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Indonesia has the third highest number of tuberculosis (TB) patients infected with Mycobacterium tuberculosis (MTB) Lineage 1 (L1). Most of these MTB L1 cases can be found in Indonesia's remote easternmost province of Papua, one of Indonesia's most underdeveloped provinces with a particularly high burden for TB. In this study, we sequenced and described 42 MTB L1 isolates from a well-characterized cohort of patients. We found a genetically diverse MTB L1 population with no association between pathogen genetic relatedness and place of residence or pathogen genetic relatedness and patient ethnicity, which could reflect mixing between different locales and ethnicities or our low sampling fraction. Only a small number showed genetic variants associated with drug resistance (5/42, 11.9 %), probably due to a lack of effective treatment programs. The Papuan isolates showed similarities to other Island Southeast Asian Countries due to the high proportion of L1.2.1.2.1 (30/42, 71.4 %), especially East Timor and the Philippines. This study fills a research gap of MTB L1 in Indonesian Papua and should serve as a stepping stone for further research in the region., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Challenges in classifying recurrent Plasmodium vivax infections constrain surveillance of antimalarial efficacy and transmission. Recurrent infections may arise from activation of dormant liver stages (relapse), blood-stage treatment failure (recrudescence) or reinfection. Molecular inference of familial relatedness (identity-by-descent or IBD) can help resolve the probable origin of recurrences. As whole genome sequencing of P. vivax remains challenging, targeted genotyping methods are needed for scalability. We describe a P. vivax marker discovery framework to identify and select panels of microhaplotypes (multi-allelic markers within small, amplifiable segments of the genome) that can accurately capture IBD. We evaluate panels of 50-250 microhaplotypes discovered in a global set of 615 P. vivax genomes. A candidate global 100-microhaplotype panel exhibits high marker diversity in the Asia-Pacific, Latin America and horn of Africa (median H E  = 0.70-0.81) and identifies 89% of the polyclonal infections detected with genome-wide datasets. Data simulations reveal lower error in estimating pairwise IBD using microhaplotypes relative to traditional biallelic SNP barcodes. The candidate global panel also exhibits high accuracy in predicting geographic origin and captures local infection outbreak and bottlenecking events. Our framework is open-source enabling customised microhaplotype discovery and selection, with potential for porting to other species or data resources., (© 2024. The Author(s).)

The SARS-CoV-2 infection that caused the COVID-19 pandemic has become a significant public health concern. New variants with distinct mutations have emerged, potentially impacting its infectivity, immune evasion capacity, and vaccine response. A whole-genome sequencing study of 292 SARS-CoV-2 isolates collected from selected regions of Indonesia between January and October 2021 was performed to identify the distribution of SARS-CoV-2 variants and common mutations in Indonesia. During January-April 2021, Indonesian lineages B.1.466.2 and B.1.470 dominated, but from May 2021, Delta's AY.23 lineage outcompeted them. An analysis of 7515 published sequences from January 2021 to June 2022 revealed a decline in Delta in November 2021, followed by the emergence of Omicron variants in December 2021. We identified C241T (5'UTR), P314L (NSP12b), F106F (NSP3), and D614G (Spike) mutations in all sequences. The other common substitutions included P681R (76.4%) and T478K (60%) in Spike, D377Y in Nucleocapsid (61%), and I82T in Membrane (60%) proteins. Breakthrough infection and prolonged viral shedding cases were associated with Delta variants carrying the Spike T19R, G142D, L452R, T478K, D614G, P681R, D950N, and V1264L mutations. The dynamic of SARS-CoV-2 variants in Indonesia highlights the importance of continuous genomic surveillance in monitoring and identifying potential strains leading to disease outbreaks.

Ethiopia has the greatest burden of Plasmodium vivax in Africa, but little is known about the epidemiological landscape of parasites across the country. We analysed the genomic diversity of 137 P. vivax isolates collected nine Ethiopian districts from 2012 to 2016. Signatures of selection were detected by cross-country comparisons with isolates from Thailand (n = 104) and Indonesia (n = 111), representing regions with low and high chloroquine resistance respectively. 26% (35/137) of Ethiopian infections were polyclonal, and 48.5% (17/35) of these comprised highly related clones (within-host identity-by-descent > 25%), indicating frequent co-transmission and superinfection. Parasite gene flow between districts could not be explained entirely by geographic distance, with economic and cultural factors hypothesised to have an impact on connectivity. Amplification of the duffy binding protein gene (pvdbp1) was prevalent across all districts (16-75%). Cross-population haplotype homozygosity revealed positive selection in a region proximal to the putative chloroquine resistance transporter gene (pvcrt-o). An S25P variant in amino acid transporter 1 (pvaat1), whose homologue has recently been implicated in P. falciparum chloroquine resistance evolution, was prevalent in Ethiopia (96%) but not Thailand or Indonesia (35-53%). The genomic architecture in Ethiopia highlights circulating variants of potential public health concern in an endemic setting with evidence of stable transmission., (© 2023. The Author(s).)

