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Abstract The Philippines is a high-incidence country for tuberculosis, with the increasing prevalence of multi- (MDR-TB) and extensively-drug (XDR-TB) resistant Mycobacterium tuberculosis strains posing difficulties to disease control. Understanding the genetic diversity of circulating strains can provide insights into underlying drug resistance mutations and transmission dynamics, thereby assisting the design of diagnostic tools, including those using next generation sequencing (NGS) platforms. By analysing genome sequencing data of 732 isolates from Philippines drug-resistance survey collections spanning from 2011 to 2019, we found that the majority belonged to lineages L1 (531/732; 72.5%) and L4 (European-American; n = 174; 23.8%), with the Manila strain (L1.2.1.2.1) being the most prominent (475/531). Approximately two-thirds of isolates were found to be at least MDR-TB (483/732; 66.0%), and potential XDR-TB genotypic resistance was observed (3/732; 0.4%), highlighting an emerging problem in the country. Genotypic resistance was highly concordant with laboratory drug susceptibility testing. By finding isolates with (near-)identical genomic variation, five major clusters containing a total of 114 isolates were identified: all containing either L1 or L4 isolates with at least MDR-TB resistance and spanning multiple years of collection. Closer inspection of clusters revealed transmission in prisons, some involving isolates with XDR-TB, and mutations linked to third-line drug bedaquiline. We have also identified previously unreported mutations linked to resistance for isoniazid, rifampicin, ethambutol, and fluoroquinolones. Overall, this study provides important insights into the genetic diversity, transmission and circulating drug resistance mutations of M. tuberculosis in the Philippines, thereby informing clinical and surveillance decision-making, which is increasingly using NGS platforms.

Abstract Whole genome sequencing (WGS) of Mycobacterium tuberculosis offers valuable insights for tuberculosis (TB) control. High throughput platforms like Illumina and Oxford Nanopore Technology (ONT) are increasingly used globally, although ONT is known for higher error rates and is less established for genomic studies. Here we present a study comparing the sequencing outputs of both Illumina and ONT platforms, analysing DNA from 59 clinical isolates in highly endemic TB regions of Thailand. The resulting sequence data were used to profile the M. tuberculosis pairs for their lineage, drug resistance and presence in transmission chains, and were compared to publicly available WGS data from Thailand (n = 1456). Our results revealed isolates that are predominantly from lineages 1 and 2, with consistent drug resistance profiles, including six multidrug-resistant strains; however, analysis of ONT data showed longer phylogenetic branches, emphasising the technologies higher error rate. An analysis incorporating the larger dataset identified fifteen of our samples within six potential transmission clusters, including a significant clade of 41 multi-drug resistant isolates. ONT's extended sequences also revealed strain-specific structural variants in pe/ppe genes (e.g. ppe50), which are candidate loci for vaccine development. Despite some limitations, our results show that ONT sequencing is a promising approach for TB genomic research, supporting precision medicine and decision-making in areas with less developed infrastructure, which is crucial for tackling the disease’s significant regional burden.

Analysis of genome sequencing data from >100,000 genomes of Mycobacterium tuberculosis complex using TB-Annotator software revealed a previously unknown lineage, proposed name L10, in central Africa. Phylogenetic reconstruction suggests L10 could represent a missing link in the evolutionary and geographic migration histories of M. africanum.

