was, in fact, Anopheles sundaicus. This review also identified that An. vagus limosus and An. vagus vagus were nearly identical to the ITS2 sequence. CONCLUSION: Literature review studies revealed that An. vagus is conspecific despite the distinct morphological characteristic of An. vagus and An. limosus. Further information using another barcoding tool, such as mitochondrial COI and ND6 and experimental cross-mating between the An. vagus and An. limosus may provide additional evidence for the status of An. vagus as a species complex.

Impact of a spatial repellent intervention on Anopheles kdr insecticide resistance allele in Sumba, Indonesia.

BACKGROUND: The emergence of insecticide resistance and outdoor transmission in malaria-endemic areas underlines the urgent need to develop innovative tools, such as spatial repellents (SR), that may circumvent this residual transmission. With limited options for effective insecticides, regular resistance monitoring is warranted for selecting and using appropriate tools. This study evaluates the pyrethroid knockdown resistance (kdr) allele before and after implementing a transfluthrin-based spatial repellent (SR) intervention in placebo-treated clusters. METHODS: This study looks at the frequency distribution of the kdr allele in Sumba Island from June 2015 to August 2018. Insecticide susceptibility tests were carried out on female Anopheles sp. aged 3-5 days against permethrin 21.5 µg/ml, deltamethrin 12.5 µg/ml, and transfluthrin 10 µg/ml using CDC bottle assay. PCR sequencing of representative samples from adult mosquito collections and insecticide tests revealed the presence of kdr mutations (L1014F and L1014S) in the VGSC gene. RESULTS: A total of 12 Anopheles species, Anopheles tesselatus, Anopheles. aconitus, Anopheles barbirostris, Anopheles kochi, Anopheles annularis, Anopheles maculatus, Anopheles sundaicus, Anopheles flavirostris, Anopheles balabacensis, Anopheles indefinitus, Anopheles subpictus, and Anopheles vagus were analysed. Anopheles vagus and An. sundaicus predominated in the larval populations. Susceptibility assays for all insecticides identified fully susceptible phenotypes in all species examined. Anopheles increasing frequency of kdr mutant alleles during the 3 year SR deployment was observed in both SR-treated and placebo areas, a statistically significant increase occurred in each arm. However, it is unclear how significant SR is in causing the increase in mutant alleles. The L1014S, knockdown resistance east type (kdr-e) allele was detected for the first time among the mosquito samples in this study. The L1014F, knockdown resistance west type (kdr-w) allele and heteroduplex form (wild-type-mutant) were found in almost all Anopheles species examined, including An. vagus, An. aconitus, An. subpictus, An. tesselatus, An. annularis, An. flavirostris and An. sundaicus. CONCLUSION: The presence of fully susceptible phenotypes over time, along with an increase in the frequency distribution of the L1014F/S mutations post-intervention, suggest drivers of resistance external to the study, including pyrethroid use in agriculture and long-lasting insecticidal nets (LLINs). However, this does not negate possible SR impacts that support resistance. More studies that enable the comprehension of possible SR-based drivers of resistance in mosquitoes need to be conducted.

Comparison between Neutralization Capacity of Antibodies Elicited by COVID-19 Natural Infection and Vaccination in Indonesia: A Prospective Cohort.

BACKGROUND: To fight the COVID-19 pandemic, immunity against SARS-CoV-2 should be achieved not only through natural infection but also by vaccination. The effect of COVID-19 vaccination on previously infected persons is debatable. METHODS: A prospective cohort was undergone to collect sera from unvaccinated survivors and vaccinated persons-with and without COVID-19 pre-infection. The sera were analyzed for the anti-receptor binding domain (RBD) titers by ELISA and for the capacity to neutralize the pseudovirus of the Wuhan-Hu-1 strain by luciferase assays. RESULTS: Neither the antibody titers nor the neutralization capacity was significantly different between the three groups. However, the correlation between the antibody titers and the percentage of viral neutralization derived from sera of unvaccinated survivors was higher than that from vaccinated persons with pre-infection and vaccinated naïve individuals (Spearman correlation coefficient (r) = -0.8558; 95% CI, -0.9259 to -0.7288), p < 0.0001 vs. -0.7855; 95% CI, -0.8877 to -0.6096, p < 0.0001 and -0.581; 95% CI, -0.7679 to -0.3028, p = 0.0002, respectively), indicating the capacity to neutralize the virus is most superior by infection alone. CONCLUSIONS: Vaccines induce anti-RBD titers as high as the natural infection with lower neutralization capacity, and it does not boost immunity in pre-infected persons.