Colombia aims to eliminate malaria by 2030 but remains one of the highest burden countries in the Americas. Plasmodium vivax contributes half of all malaria cases, with its control challenged by relapsing parasitaemia, drug resistance and cross-border spread. Using 64 Colombian P. vivax genomes collected between 2013 and 2017, we explored diversity and selection in two major foci of transmission: Chocó and Córdoba. Open-access data from other countries were used for comparative assessment of drug resistance candidates and to assess cross-border spread. Across Colombia, polyclonal infections were infrequent (12%), and infection connectivity was relatively high (median IBD = 5%), consistent with low endemicity. Chocó exhibited a higher frequency of polyclonal infections (23%) than Córdoba (7%), although the difference was not significant (P = 0.300). Most Colombian infections carried double pvdhfr (95%) and single pvdhps (71%) mutants, but other drug resistance mutations were less prevalent (< 10%). There was no evidence of selection at the pvaat1 gene, whose P. falciparum orthologue has recently been implicated in chloroquine resistance. Global population comparisons identified other putative adaptations. Within the Americas, low-level connectivity was observed between Colombia and Peru, highlighting potential for cross-border spread. Our findings demonstrate the potential of molecular data to inform on infection spread and adaptation., (© 2023. The Author(s).)

Traditionally, patient travel history has been used to distinguish imported from autochthonous malaria cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative method to identify, and map imported cases. Using machine learning approaches incorporating hierarchical fixation index and decision tree analyses applied to 799 P. vivax genomes from 21 countries, we identified 33-SNP, 50-SNP and 55-SNP barcodes (GEO33, GEO50 and GEO55), with high capacity to predict the infection's country of origin. The Matthews correlation coefficient (MCC) for an existing, commonly applied 38-SNP barcode (BR38) exceeded 0.80 in 62% countries. The GEO panels outperformed BR38, with median MCCs > 0.80 in 90% countries at GEO33, and 95% at GEO50 and GEO55. An online, open-access, likelihood-based classifier framework was established to support data analysis (vivaxGEN-geo). The SNP selection and classifier methods can be readily amended for other use cases to support malaria control programs., (© 2022. The Author(s).)

Although the power of genetic surveillance tools has been acknowledged widely, there is an urgent need in malaria endemic countries for feasible and cost-effective tools to implement in national malaria control programs (NMCPs) that can generate evidence to guide malaria control and elimination strategies, especially in the case of Plasmodium vivax . Several genetic surveillance applications ('use cases') have been identified to align research, technology development, and public health efforts, requiring different types of molecular markers. Here we present a new highly-multiplexed deep sequencing assay (Pv AmpliSeq). The assay targets the 33-SNP vivaxGEN-geo panel for country-level classification, and a newly designed 42-SNP within-country barcode for analysis of parasite dynamics in Vietnam and 11 putative drug resistance genes in a highly multiplexed NGS protocol with easy workflow, applicable for many different genetic surveillance use cases. The Pv AmpliSeq assay was validated using: 1) isolates from travelers and migrants in Belgium, and 2) routine collections of the national malaria control program at sentinel sites in Vietnam. The assay targets 229 amplicons and achieved a high depth of coverage (mean 595.7 ± 481) and high accuracy (mean error-rate of 0.013 ± 0.007). P. vivax parasites could be characterized from dried blood spots with a minimum of 5 parasites/µL and 10% of minority-clones. The assay achieved good spatial specificity for between-country prediction of origin using the 33-SNP vivaxGEN-geo panel that targets rare alleles specific for certain countries and regions. A high resolution for within-country diversity in Vietnam was achieved using the designed 42-SNP within-country barcode that targets common alleles (median MAF 0.34, range 0.01-0.49. Many variants were detected in (putative) drug resistance genes, with different predominant haplotypes in the pvmdr1 and pvcrt genes in different provinces in Vietnam. The capacity of the assay for high resolution identity-by-descent (IBD) analysis was demonstrated and identified a high rate of shared ancestry within Gia Lai Province in the Central Highlands of Vietnam, as well as between the coastal province of Binh Thuan and Lam Dong. Our approach performed well in geographically differentiating isolates at multiple spatial scales, detecting variants in putative resistance genes, and can be easily adjusted to suit the needs in other settings in a country or region. We prioritize making this tool available to researchers and NMCPs in endemic countries to increase ownership and ensure data usage for decision-making and malaria policy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kattenberg, Nguyen, Nguyen, Sauve, Nguyen, Chopo-Pizarro, Trimarsanto, Monsieurs, Guetens, Nguyen, Esbroeck, Auburn, Nguyen and Rosanas-Urgell.)