Abstract Despite Plasmodium ovale curtisi (Poc) and wallikeri (Pow) being important human-infecting malaria parasites that are widespread across Africa and Asia, little is known about their genome diversity. Morphologically identical, Poc and Pow are indistinguishable and commonly misidentified. Recent rises in the incidence of Poc/Pow infections have renewed efforts to address fundamental knowledge gaps in their biology, and to develop diagnostic tools to understand their epidemiological dynamics and malaria burden. A major roadblock has been the incompleteness of available reference assemblies (PocGH01, PowCR01; ~ 33.5 Mbp). Here, we applied multiple sequencing platforms and advanced bioinformatics tools to generate new reference genomes, Poc221 (South Sudan; 36.0 Mbp) and Pow222 (Nigeria; 34.3 Mbp), with improved nuclear genome contiguity (> 4.2 Mbp), annotation and completeness (> 99% Plasmodium spp., single copy orthologs). Subsequent sequencing of 6 Poc and 15 Pow isolates from Africa revealed a total of 22,517 and 43,855 high-quality core genome SNPs, respectively. Genome-wide levels of nucleotide diversity were determined to be 2.98 × 10–4 (Poc) and 3.43 × 10–4 (Pow), comparable to estimates for other Plasmodium species. Overall, the new reference genomes provide a robust foundation for dissecting the biology of Poc/Pow, their population structure and evolution, and will contribute to uncovering the recombination barrier separating these species.

Abstract Background Malaria continues to be a major threat to global public health. Whole genome sequencing (WGS) of the underlying Plasmodium parasites has provided insights into the genomic epidemiology of malaria. Genome sequencing is rapidly gaining traction as a diagnostic and surveillance tool for clinical settings, where the profiling of co-infections, identification of imported malaria parasites, and detection of drug resistance are crucial for infection control and disease elimination. To support this informatically, we have developed the Malaria-Profiler tool, which rapidly (within minutes) predicts Plasmodium species, geographical source, and resistance to antimalarial drugs directly from WGS data. Results The online and command line versions of Malaria-Profiler detect ~ 250 markers from genome sequences covering Plasmodium speciation, likely geographical source, and resistance to chloroquine, sulfadoxine-pyrimethamine (SP), and other anti-malarial drugs for P. falciparum, but also providing mutations for orthologous resistance genes in other species. The predictive performance of the mutation library was assessed using 9321 clinical isolates with WGS and geographical data, with most being single-species infections (P. falciparum 7152/7462, P. vivax 1502/1661, P. knowlesi 143/151, P. malariae 18/18, P. ovale ssp. 5/5), but co-infections were identified (456/9321; 4.8%). The accuracy of the predicted geographical profiles was high to both continental (96.1%) and regional levels (94.6%). For P. falciparum, markers were identified for resistance to chloroquine (49.2%; regional range: 24.5% to 100%), sulfadoxine (83.3%; 35.4– 90.5%), pyrimethamine (85.4%; 80.0–100%) and combined SP (77.4%). Markers associated with the partial resistance of artemisinin were found in WGS from isolates sourced from Southeast Asia (30.6%). Conclusions Malaria-Profiler is a user-friendly tool that can rapidly and accurately predict the geographical regional source and anti-malarial drug resistance profiles across large numbers of samples with WGS data. The software is flexible with modifiable bioinformatic pipelines. For example, it is possible to select the sequencing platform, display specific variants, and customise the format of outputs. With the increasing application of next-generation sequencing platforms on Plasmodium DNA, Malaria-Profiler has the potential to be integrated into point-of-care and surveillance settings, thereby assisting malaria control. Malaria-Profiler is available online (bioinformatics.lshtm.ac.uk/malaria-profiler) and as standalone software ( https://github.com/jodyphelan/malaria-profiler ).

Abstract Vector control strategies have been successful in reducing the number of malaria cases and deaths globally, but the spread of insecticide resistance represents a significant threat to disease control. Insecticide resistance has been reported across Anopheles (An.) vector populations, including species within the An. funestus group. These mosquitoes are responsible for intense malaria transmission across sub-Saharan Africa, including in the Democratic Republic of the Congo (DRC), a country contributing > 12% of global malaria infections and mortality events. To support the continuous efficacy of vector control strategies, it is essential to monitor insecticide resistance using molecular surveillance tools. In this study, we developed an amplicon sequencing (“Amp-seq”) approach targeting An. funestus, and using multiplex PCR, dual index barcoding, and next-generation sequencing for high throughput and low-cost applications. Using our Amp-seq approach, we screened 80 An. funestus field isolates from the DRC across a panel of nine genes with mutations linked to insecticide resistance (ace-1, CYP6P4, CYP6P9a, GSTe2, vgsc, and rdl) and mosquito speciation (cox-1, mtND5, and ITS2). Amongst the 18 non-synonymous mutations detected, was N485I, in the ace-1 gene associated with carbamate resistance. Overall, our panel represents an extendable and much-needed method for the molecular surveillance of insecticide resistance in An. funestus populations.