Case Series of Primaquine-Induced Haemolytic Events in Controlled Trials with G6PD Screening.

Primaquine for radical cure of Plasmodium vivax malaria poses a potentially life-threatening risk of haemolysis in G6PD-deficient patients. Herein, we review five events of acute haemolytic anaemia following the administration of primaquine in four malaria trials from Indonesia, the Solomon Islands, and Vietnam. Five males aged 9 to 48 years were improperly classified as G6PD-normal by various screening procedures and included as subjects in trials of anti-relapse therapy with daily primaquine. Routine safety monitoring by physical examination, urine inspection, and blood haemoglobin (Hb) assessment were performed in all those trials. Early signs of acute haemolysis, i.e., dark urine and haemoglobin drop >20%, occurred only after day 3 and as late as day 8 of primaquine dosing. All patients were hospitalized and fully recovered, all but one following blood transfusion rescue. Hb nadir was 4.7 to 7.9 g/dL. Hospitalization was for 1 to 7 days. Hb levels returned to baseline values 3 to 10 days after transfusion. Failed G6PD screening procedures in these trials led G6PD-deficient patients to suffer harmful exposures to primaquine. The safe application of primaquine anti-relapse therapy requires G6PD screening and anticipation of its failure with a means of prompt detection and rescue from the typically abrupt haemolytic crisis.

Diversity of Anopheles species and zoonotic malaria vector of the Buton Utara Wildlife Sanctuary, Southeast Sulawesi, Indonesia.

BACKGROUND: The recent deforestation for agricultural, mining, and human re-settlement has significantly reduced the habitat of many non-human primates (NHPs) in Indonesia and intensifies interaction between the NHPs and humans and thus opening the possibility of pathogen spill-over. The emergence of zoonotic malaria, such as Plasmodium knowlesi, poses an immense threat to the current malaria control and elimination that aims for the global elimination of malaria by 2030. As malaria in humans and NHPs is transmitted by the female Anopheles mosquito, malaria vector control is very important to mitigate the spill-over of the malaria parasite to humans. The present study aims to explore the Anopheles species diversity in human settlements adjacent to the wildlife sanctuary forest in Buton Utara Regency, Southeast Sulawesi, Indonesia, and identify the species that potentially transmit the pathogen from monkey to human in the area. METHODS: Mosquito surveillance was conducted using larval and adult collection, and the collected mosquitoes were identified morphologically and molecularly using the barcoding markers, cytochrome oxidase subunit I (COI), and internal transcribed species 2 (ITS2) genes. Plasmodium sporozoite carriage was conducted on mosquitoes collected through human landing catch (HLC) and human-baited double net trap (HDNT). RESULTS: The results revealed several Anopheles species, such as Anopheles flavirostris (16.6%), Anopheles sulawesi (3.3%), Anopheles maculatus (3.3%), Anopheles koliensis (1.2%), and Anopheles vagus (0.4%). Molecular analysis of the sporozoite carriage using the primate-specific malaria primers identified An. sulawesi, a member of the Leucosphyrus group, carrying Plasmodium inui sporozoite. CONCLUSIONS: This study indicates that the transmission of zoonotic malaria in the area is possible and alerts to the need for mitigation efforts through a locally-tailored vector control intervention and NHPs habitat conservation.

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.

The potential for zoonotic malaria transmission in five areas of Indonesia inhabited by non-human primates.