Background: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide and genetic mutation plays a vital role in CRC development. A previous study has suggested that genetic alterations among Indonesian patients with CRC might differ from those known in developed countries. This study aimed to describe the genomic profiles of Indonesian patients with CRC. Methods: A total of 13 patients were recruited for this study from May to July 2019. Tissue samples were collected, and genomic DNA was extracted from the samples. AmpliSeq for Illumina Cancer HotSpot Panel v2 Next-generation sequencing was used for DNA sequencing and a genome analysis toolkit was used for local realignment around the discovered variants. Results: A total of 45 genes comprising 391 single nucleotide variants (SNVs) with a depth >10 were observed. The genes with the most variants were STK11, SMAD4, EGFR, and ERBB4 and the genes with the most non-synonymous variants were SMAD4, TP53, FGFR3, CDKN2A, and STK11. Genes and SNVs in at least 90% of all samples consisted of 43 genes comprising 286 variants. Genes with the most non-synonymous SNVs were EGFR, SMO, FGFR3, TP53, STK11, CDKN2A. Genes related to the chromosomal instability pathway, such as TP53, SMAD4, KRAS, and APC, are also found in the analysis. Conclusions: Our findings showed that all patients with CRC in this study had genetic mutations in the chromosomal instability pathway. Analysis of genetic mutation of Indonesian patients with CRC might be crucial for advanced targeted therapy and for better clinical outcomes., Competing Interests: No competing interests were disclosed., (Copyright: © 2023 Abdullah M et al.)

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr , dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 MalariaGEN et al.)

The Asia-Pacific region faces formidable challenges in achieving malaria elimination by the proposed target in 2030. Molecular surveillance of Plasmodium parasites can provide important information on malaria transmission and adaptation, which can inform national malaria control programmes (NMCPs) in decision-making processes. In November 2019 a parasite genotyping workshop was held in Jakarta, Indonesia, to review molecular approaches for parasite surveillance and explore ways in which these tools can be integrated into public health systems and inform policy. The meeting was attended by 70 participants from 8 malaria-endemic countries and partners of the Asia Pacific Malaria Elimination Network. The participants acknowledged the utility of multiple use cases for parasite genotyping including: quantifying the prevalence of drug resistant parasites, predicting risks of treatment failure, identifying major routes and reservoirs of infection, monitoring imported malaria and its contribution to local transmission, characterizing the origins and dynamics of malaria outbreaks, and estimating the frequency of Plasmodium vivax relapses. However, the priority of each use case varies with different endemic settings. Although a one-size-fits-all approach to molecular surveillance is unlikely to be applicable across the Asia-Pacific region, consensus on the spectrum of added-value activities will help support data sharing across national boundaries. Knowledge exchange is needed to establish local expertise in different laboratory-based methodologies and bioinformatics processes. Collaborative research involving local and international teams will help maximize the impact of analytical outputs on the operational needs of NMCPs. Research is also needed to explore the cost-effectiveness of genetic epidemiology for different use cases to help to leverage funding for wide-scale implementation. Engagement between NMCPs and local researchers will be critical throughout this process.

Plasmodium vivax is the most widespread and difficult to treat cause of human malaria. The development of vaccines against the blood stages of P. vivax remains a key objective for the control and elimination of vivax malaria. Erythrocyte binding-like (EBL) protein family members such as Duffy binding protein (PvDBP) are of critical importance to erythrocyte invasion and have been the major target for vivax malaria vaccine development. In this study, we focus on another member of EBL protein family, P. vivax erythrocyte binding protein (PvEBP). PvEBP was first identified in Cambodian (C127) field isolates and has subsequently been showed its preferences for binding reticulocytes which is directly inhibited by antibodies. We analysed PvEBP sequence from 316 vivax clinical isolates from eight countries including China (n = 4), Ethiopia (n = 24), Malaysia (n = 53), Myanmar (n = 10), Papua New Guinea (n = 16), Republic of Korea (n = 10), Thailand (n = 174), and Vietnam (n = 25). PvEBP gene exhibited four different phenotypic clusters based on the insertion/deletion (indels) variation. PvEBP-RII (179-479 aa.) showed highest polymorphism similar to other EBL family proteins in various Plasmodium species. Whereas even though PvEBP-RIII-V (480-690 aa.) was the most conserved domain, that showed strong neutral selection pressure for gene purifying with significant population expansion. Antigenicity of both of PvEBP-RII (16.1%) and PvEBP-RIII-V (21.5%) domains were comparatively lower than other P. vivax antigen which expected antigens associated with merozoite invasion. Total IgG recognition level of PvEBP-RII was stronger than PvEBP-RIII-V domain, whereas total IgG inducing level was stronger in PvEBP-RIII-V domain. These results suggest that PvEBP-RII is mainly recognized by natural IgG for innate protection, whereas PvEBP-RIII-V stimulates IgG production activity by B-cell for acquired immunity. Overall, the low antigenicity of both regions in patients with vivax malaria likely reflects genetic polymorphism for strong positive selection in PvEBP-RII and purifying selection in PvEBP-RIII-V domain. These observations pose challenging questions to the selection of EBP and point out the importance of immune pressure and polymorphism required for inclusion of PvEBP as a vaccine candidate., Competing Interests: The authors have declared that no competing interests exist.