The integration of next-generation sequencing into the identification and characterization of resistant and virulent strains as well as the routine surveillance of foodborne pathogens such as Salmonella enterica have not yet been accomplished in the Philippines. This study investigated the antimicrobial profiles, virulence, and susceptibility of the 105 S. enterica isolates from swine and chicken samples obtained from slaughterhouses and public wet markets in Metropolitan Manila using whole-genome sequence analysis. Four predominant serovars were identified in genotypic serotyping, namely, Infantis (26.7%), Anatum (19.1%), Rissen (18.1%), and London (13.3%). Phenotypic antimicrobial resistance (AMR) profiling revealed that 65% of the isolates were resistant to at least one antibiotic, 37% were multidrug resistant (MDR), and 57% were extended-spectrum β-lactamase producers. Bioinformatic analysis revealed that isolates had resistance genes and plasmids belonging to the Col and Inc plasmid families that confer resistance against tetracycline (64%), sulfonamide (56%), and streptomycin (56%). Further analyses revealed the presence of 155 virulence genes, 42 of which were serovar-specific. The virulence genes primarily code for host immune system modulators, iron acquisition enzyme complexes, host cell invasion proteins, as well as proteins that allow intracellular and intramacrophage survival. This study showed that virulent MDR S. enterica and several phenotypic and genotypic AMR patterns were present in the food chain. It serves as a foundation to understand the current AMR status in the Philippines food chain and to prompt the creation of preventative measures and efficient treatments against foodborne pathogens.

Abstract Flaviviruses are a diverse group of RNA viruses, which include the etiological agents of Zika, dengue and yellow fever that are transmitted by mosquitoes. Flaviviruses do not encode reverse transcriptase and cannot reverse transcribe into DNA, yet DNA sequences of flaviviruses are found both integrated in the chromosomes of Aedes aegypti mosquitoes and as extrachromosomal sequences. We have previously examined the Ae. aegypti reference genome to identify flavivirus integrations and analyzed conservation of these sequences among whole-genome data of 464 Ae. aegypti collected across 10 countries globally. Here, we extended this analysis by identifying flavivirus sequences in these samples independently of the Ae. aegypti reference assembly. Our aim was to identify the complete set of viral sequences, including those absent in the reference genome, and their geographical distribution. We compared the identified sequences using BLASTn and applied machine learning methods to identify clusters of similar sequences. Apart from clusters of sequences that correspond to the four viral integration events that we had previously described, we identified 19 smaller clusters. The only cluster with a strong geographic association consisted of Cell-fusing agent virus-like sequences specific to Thailand. The remaining clusters did not have a geographic association and mostly consisted of near identical short sequences without strong similarity to any known flaviviral genomes. The short read sequencing data did not permit us to determine whether identified sequences were extrachromosomal or integrated into Ae. aegypti chromosomes. Our results suggest that Liverpool strain and field Ae. aegypti mosquitoes have a similar variety of conserved flaviviral DNA, whose functional role should be investigated in follow-up studies. Graphical Abstract