BACKGROUND: Indonesia is home to many species of non-human primates (NHPs). Deforestation, which is still ongoing in Indonesia, has substantially reduced the habitat of NHPs in the republic. This has led to an intensification of interactions between NHPs and humans, which opens up the possibility of pathogen spillover. The aim of the present study was to determine the prevalence of malarial parasite infections in NHPs in five provinces of Indonesia in 2022. Species of the genus Anopheles that can potentially transmit malarial pathogens to humans were also investigated. METHODS: An epidemiological survey was conducted by capturing NHPs in traps installed in several localities in the five provinces, including in the surroundings of a wildlife sanctuary. Blood samples were drawn aseptically after the NHPs had been anesthetized; the animals were released after examination. Blood smears were prepared on glass slides, and dried blood spot tests on filter paper. Infections with Plasmodium spp. were determined morphologically from the blood smears, which were stained with Giemsa solution, and molecularly through polymerase chain reaction and DNA sequencing using rplU oligonucleotides. The NHPs were identified to species level by using the mitochondrial cytochrome c oxidase subunit I gene and the internal transcribed spacer 2 gene as barcoding DNA markers. Mosquito surveillance included the collection of larvae from breeding sites and that of adults through the human landing catch (HLC) method together with light traps. RESULTS: Analysis of the DNA extracted from the dried blood spot tests of the 110 captured NHPs revealed that 50% were positive for Plasmodium, namely Plasmodium cynomolgi, Plasmodium coatneyi, Plasmodium inui, Plasmodium knowlesi and Plasmodium sp. Prevalence determined by microscopic examination of the blood smears was 42%. Species of the primate genus Macaca and family Hylobatidae were identified by molecular analysis. The most common mosquito breeding sites were ditches, puddles and natural ponds. Some of the Anopheles letifer captured through HLC carried sporozoites of malaria parasites that can cause the disease in primates. CONCLUSIONS: The prevalence of malaria in the NHPs was high. Anopheles letifer, a potential vector of zoonotic malaria, was identified following its collection in Central Kalimantan by the HLC method. In sum, the potential for the transmission of zoonotic malaria in several regions of Indonesia is immense.

Seroprevalence of Chikungunya in an Asymptomatic Adult Population in North and South Sulawesi, Indonesia.

Chikungunya (CHIK) is an emerging and reemerging infectious disease of public health importance in Indonesia. Information on the asymptomatic and true burden of CHIK virus (CHIKV) infections is limited. We assayed 1,092 healthy population samples, collected in North and South Sulawesi between 2019 and 2020, for antibodies against CHIKV. Blood samples were screened by IgM and IgG ELISAs and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay. CHIKV IgG seroprevalence in North and South Sulawesi was 53.2% and 53.9%, respectively. The overall prevalence of anti-CHIKV IgM antibody was 12.9%. Molecular testing of blood donors revealed 0.66% (2/300) were positive for CHIKV qRT-PCR. Our study provides new insights into the CHIKV endemicity situation in the eastern part of Indonesia and warrants the need for further systematic surveillance considering there is no treatment or vaccine for CHIK infection.

Effect of geranylated dihydrochalcone from Artocarpus altilis leaves extract on Plasmodium falciparum ultrastructural changes and mitochondrial malate: Quinone oxidoreductase.

Nearly half of the world's population is at risk of being infected by Plasmodium falciparum, the pathogen of malaria. Increasing resistance to common antimalarial drugs has encouraged investigations to find compounds with different scaffolds. Extracts of Artocarpus altilis leaves have previously been reported to exhibit in vitro antimalarial activity against P. falciparum and in vivo activity against P. berghei. Despite these initial promising results, the active compound from A. altilis is yet to be identified. Here, we have identified 2-geranyl-2', 4', 3, 4-tetrahydroxy-dihydrochalcone (1) from A. altilis leaves as the active constituent of its antimalarial activity. Since natural chalcones have been reported to inhibit food vacuole and mitochondrial electron transport chain (ETC), the morphological changes in food vacuole and biochemical inhibition of ETC enzymes of (1) were investigated. In the presence of (1), intraerythrocytic asexual development was impaired, and according to the TEM analysis, this clearly affected the ultrastructure of food vacuoles. Amongst the ETC enzymes, (1) inhibited the mitochondrial malate: quinone oxidoreductase (PfMQO), and no inhibition could be observed on dihydroorotate dehydrogenase (DHODH) as well as bc1 complex activities. Our study suggests that (1) has a dual mechanism of action affecting the food vacuole and inhibition of PfMQO-related pathways in mitochondria.