Genetic epidemiology can provide important insights into parasite transmission that can inform public health interventions. The current study compared long-term changes in the genetic diversity and structure of co-endemic Plasmodium falciparum and P. vivax populations. The study was conducted in Papua Indonesia, where high-grade chloroquine resistance in P. falciparum and P. vivax led to a universal policy of Artemisinin-based Combination Therapy (ACT) in 2006. Microsatellite typing and population genetic analyses were undertaken on available isolates collected between 2004 and 2017 from patients with uncomplicated malaria (n = 666 P. falciparum and n = 615 P. vivax). The proportion of polyclonal P. falciparum infections fell from 28% (38/135) before policy change (2004-2006) to 18% (22/125) at the end of the study (2015-2017); p<0.001. Over the same period, polyclonal P. vivax infections fell from 67% (80/119) to 35% (33/93); p<0.001. P. falciparum strains persisted for up to 9 years compared to 3 months for P. vivax, reflecting higher rates of outbreeding in the latter. Sub-structure was observed in the P. falciparum population, but not in P. vivax, confirming different patterns of outbreeding. The P. falciparum population exhibited 4 subpopulations that changed in frequency over time. Notably, a sharp rise was observed in the frequency of a minor subpopulation (K2) in the late post-ACT period, accounting for 100% of infections in late 2016-2017. The results confirm epidemiological evidence of reduced P. falciparum and P. vivax transmission over time. The smaller change in P. vivax population structure is consistent with greater outbreeding associated with relapsing infections and highlights the need for radical cure to reduce recurrent infections. The study emphasizes the challenge in disrupting P. vivax transmission and demonstrates the potential of molecular data to inform on the impact of public health interventions., Competing Interests: The authors have declared that no competing interests exist.

The Horn of Africa harbors the largest reservoir of Plasmodium vivax in the continent. Most of sub-Saharan Africa has remained relatively vivax-free due to a high prevalence of the human Duffy-negative trait, but the emergence of strains able to invade Duffy-negative reticulocytes poses a major public health threat. We undertook the first population genomic investigation of P. vivax from the region, comparing the genomes of 24 Ethiopian isolates against data from Southeast Asia to identify important local adaptions. The prevalence of the Duffy binding protein amplification in Ethiopia was 79%, potentially reflecting adaptation to Duffy negativity. There was also evidence of selection in a region upstream of the chloroquine resistance transporter, a putative chloroquine-resistance determinant. Strong signals of selection were observed in genes involved in immune evasion and regulation of gene expression, highlighting the need for a multifaceted intervention approach to combat P. vivax in the region., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

The hyper-endemicity of dengue in Indonesia poses a significant threat of dengue virus (DENV) vertical transmission during pregnancy. A 29-year-old female at 38 weeks of pregnancy presented to hospital with acute fever and later confirmed with DENV infection. Due to signs of fetal distress, the neonate was delivered by emergency caesarean section. The mother developed a dengue critical phase post-caesarean with excessive bleeding and required blood transfusion. During the 6th day of life, the neonate was diagnosed and later confirmed with dengue. Next-generation sequencing of DENV RNA isolated directly from sera of both mother and neonate revealed identical DENV-2 whole-genome sequences. Plaque reduction neutralization test (PRNT) detected anti-dengue antibodies in both mother and neonate. Altogether, our data confirmed the occurrence of vertical transmission. Dengue vertical transmission during pregnancy may lead to severe manifestation, hence early diagnosis, close monitoring, and prompt intervention are critical., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Placental malaria (PM) causes placental insufficiency, leading to reduced birth weight (BW). Placental mtDNA copy number (mtDNA-CN) and relative telomere length (RTL) have been described as potential biomarkers for placental insufficiency and intrauterine growth restriction (IUGR). We investigated their associations with BW in women with PM from malaria-endemic region of Papua, Indonesia. MtDNA-CN and RTL were determined in 50 placentas by quantitative real-time PCR. Increased placental mtDNA-CN was associated with reduced BW (coef = -193.71, p = 0.016), particularly in preterm group (coef = -374.21, p < 0.001). RTL did not associate with BW. Increased placental mtDNA-CN indicates a compensatory mechanism to reduced BW in women with PM., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Transcription factor 7-like 2 (TCF7L2) protein plays an important role in glucose and lipid metabolisms. Single nucleotide polymorphisms (SNPs) in the TCF7L2 gene contribute to increased fasting plasma glucose (FPG) and body mass index (BMI), and altered lipid concentrations in various population. We investigated whether the TCF7L2 SNPs were associated with obesity, high FPG and altered lipid profile in the Balinese. A total of 608 Balinese from rural and urban Bali, Indonesia, were recruited. Triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC) and FPG were measured, and BMI was calculated. Ratios of TG/HDL-C, LDL-C/HDL-C, and TC/HDL-C were determined. Genotyping of SNPs rs7903146, rs10885406, and rs12255372 were done in all samples. Genetic association analyses under a dominant model showed that the rs7903146 (OR 5.50, 95% CI 2.34-12.91, p = 8.5 × 10 -5 ), rs12255372 (OR 4.15, 95% CI 1.66-10.33, p = 0.003) and rs10885406 (OR 2.43, 95% CI 1.39-4.25, p = 0.003) were significantly associated with high TC/HDL-C ratio. The rs10885406 also presented a significant association with high TG (OR 2.21, 95% CI 1.29-3.81, p = 0.004) and low HDL-C (OR 3.02, 95% CI 1.58-5.80, p = 0.001) concentrations, as well as high TG/HDL-C ratio (OR 1.95, 95% CI 1.16-3.27, p = 0.013). None of the SNPs exhibited significant association with obesity or high FPG. SNPs in the TCF7L2 gene are associated with altered lipid profile in the Balinese.