Abstract Malaria has exhibited the strongest known selective pressure on the human genome in recent history and is the evolutionary driving force behind genetic conditions, such as sickle-cell disease, glucose-6-phosphatase deficiency, and some other erythrocyte defects. Genomic studies (e.g., The 1000 Genomes project) have provided an invaluable baseline for human genetics, but with an estimated two thousand ethno-linguistic groups thought to exist across the African continent, our understanding of the genetic differences between indigenous populations and their implications on disease is still limited. Low-cost sequencing-based approaches make it possible to target specific molecular markers and genes of interest, leading to potential insights into genetic diversity. Here we demonstrate the versatility of custom dual-indexing technology and Illumina next generation sequencing to generate a genetic profile of human polymorphisms associated with malaria pathology. For 100 individuals diagnosed with severe malaria in Northeast Tanzania, variants were successfully characterised on the haemoglobin subunit beta (HBB), glucose-6-phosphate dehydrogenase (G6PD), atypical chemokine receptor 1 (ACKR1) genes, and the intergenic Dantu genetic blood variant, then validated using pre-existing genotyping data. High sequencing coverage was observed across all amplicon targets in HBB, G6PD, ACKR1, and the Dantu blood group, with variants identified at frequencies previously observed within this region of Tanzania. Sequencing data exhibited high concordance rates to pre-existing genotyping data (> 99.5%). Our work demonstrates the potential utility of amplicon sequencing for applications in human genetics, including to personalise medicine and understand the genetic diversity of loci linked to important host phenotypes, such as malaria susceptibility.

Abstract Malaria control initiatives require rapid and reliable methods for the detection and monitoring of molecular markers associated with antimalarial drug resistance in Plasmodium falciparum parasites. Ngodhe island, Kenya, presents a unique malaria profile, with lower P. falciparum incidence rates than the surrounding region, and a high proportion of sub-microscopic and low-density infections. Here, using custom dual-indexing and Illumina next generation sequencing, we generate resistance profiles on seventy asymptomatic and low-density P. falciparum infections from a mass drug administration program implemented on Ngodhe island between 2015 and 2016. Our assay encompasses established molecular markers on the Pfcrt, Pfmdr1, Pfdhps, Pfdhfr, and Pfk13 genes. Resistance markers for sulfadoxine-pyrimethamine were identified at high frequencies, including a quintuple mutant haplotype (Pfdhfr/Pfdhps: N51I, C59R, S108N/A437G, K540E) identified in 62.2% of isolates. The Pfdhps K540E biomarker, used to inform decision making for intermittent preventative treatment in pregnancy, was identified in 79.2% of isolates. Several variants on Pfmdr1, associated with reduced susceptibility to quinolones and lumefantrine, were also identified (Y184F 47.1%; D1246Y 16.0%; N86 98%). Overall, we have presented a low-cost and extendable approach that can provide timely genetic profiles to inform clinical and surveillance activities, especially in settings with abundant low-density infections, seeking malaria elimination.

Abstract Since its first detection in 2012 in Djibouti, Anopheles stephensi has invaded and established in the Horn of Africa, and more recently Nigeria. The expansion of this vector poses a significant threat to malaria control and elimination efforts. Integrated vector management is the primary strategy used to interrupt disease transmission; however, growing insecticide resistance is threatening to reverse gains in global malaria control. We present a next-generation amplicon-sequencing approach, for high-throughput monitoring of insecticide resistance genes (ace1, GSTe2, vgsc and rdl), species identification and characterization of genetic diversity (its2 and cox1) in An. stephensi. Ninety-five An. stephensi mosquitoes, collected in Ethiopia, were screened, identifying 104 SNPs, including the knock-down mutation L958F (L1014F in Musca domestica), and for the first time in this vector species, the A296S substitution (A301S in Drosophila melanogaster) in the rdl locus. Two other amino acid substitutions (ace1-N177D, GSTe2-V189L) were also identified but have not been previously implicated in insecticide resistance. Genetic diversity in the mitochondrial cox1 gene revealed shared haplotypes between Ethiopian An. stephensi with samples from Pakistan, Sudan, and Djibouti. Overall, we present a reliable, cost-effective strategy using amplicon-sequencing to monitor known insecticide resistance mutations, with the potential to identify new genetic variants, to assist in the high-throughput surveillance of insecticide resistance in An. stephensi populations.