Drug resistance of Plasmodium falciparum and Plasmodium vivax isolates in Indonesia.

This review article aims to investigate the genotypic profiles of Plasmodium falciparum and Plasmodium vivax isolates collected across a wide geographic region and their association with resistance to anti-malarial drugs used in Indonesia. A systematic review was conducted between 1991 and date. Search engines, such as PubMed, Science Direct, and Google Scholar, were used for articles published in English and Indonesian to search the literature. Of the 471 initially identified studies, 61 were selected for 4316 P. falciparum and 1950 P. vivax individual infections. The studies included 23 molecular studies and 38 therapeutic efficacy studies. K76T was the most common pfcrt mutation. K76N (2.1%) was associated with the haplotype CVMNN. By following dihydroartemisinin-piperaquine (DHA-PPQ) therapy, the mutant pfmdr1 alleles 86Y and 1034C were selected. Low prevalence of haplotype N86Y/Y184/D1246Y pfmdr1 reduces susceptibility to AS-AQ. SNP mutation pvmdr1 Y976F reached 96.1% in Papua and East Nusa Tenggara. Polymorphism analysis in the pfdhfr gene revealed 94/111 (84.7%) double mutants S108N/C59R or S108T/A16V in Central Java. The predominant pfdhfr haplotypes (based on alleles 16, 51, 59,108, 164) found in Indonesia were ANCNI, ANCSI, ANRNI, and ANRNL. Some isolates carried A437G (35.3%) or A437G/K540E SNPs (26.5%) in pfdhps. Two novel pfdhps mutant alleles, I588F/G and K540T, were associated with six pfdhps haplotypes. The highest prevalence of pvdhfr quadruple mutation (F57L/S58R/T61M/S117T) (61.8%) was detected in Papua. In pvdhps, the only polymorphism before and after 2008 was 383G mutation with 19% prevalence. There were no mutations in the pfk13 gene reported with validated and candidate or associated k13 mutation. An increased copy number of pfpm2, associated with piperaquine resistance, was found only in cases of reinfection. Meanwhile, mutation of pvk12 and pvpm4 I165V is unlikely associated with ART and PPQ drug resistance. DHA-PPQ is still effective in treating uncomplicated falciparum and vivax malaria. Serious consideration should be given to interrupt local malaria transmission and dynamic patterns of resistance to anti-malarial drugs to modify chemotherapeutic policy treatment strategies. The presence of several changes in pfk13 in the parasite population is of concern and highlights the importance of further evaluation of parasitic ART susceptibility in Indonesia.

Human behavior determinants of exposure to Anopheles vectors of malaria in Sumba, Indonesia.

Malaria vector control interventions in Sumba, Indonesia, have not been able to eliminate malaria. Human drivers of exposure to Anopheles bites were investigated as part of a larger clinical trial evaluating the impact of a spatial repellent product on malaria incidence. Human behavioral observations (HBOs) evaluating temporal and spatial presence, sleeping behaviors, and insecticide treated net (ITN) use, were collected parallel to entomological collections-indoor and outdoor human landing catches (HLCs), and house hold surveys. Data demonstrates that mosquito access to humans, enabled by structurally open houses, is evident by the similar entomological landing rates both inside and outside households. The presence of animals inside houses was associated with increased mosquito entry-however, the number of humans present inside houses was not related to increased mosquito landing. Analyzing mosquito landing rates with human behavior data enables the spatial and temporal estimation of exposure to Anopheles bites, accounting for intervention (ITN) presence and usage. Human behavior adjusted exposure to Anopheles bites was found to be highest in the early in the evening, but continued at lower levels throughout the night. Over the night, most exposure (53%) occurred when people were indoors and not under the protection of nets (asleep or awake) followed by exposure outside (44%). Characterized gaps in protection are outdoor exposure as well as exposure indoors-when awake, and when asleep and not using ITNs. Interestingly, in the primary trial, even though there was not a significant impact of the spatial repellent on vector biting rates by themselves (16%), when factoring in human behavior, there was approximately 28% less exposure in the intervention arm than in the placebo arm. The treated arm had less human behavior adjusted bites in all spaces evaluated though there was proportionally higher exposure indoors. This analysis points to the importance of using HBOs both towards understanding gaps in protection as well as how interventions are evaluated. To mitigate ongoing transmission, understanding context specific spatial and temporal exposure based on the interactions of vectors, humans and interventions would be vital for a directed evidence-based control or elimination strategy.