Influenza viruses are by nature unstable with high levels of mutations. The sequential accumulation of mutations in the surface glycoproteins allows the virus to evade the neutralizing antibodies. The consideration of the tropics as the influenza reservoir where viral genetic and antigenic diversity are continually generated and reintroduced into temperate countries makes the study of influenza virus evolution in Indonesia essential. A total of 100 complete coding sequences (CDS) of Hemagglutinin (HA) and Neuraminidase (NA) genes of H3N2 virus were obtained from archived samples of Influenza-Like Illness (ILI) surveillance collected from 2008 to 2010. Our evolutionary and phylogenetic analyses provide insight into the dynamic changes of Indonesian H3N2 virus from 2008 to 2010. Obvious antigenic drift with typical 'ladder-like' phylogeny was observed with multiple lineages found in each year, suggesting co-circulation of H3N2 strains at different time periods. The mutational pattern of the Indonesian H3N2 virus was not geographically related as relatively low levels of mutations with similar pattern of relative genetic diversity were observed in various geographical origins. This study reaffirms that the existence of a particular lineage is most likely the result of adaptation or competitive exclusion among different host populations and combination of stochastic ecological factors, rather than its geographical origin alone., Competing Interests: The authors have declared that no competing interests exist.

The incidence of Plasmodium vivax infection has declined markedly in Malaysia over the past decade despite evidence of high-grade chloroquine resistance. Here we investigate the genetic changes in a P. vivax population approaching elimination in 51 isolates from Sabah, Malaysia and compare these with data from 104 isolates from Thailand and 104 isolates from Indonesia. Sabah displays extensive population structure, mirroring that previously seen with the emergence of artemisinin-resistant P. falciparum founder populations in Cambodia. Fifty-four percent of the Sabah isolates have identical genomes, consistent with a rapid clonal expansion. Across Sabah, there is a high prevalence of loci known to be associated with antimalarial drug resistance. Measures of differentiation between the three countries reveal several gene regions under putative selection in Sabah. Our findings highlight important factors pertinent to parasite resurgence and molecular cues that can be used to monitor low-endemic populations at the end stages of P. vivax elimination.

Dengue has caused a significant public health impact globally. With the diverse genetic of the causative viruses, analysis of dengue virus (DENV) genomes is important to supplement epidemiological data with information that can be used to reconstruct the history of epidemics in time and space. We have reported the clinical and virological characteristics of dengue in Surabaya, Indonesia and revealed the presence of all four DENV serotypes and the predominance of DENV-1. The further classification of Surabaya DENV-1 into two different genotypes warrants in-depth genomic analysis to study the dynamics of both genotypes and their contribution to virus evolution, virus transmission, and disease. We performed full-length genome sequencing to nine isolates' representatives from DENV-1 Genotype I and Genotype IV. Phylogenetic and evolutionary analyses suggested the more recent introduction of Genotype I viruses compared to the more endemic Genotype IV. Comparative analysis of Surabaya DENV-1 genomes and other sequences available publicly revealed that the majority of the DENV-1 codons were under strong purifying selection, while seven codon sites identified to be under positive selection. We highlight a unique codon site under the positive pressure in the NS1 gene of DENV-1. Our results provide additional genomic data of DENV from Indonesia that may contribute to the better understanding of dengue disease dynamics.