IntroductionAround 10% of the coding potential of Mycobacterium tuberculosisis constituted by two poorly understood gene families, the pe and ppe loci, thought to be involved in host-pathogen interactions. Their repetitive nature and high GC content have hindered sequence analysis, leading to exclusion from whole-genome studies. Understanding the genetic diversity of pe/ppe families is essential to facilitate their potential translation into tools for tuberculosis prevention and treatment.MethodsTo investigate the genetic diversity of the 169 pe/ppe genes, we performed a sequence analysis across 73 long-read assemblies representing seven different lineages of M. tuberculosis and M. bovis BCG. Individual pe/ppe gene alignments were extracted and diversity and conservation across the different lineages studied.ResultsThe pe/ppe genes were classified into three groups based on the level of protein sequence conservation relative to H37Rv, finding that >50% were conserved, with indels in pe_pgrs and ppe_mptr sub-families being major drivers of structural variation. Gene rearrangements, such as duplications and gene fusions, were observed between pe and pe_pgrs genes. Inter-lineage diversity revealed lineage-specific SNPs and indels.DiscussionThe high level of pe/ppe genes conservation, together with the lineage-specific findings, suggest their phylogenetic informativeness. However, structural variants and gene rearrangements differing from the reference were also identified, with potential implications for pathogenicity. Overall, improving our knowledge of these complex gene families may have insights into pathogenicity and inform the development of much-needed tools for tuberculosis control.

Abstract Resistance to isoniazid (INH) and rifampicin (RIF) first-line drugs in Mycobacterium tuberculosis (Mtb), together called multi-drug resistance, threatens tuberculosis control. Resistance mutations in katG (for INH) and rpoB (RIF) genes often come with fitness costs. To overcome these costs, Mtb compensatory mutations have arisen in rpoC/rpoA (RIF) and ahpC (INH) loci. By leveraging the presence of known compensatory mutations, we aimed to detect novel resistance mutations occurring in INH and RIF target genes. Across ~ 32 k Mtb isolates with whole genome sequencing (WGS) data, there were 6262 (35.7%) with INH and 5435 (30.7%) with RIF phenotypic resistance. Known mutations in katG and rpoB explained ~ 99% of resistance. However, 188 (0.6%) isolates had ahpC compensatory mutations with no known resistance mutations in katG, leading to the identification of 31 putative resistance mutations in katG, each observed in at least 3 isolates. These putative katG mutations can co-occur with other INH variants (e.g., katG-Ser315Thr, fabG1 mutations). For RIF, there were no isolates with rpoC/rpoA compensatory mutations and unknown resistance mutations. Overall, using WGS data we identified putative resistance markers for INH that could be used for genotypic drug-resistance profiling. Establishing the complete repertoire of Mtb resistance mutations will assist the clinical management of tuberculosis.

Atypical Escherichia coli forms exhibit unusual characteristics compared to typical strains. The H2S-producing variants of some atypical E. coli strains cause a wide range of illnesses in humans and animals. However, there are sparse reports on such strains worldwide. We performed whole-genome sequencing (WGS) and detailed characterization of four H2S-producing E. coli variants from poultry and human clinical sources in Dhaka, Bangladesh. All four isolates were confirmed as E. coli using biochemical tests and genomic analysis, and were multidrug-resistant (MDR). WGS analysis including an additional Chinese strain, revealed diverse STs among the five H2S-producing E. coli genomes, with clonal complex ST10 being detected in 2 out of 5 genomes. The predominant phylogroup detected was group A (n = 4/5). The blaTEM1B (n = 5/5) was the most predominant extended-spectrum beta-lactamase (ESBL) gene, followed by different alleles of blaCTX-M (blaCTX-M -55,-65,-123; n = 3/5). Multiple plasmid replicons were detected, with IncX being the most common. One E. coli strain was classified as enteropathogenic E. coli. The genomes of all five isolates harbored five primary and four secondary function genes related to H2S production. These findings suggest the potential of these isolates to cause disease and spread antibiotic resistance. Therefore, such atypical E. coli forms should be included in differential diagnosis to understand the pathogenicity, antimicrobial resistance and evolution of H2S-producing E. coli.