Use of mobile data collection systems within large-scale epidemiological field trials: findings and lessons-learned from a vector control trial in Iquitos, Peru.

Vector-borne diseases are among the most burdensome infectious diseases worldwide with high burden to health systems in developing regions in the tropics. For many of these diseases, vector control to reduce human biting rates or arthropod populations remains the primary strategy for prevention. New vector control interventions intended to be marketed through public health channels must be assessed by the World Health Organization for public health value using data generated from large-scale trials integrating epidemiological endpoints of human health impact. Such phase III trials typically follow large numbers of study subjects to meet necessary power requirements for detecting significant differences between treatment arms, thereby generating substantive and complex datasets. Data is often gathered directly in the field, in resource-poor settings, leading to challenges in efficient data reporting and/or quality assurance. With advancing technology, mobile data collection (MDC) systems have been implemented in many studies to overcome these challenges. Here we describe the development and implementation of a MDC system during a randomized-cluster, placebo-controlled clinical trial evaluating the protective efficacy of a spatial repellent intervention in reducing human infection with Aedes-borne viruses (ABV) in the urban setting of Iquitos, Peru, as well as the data management system that supported it. We discuss the benefits, remaining capacity gaps and the key lessons learned from using a MDC system in this context in detail.

Genome-wide functional screening of drug-resistance genes in Plasmodium falciparum.

The global spread of drug resistance is a major obstacle to the treatment of Plasmodium falciparum malaria. The identification of drug-resistance genes is an essential step toward solving the problem of drug resistance. Here, we report functional screening as a new approach with which to identify drug-resistance genes in P. falciparum. Specifically, a high-coverage genomic library of a drug-resistant strain is directly generated in a drug-sensitive strain, and the resistance gene is then identified from this library using drug screening. In a pilot experiment using the strain Dd2, the known chloroquine-resistant gene pfcrt is identified using the developed approach, which proves our experimental concept. Furthermore, we identify multidrug-resistant transporter 7 (pfmdr7) as a novel candidate for a mefloquine-resistance gene from a field-isolated parasite; we suggest that its upregulation possibly confers the mefloquine resistance. These results show the usefulness of functional screening as means by which to identify drug-resistance genes.

Host Factors in the Natural History of Chronic Hepatitis B: Role of Genetic Determinants.