Although neurological manifestations associated with dengue viruses (DENV) infection have been reported, there is very limited information on the genetic characteristics of neurotropic DENV. Here we describe the isolation and complete genome analysis of DENV serotype 3 (DENV-3) from cerebrospinal fluid of an encephalitis paediatric patient in Jakarta, Indonesia. Next-generation sequencing was employed to deduce the complete genome of the neurotropic DENV-3 isolate. Based on complete genome analysis, two unique and nine uncommon amino acid changes in the protein coding region were observed in the virus. A phylogenetic tree and molecular clock analysis revealed that the neurotropic virus was a member of Sumatran-Javan clade of DENV-3 genotype I and shared a common ancestor with other isolates from Jakarta around 1998. This is the first report of neurotropic DENV-3 complete genome analysis, providing detailed information on the genetic characteristics of this virus.

The surveillance of malaria is generally undertaken on the assumption that samples passively collected at health facilities are comparable to or representative of the broader Plasmodium reservoir circulating in the community. Further characterization and comparability of the hidden asymptomatic parasite reservoir are needed to inform on the potential impact of sampling bias. This study explores the impact of sampling strategy on molecular surveillance by comparing the genetic make-up of Plasmodium falciparum and Plasmodium vivax isolates collected by passive versus active case detection. Sympatric isolates of P. falciparum and P. vivax were collected from a large community survey and ongoing clinical surveillance studies undertaken in the hypomesoendemic setting of Mimika District (Papua, Indonesia). Plasmodium falciparum isolates were genotyped at nine microsatellite loci and P. vivax at eight loci. Measures of diversity and differentiation were used to compare different patient and parasitological sample groups. The results demonstrated that passively detected cases (symptomatic) had comparable population diversity to those circulating in the community (asymptomatic) in both species. In addition, asymptomatic patent infections were as diverse as subpatent infections. However, a significant difference in multiplicity of infection (MOI) and percentage of polyclonal infections was observed between actively and passively detected P. vivax cases (mean MOI: 1.7 ± 0.7 versus 1.4 ± 1.4, respectively; P = 0.001). The study findings infer that, in hypomesoendemic settings, passive sampling is appropriate for molecular parasite surveillance strategies using the predominant clone in any given infection; however, the findings suggest caution when analyzing complexity of infection. Further evaluation is required in other endemic settings.

Dengue has affected Indonesia for the last five decades and become a major health problem in many cities in the country. Jakarta, the capital of Indonesia, reports dengue cases annually, with several outbreaks documented. To gain information on the dynamic and evolutionary history of dengue virus (DENV) in Jakarta, we conducted phylogenetic and evolutionary analyses of DENV isolated in 2009. Three hundred thirty-three dengue-suspected patients were recruited. Our data revealed that dengue predominantly affected young adults, and the majority of cases were due to secondary infection. A total of 171 virus isolates were successfully serotyped. All four DENV serotypes were circulating in the city, and DENV-1 was the predominant serotype. The DENV genotyping of 17 isolates revealed the presence of Genotypes I and IV in DENV-1, while DENV-2 isolates were grouped into the Cosmopolitan genotype. The grouping of isolates into Genotype I and II was seen for DENV-3 and DENV-4, respectively. Evolutionary analysis revealed the relatedness of Jakarta isolates with other isolates from other cities in Indonesia and isolates from imported cases in other countries. We revealed the endemicity of DENV and the role of Jakarta as the potential source of imported dengue cases in other countries. Our study provides genetic information regarding DENV from Jakarta, which will be useful for upstream applications, such as the study of DENV epidemiology and evolution and transmission dynamics.