Abstract Artificial intelligence (AI)-based approaches can now use electrocardiograms (ECGs) to provide expert-level performance in detecting heart abnormalities and diagnosing disease. Additionally, patient age predicted from ECGs by AI models has shown great potential as a biomarker for cardiovascular age, where recent work has found its deviation from chronological age (“delta age”) to be associated with mortality and co-morbidities. However, despite being crucial for understanding underlying individual risk, the genetic underpinning of delta age is unknown. In this work we performed a genome-wide association study using UK Biobank data (n=34,432) and identified eight loci associated with delta age ( $$p\le 5 \times 10^{-8}$$ p ≤ 5 × 10 - 8 ), including genes linked to cardiovascular disease (CVD) (e.g. SCN5A) and (heart) muscle development (e.g. TTN). Our results indicate that the genetic basis of cardiovascular ageing is predominantly determined by genes directly involved with the cardiovascular system rather than those connected to more general mechanisms of ageing. Our insights inform the epidemiology of CVD, with implications for preventative and precision medicine.

Abstract Malaria, caused by Plasmodium parasites, is a major global health challenge. Whole genome sequencing (WGS) of Plasmodium falciparum and Plasmodium vivax genomes is providing insights into parasite genetic diversity, transmission patterns, and can inform decision making for clinical and surveillance purposes. Advances in sequencing technologies are helping to generate timely and big genomic datasets, with the prospect of applying Artificial Intelligence analytical techniques (e.g., machine learning) to support programmatic malaria control and elimination. Here, we assess the potential of applying deep learning convolutional neural network approaches to predict the geographic origin of infections (continents, countries, GPS locations) using WGS data of P. falciparum (n = 5957; 27 countries) and P. vivax (n = 659; 13 countries) isolates. Using identified high-quality genome-wide single nucleotide polymorphisms (SNPs) (P. falciparum: 750 k, P. vivax: 588 k), an analysis of population structure and ancestry revealed clustering at the country-level. When predicting locations for both species, classification (compared to regression) methods had the lowest distance errors, and > 90% accuracy at a country level. Our work demonstrates the utility of machine learning approaches for geo-classification of malaria parasites. With timelier WGS data generation across more malaria-affected regions, the performance of machine learning approaches for geo-classification will improve, thereby supporting disease control activities.

Klebsiella pneumoniae is recognized as an urgent public health threat because of the emergence of difficult-to-treat (DTR) strains and hypervirulent clones, resulting in infections with high morbidity and mortality rates. Despite its prominence, little is known about the genomic epidemiology of K. pneumoniae in resource-limited settings like Bangladesh. We sequenced genomes of 32 K. pneumoniae strains isolated from patient samples at the International Center for Diarrhoeal Disease Research, Bangladesh (icddr,b). Genome sequences were examined for their diversity, population structure, resistome, virulome, MLST, O and K antigens and plasmids. Our results revealed the presence of two K. pneumoniae phylogroups, namely KpI (K. pneumoniae) (97%) and KpII (K. quasipneumoniae) (3%). The genomic characterization revealed that 25% (8/32) of isolates were associated with high-risk multidrug-resistant clones, including ST11, ST14, ST15, ST307, ST231 and ST147. The virulome analysis confirmed the presence of six (19%) hypervirulent K. pneumoniae (hvKp) and 26 (81%) classical K. pneumoniae (cKp) strains. The most common ESBL gene identified was blaCTX-M-15 (50%). Around 9% (3/32) isolates exhibited a difficult-to-treat phenotype, harboring carbapenem resistance genes (2 strains harbored blaNDM-5 plus blaOXA-232, one isolate blaOXA-181). The most prevalent O antigen was O1 (56%). The capsular polysaccharides K2, K20, K16 and K62 were enriched in the K. pneumoniae population. This study suggests the circulation of the major international high-risk multidrug-resistant and hypervirulent (hvKp) K. pneumoniae clones in Dhaka, Bangladesh. These findings warrant immediate appropriate interventions, which would otherwise lead to a high burden of untreatable life-threatening infections locally.