Background: The host immune system plays an important role in hepatitis B virus (HBV) infection manifestation. Genetic polymorphisms of several inflammatory cytokines, including TNF-α and IL-10, have been associated with chronic hepatitis B (CHB) progression, although with contradicting results. CHB progression can be categorized into four phases, immune tolerance (IT), immune clearance (IC), low/no replicative (LR), and e-negative hepatitis (ENH), with HBeAg seroconversion as an important milestone. Here, we determined the association of TNF-α (rs1800629) and IL-10 (rs1800896 and rs1800872) SNPs in the context of CHB natural history progression, particularly to HBeAg seroconversion, in Indonesian CHB patients. Methods: A total of 287 subjects were recruited and categorized into distinct CHB phases based on HBeAg, viral load, and ALT levels. TNF-α and IL-10 SNPs were determined using PCR-RFLP and confirmed with direct sequencing. The association between SNP genotypes with CHB dynamics was determined using logistic regression presented as odds ratio (OR) with 95% CI. Results: No significant association was found between IL-10 -592A/C polymorphism and progression of IT and IC to LR, IT and IC to ENH, and LR to ENH phases in all the gene models. IL-10 rs1800896 and TNF-α rs1800629 could not be analyzed using logistic regression. Subjects' age (≥40 years old) was significantly associated with IT and IC to LR (OR: 2.191, 95% CI 1.067-4.578, P = 0.034), IT and IC to ENH (OR: 7.460, 95% CI 3.316-18.310, P < 0.001), and LR to ENH (OR: 5.252, 95% CI 2.010-14.858, P = 0.001). Male gender was associated with LR to ENH (OR: 4.077, 95% CI 1.605-11.023, P = 0.004). Conclusions: Age and male gender were associated with CHB phase progression instead of the TNF-α and IL-10 polymorphisms. It would be beneficial to study not only the effect of host determinants but also the viral factor to understand the mechanisms of CHB phase progression.

Treatment with specific and pan-plasma membrane calcium ATPase (PMCA) inhibitors reduces malaria parasite growth in vitro and in vivo.

BACKGROUND: Rapid emergence of Plasmodium resistance to anti-malarial drug mainstays has driven a continual effort to discover novel drugs that target different biochemical pathway (s) during infection. Plasma membrane Calcium + 2 ATPase (PMCA4), a novel plasma membrane protein that regulates Calcium levels in various cells, namely red blood cell (RBC), endothelial cell and platelets, represents a new biochemical pathway that may interfere with susceptibility to malaria and/or severe malaria. METHODS: This study identified several pharmacological inhibitors of PMCA4, namely ATA and Resveratrol, and tested for their anti-malarial activities in vitro and in vivo using the Plasmodium falciparum 3D7 strain, the Plasmodium berghei ANKA strain, and Plasmodium yoelii 17XL strain as model. RESULTS: In vitro propagation of P. falciparum 3D7 strain in the presence of a wide concentration range of the inhibitors revealed that the parasite growth was inhibited in a dose-dependent manner, with IC50s at 634 and 0.231 µM, respectively. RESULTS: The results confirmed that both compounds exhibit moderate to potent anti-malarial activities with the strongest parasite growth inhibition shown by resveratrol at 0.231 µM. In vivo models using P. berghei ANKA for experimental cerebral malaria and P. yoelii 17XL for the effect on parasite growth, showed that the highest dose of ATA, 30 mg/kg BW, increased survival of the mice. Likewise, resveratrol inhibited the parasite growth following 4 days intraperitoneal injection at the dose of 100 mg/kg BW. CONCLUSION: The findings indicate that the PMCA4 of the human host may be a potential target for novel anti-malarials, either as single drug or in combination with the currently available effective anti-malarials.

Impact of a spatial repellent product on Anopheles and non-Anopheles mosquitoes in Sumba, Indonesia.