Zika virus (ZIKV) JMB-185 strain was isolated from a febrile patient in Jambi, Indonesia in 2014. To understand its genetic characteristics, we performed whole genome sequencing using the Ion Torrent PGM platform on the supernatant of the first passage. The phylogenetic analysis showed that the isolate was not closely related to the Brazilian ZIKV associated with microcephaly or isolates from the recent Singapore Zika outbreak. Molecular evolution analysis indicated that JMB-185 strain may have been circulating in the Southeast Asia region, including Indonesia since 2000. We observed high nucleotide sequence identity between Indonesia, Thailand, Singapore, and American strains although unique amino acid substitutions were also observed. This report provides information on the genomic characteristics of Indonesian ZIKV which may be used for further studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Plasmodium vivax malaria remains a major public health burden in Myanmar. Resistance to chloroquine (CQ), the first-line treatment for P. vivax, has been reported in the country and has potential to undermine local control efforts., Methods: Patients over 6 years of age with uncomplicated P. vivax mono-infection were enrolled into clinical efficacy studies in Myawaddy in 2014 and Kawthoung in 2012. Study participants received a standard dose of CQ (25 mg/kg over 3 days) followed by weekly review until day 28. Pvmdr1 copy number (CN) and microsatellite diversity were assessed on samples from the patients enrolled in the clinical study and additional cross-sectional surveys undertaken in Myawaddy and Shwegyin in 2012., Results: A total of 85 patients were enrolled in the CQ clinical studies, 25 in Myawaddy and 60 in Kawthoung. One patient in Myawaddy (1.2%) had an early treatment failure and two patients (2.3%) in Kawthoung presented with late treatment failures on day 28. The day 28 efficacy was 92.0% (95% CI 71.6-97.9) in Myawaddy and 98.3% (95% CI 88.7-99.8) in Kawthoung. By day 2, 92.2% (23/25) in Myawaddy and 85.0% (51/60) in Kawthoung were aparasitaemic. Genotyping and pvmdr1 CN assessment was undertaken on 43, 52 and 46 clinical isolates from Myawaddy, Kawthoung and Shwegyin respectively. Pvmdr1 amplification was observed in 3.2% (1/31) of isolates in Myawaddy, 0% (0/49) in Kawthoung and 2.5% (1/40) in Shwegyin. Diversity was high in all sites (H E 0.855-0.876), with low inter-population differentiation (F ST 0.016-0.026, P < 0.05)., Conclusions: Treatment failures after chloroquine were observed following chloroquine monotherapy, with pvmdr1 amplification present in both Myawaddy and Shwegyin. The results emphasize the importance of ongoing P. vivax drug resistance surveillance in Myanmar, particularly given the potential connectivity between parasite population at different sites.

Dengue disease is still a major health problem in Indonesia. Surabaya, the second largest city in the country, is endemic for dengue. We report here on dengue disease in Surabaya, investigating the clinical manifestations, the distribution of dengue virus (DENV) serotypes, and the relationships between clinical manifestations and the genetic characteristics of DENV. A total of 148 patients suspected of having dengue were recruited during February-August 2012. One hundred one (68%) of them were children, and 47 (32%) were adults. Dengue fever (DF) and Dengue hemorrhagic fever (DHF) were equally manifested in all of the patients. We performed DENV serotyping on all of the samples using real-time RT-PCR. Of 148, 79 (53%) samples were detected as DENV positive, with DENV-1 as the predominant serotype (73%), followed by DENV-2 (8%), DENV-4 (8%), and DENV-3 (6%), while 5% were mixed infections. Based on the Envelope gene sequences, we performed phylogenetic analyses of 24 isolates to genotype the DENV circulating in Surabaya in 2012, and the analysis revealed that DENV-1 consisted of Genotypes I and IV, DENV-2 was of the Cosmopolitan genotype, the DENV-3 viruses were of Genotype I, and DENV-4 was detected as Genotype II. We correlated the infecting DENV serotypes with clinical manifestations and laboratory parameters; however, no significant correlations were found. Amino acid analysis of Envelope protein did not find any unique mutations related to disease severity.

Background: Genetic analyses of Plasmodium have potential to inform on transmission dynamics, but few studies have evaluated this on a local spatial scale. We used microsatellite genotyping to characterise the micro-epidemiology of P. vivax and P. falciparum diversity to inform malaria control strategies in Timika, Papua Indonesia., Methods: Genotyping was undertaken on 713 sympatric P. falciparum and P. vivax isolates from a cross-sectional household survey and clinical studies conducted in Timika. Standard population genetic measures were applied, and the data was compared to published data from Kalimantan, Bangka, Sumba and West Timor., Results: Higher diversity (HE = 0.847 vs 0.625; p = 0.017) and polyclonality (46.2% vs 16.5%, p<0.001) were observed in P. vivax versus P. falciparum. Distinct P. falciparum substructure was observed, with two subpopulations, K1 and K2. K1 was comprised solely of asymptomatic infections and displayed greater relatedness to isolates from Sumba than to K2, possibly reflecting imported infections., Conclusions: The results demonstrate the greater refractoriness of P. vivax versus P. falciparum to control measures, and risk of distinct parasite subpopulations persisting in the community undetected by passive surveillance. These findings highlight the need for complimentary new surveillance strategies to identify transmission patterns that cannot be detected with traditional malariometric methods.