BACKGROUND: The East Nusa Tenggara province, Indonesia, contributed to 5% of malaria cases nationally in 2020, with other mosquito-borne diseases, such as dengue and filariasis also being endemic. Monitoring of spatial and temporal vector species compositions and bionomic traits is an efficient method for generating evidence towards intervention strategy optimization and meeting disease elimination goals. METHODS: The impact of a spatial repellent (SR) on human biting mosquitoes was evaluated as part of a parent cluster-randomized, double-blinded, placebo-controlled trial, in Sumba, East Nusa Tenggara. A 10-month (June 2015-March 2016) baseline study was followed by a 24-month intervention period (April 2016 to April 2018)-where half the clusters were randomly assigned either a passive transfluthrin emanator or a placebo control. RESULTS: Human-landing mosquito catches documented a reduction in landing rates related to the SR. Overall, there was a 16.4% reduction (21% indoors, and 11.3% outdoors) in human biting rates (HBR) for Anopheles. For Aedes, there was a 44.3% HBR reduction indoors and a 35.6% reduction outdoors. This reduction was 38.3% indoors and 39.1% outdoors for Armigeres, and 36.0% indoors and 32.3% outdoors for Culex species. Intervention impacts on the HBRs were not significant and are attributed to large inter-household and inter cluster variation. Anopheles flavirostris, Anopheles balabacensis and Anopheles maculatus individually impacted the overall malaria infections hazard rate with statistically significance. Though there was SR-based protection against malaria for all Anopheles species (except Anopheles sundaicus), only five (Anopheles aconitus, Anopheles kochi, Anopheles tessellatus, An. maculatus and An. sundaicus) demonstrated statistical significance. The SR numerically reduced Anopheles parity rates indoors and outdoors when compared to the placebo. CONCLUSION: Evidence demonstrating that Anopheles vectors bite both indoors and outdoors indicates that currently implemented indoor-based vector control tools may not be sufficient to eliminate malaria. The documented impact of the SR intervention on Aedes, Armigeres and Culex species points to its importance in combatting other vector borne diseases. Studies to determine the impact of spatial repellents on other mosquito-borne diseases is recommended.

Primate malaria: An emerging challenge of zoonotic malaria in Indonesia.

The emergence of zoonotic malaria in different parts of the world, including Indonesia poses a challenge to the current malaria control and elimination program that target global malaria elimination at 2030. The reported cases in human include Plasmodium knowlesi, P. cynomolgi and P. inui, in South and Southeast Asian region and P. brazilianum and P. simium in Latin America. All are naturally found in the Old and New-world monkeys, macaques spp. This review focuses on the currently available data that may represent primate malaria as an emerging challenge of zoonotic malaria in Indonesia, the distribution of non-human primates and the malaria parasites it carries, changes in land use and deforestation that impact the habitat and intensifies interaction between the non-human primate and the human which facilitate spill-over of the pathogens. Although available data in Indonesia is very limited, a growing body of evidence indicate that the challenge of zoonotic malaria is immense and alerts to the need to conduct mitigation efforts through multidisciplinary approach involving environmental management, non-human primates conservation, disease management and vector control.

An inventory of human night-biting mosquitoes and their bionomics in Sumba, Indonesia.

Mosquitoes are important vectors that transmit pathogens to human and other vertebrates. Each mosquito species has specific ecological requirements and bionomic traits that impact human exposure to mosquito bites, and hence disease transmission and vector control. A study of human biting mosquitoes and their bionomic characteristics was conducted in West Sumba and Southwest Sumba Districts, Nusa Tenggara Timur Province, Indonesia from May 2015 to April 2018. Biweekly human landing catches (HLC) of night biting mosquitoes both indoors and outdoors caught a total of 73,507 mosquito specimens (59.7% non-Anopheles, 40.3% Anopheles). A minimum of 22 Culicinae species belonging to four genera (Aedes, Armigeres, Culex, Mansonia), and 13 Anophelinae species were identified. Culex quinquefasciatus was the dominant Culicinae species, Anopheles aconitus was the principal Anopheles species inland, while An. sundaicus was dominant closer to the coast. The overall human biting rate (HBR) was 10.548 bites per person per night (bpn) indoors and 10.551 bpn outdoors. Mosquitoes biting rates were slightly higher indoors for all genera with the exception of Anopheles, where biting rates were slightly higher outdoors. Diurnal and crepuscular Aedes and Armigeres demonstrated declining biting rates throughout the night while Culex and Anopheles biting rates peaked before midnight and then declined. Both anopheline and non-anopheline populations did not have a significant association with temperature (p = 0.3 and 0.88 respectively), or rainfall (p = 0.13 and 0.57 respectively). The point distribution of HBR and seasonal variables did not have a linear correlation. Data demonstrated similar mosquito-human interactions occurring outdoors and indoors and during early parts of the night implying both indoor and outdoor disease transmission potential in the area-pointing to the need for interventions in both spaces. Integrated vector analysis frameworks may enable better surveillance, monitoring and evaluation strategies for multiple diseases.

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.