Dengue is a febrile disease caused by infection of dengue virus (DENV). Early diagnosis of dengue infection is important for better management of the disease. The DENV Non-Structural Protein 1 (NS1) antigen has been routinely used for the early dengue detection. In dengue epidemic countries such as Indonesia, clinicians are increasingly relying on the NS1 detection for confirmation of dengue infection. Various NS1 diagnostic tests are commercially available, however different sensitivities and specificities were observed in various settings. This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant proteins from isolates obtained from other geographical regions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Objectives: Plasmodium falciparum infection during pregnancy is characterised by placental inefficiency caused by infected erythrocyte sequestration. Reduced placental efficiency leads to placental intrauterine adaptation for sustaining fetal growth, which is reflected by changes in the expression of placental genes involved in intrauterine growth regulation. Therefore, we aimed to determine whether the placental weight ratio, an indicator of placental efficiency, affects the placental expression of the components of the insulin-like growth factor axis and leptin signalling pathway in P. falciparum-infected pregnant women., Methods and Study Design: A malaria case-only analysis of 50 P. falciparum-infected pregnant women in Timika, Papua, Indonesia, was conducted. The placental mRNA expression of insulin-like growth factor-I, insulin-like growth factor binding protein-1, leptin, and the long and short isoforms of the leptin receptor was measured through quantitative realtime PCR., Results: The placental weight ratio exerted a positive effect on the placental mRNA expression of insulin- like growth factor-I (coefficient=6.10, p=0.002) and the long isoform of the leptin receptor (coefficient= 4.73, p=0.015) in malaria-infected pregnant women without fever or chill symptoms., Conclusion: Our results indicate that placental adaptive responses caused by adverse intrauterine conditions in P. falciparuminfected pregnant women vary depending on the presence or absence of fever and chill symptoms.

Plasmodium vivax is now the predominant cause of malaria in the Asia-Pacific, South America and Horn of Africa. Laboratory studies of this species are constrained by the inability to maintain the parasite in continuous ex vivo culture, but genomic approaches provide an alternative and complementary avenue to investigate the parasite's biology and epidemiology. To date, molecular studies of P. vivax have relied on the Salvador-I reference genome sequence, derived from a monkey-adapted strain from South America. However, the Salvador-I reference remains highly fragmented with over 2500 unassembled scaffolds.  Using high-depth Illumina sequence data, we assembled and annotated a new reference sequence, PvP01, sourced directly from a patient from Papua Indonesia. Draft assemblies of isolates from China (PvC01) and Thailand (PvT01) were also prepared for comparative purposes. The quality of the PvP01 assembly is improved greatly over Salvador-I, with fragmentation reduced to 226 scaffolds. Detailed manual curation has ensured highly comprehensive annotation, with functions attributed to 58% core genes in PvP01 versus 38% in Salvador-I. The assemblies of PvP01, PvC01 and PvT01 are larger than that of Salvador-I (28-30 versus 27 Mb), owing to improved assembly of the subtelomeres.  An extensive repertoire of over 1200 Plasmodium interspersed repeat ( pir ) genes were identified in PvP01 compared to 346 in Salvador-I, suggesting a vital role in parasite survival or development. The manually curated PvP01 reference and PvC01 and PvT01 draft assemblies are important new resources to study vivax malaria. PvP01 is maintained at GeneDB and ongoing curation will ensure continual improvements in assembly and annotation quality., Competing Interests: No competing interests were disclosed.

Background: Malaria remains endemic at low levels in the south-eastern provinces of Iran bordering Afghanistan and Pakistan, with the majority of cases attributable to P. vivax. The national guidelines recommend chloroquine (CQ) as blood-stage treatment for uncomplicated P. vivax, but the large influx of imported cases enhances the risk of introducing CQ resistance (CQR)., Methodology and Principal Findings: The genetic diversity at pvmdr1, a putative modulator of CQR, and across nine putatively neutral short tandem repeat (STR) markers were assessed in P. vivax clinical isolates collected between April 2007 and January 2013 in Hormozgan Province, south-eastern Iran. One hundred blood samples were collected from patients with microscopy-confirmed P. vivax enrolled at one of five district clinics. In total 73 (73%) were autochthonous cases, 23 (23%) imported cases from Afghanistan or Pakistan, and 4 (4%) with unknown origin. 97% (97/100) isolates carried the F1076L mutation, but none carried the Y976F mutation. STR genotyping was successful in 71 (71%) isolates, including 57(57%) autochthonous and 11 (11%) imported cases. Analysis of population structure revealed 2 major sub-populations, K1 and K2, with further sub-structure within K2. The K1 sub-population had markedly lower diversity than K2 (HE = 0.06 vs HE = 0.82) suggesting that the sub-populations were sustained by distinct reservoirs with differing transmission dynamics, possibly reflecting local versus imported/introduced populations. No notable separation was observed between the local and imported cases although the sample size was limited., Conclusions: The contrasting low versus high diversity in the two sub-populations (K1 and K2) infers that a combination of local transmission and cross-border malaria from higher transmission regions shape the genetic make-up of the P. vivax population in south-eastern Iran. There was no molecular evidence of CQR amongst the local or imported cases, but ongoing clinical surveillance is warranted., Competing Interests: The authors have declared that no competing interests exist.

Background: Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009., Results: An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested., Conclusions: The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.