Abstract Tuberculosis, caused by Mycobacterium tuberculosis, is a high-burden disease in Pakistan, with multi-drug (MDR) and extensive-drug (XDR) resistance, complicating infection control. Whole genome sequencing (WGS) of M. tuberculosis is being used to infer lineages (strain-types), drug resistance mutations, and transmission patterns—all informing infection control and clinical decision making. Here we analyse WGS data on 535 M. tuberculosis isolates sourced across Pakistan between years 2003 and 2020, to understand the circulating strain-types and mutations related to 12 anti-TB drugs, as well as identify transmission clusters. Most isolates belonged to lineage 3 (n = 397; 74.2%) strain-types, and were MDR (n = 328; 61.3%) and (pre-)XDR (n = 113; 21.1%). By inferring close genomic relatedness between isolates (

Abstract Background Second-line drug resistance (SLD) among tuberculosis (TB) patients is a serious emerging challenge towards global control of the disease. We characterized SLD-resistance conferring-mutations among TB patients with rifampicin and/or isoniazid (RIF and/or INH) drug-resistance tested at the Uganda National TB Reference Laboratory (NTRL) between June 2017 and December 2019. Methods This was a descriptive cross-sectional secondary data analysis of 20,508 M. tuberculosis isolates of new and previously treated patients’ resistant to RIF and/or INH. DNA strips with valid results to characterise the SLD resistance using the commercial Line Probe Assay Genotype MTBDRsl Version 2.0 Assay (Hain Life Science, Nehren, Germany) were reviewed. Data were analysed with STATAv15 using cross-tabulation for frequency and proportions of known resistance-conferring mutations to injectable agents (IA) and fluoroquinolones (FQ). Results Among the eligible participants, 12,993/20,508 (63.4%) were male and median (IQR) age 32 (24–43). A total of 576/20,508 (2.8%) of the M. tuberculosis isolates from participants had resistance to RIF and/or INH. These included; 102/576 (17.7%) single drug-resistant and 474/576 (82.3%) multidrug-resistant (MDR) strains. Only 102 patients had test results for FQ of whom 70/102 (68.6%) and 01/102 (0.98%) had resistance-conferring mutations in the gyrA locus and gyrB locus respectively. Among patients with FQ resistance, gyrAD94G 42.6% (30.0–55.9) and gyrA A90V 41.1% (28.6–54.3) mutations were most observed. Only one mutation, E540D was detected in the gyrB locus. A total of 26 patients had resistance-conferring mutations to IA in whom, 20/26 77.0% (56.4–91.0) had A1401G mutation in the rrs gene locus. Conclusions Our study reveals a high proportion of mutations known to confer high-level fluoroquinolone drug-resistance among patients with rifampicin and/or isoniazid drug resistance. Utilizing routinely generated laboratory data from existing molecular diagnostic methods may aid real-time surveillance of emerging tuberculosis drug-resistance in resource-limited settings.

Aedes mosquito vectors transmit many viruses of global health concern, including dengue, chikungunya and Zika. These vector-borne viral diseases have a limited number of treatment options, and vaccines vary in their effectiveness. Consequently, integrated vector management is a primary strategy for disease control. However, the increasing emergence and spread of insecticide resistance is threatening the efficacy of vector control methods. Identifying mutations associated with resistance in vector populations is important to monitor the occurrence and evolution of insecticide resistance and inform control strategies. Rapid and cost-effective genome sequencing approaches are urgently needed. Here we present an adaptable targeted amplicon approach for cost-effective implementation within next generation sequencing platforms. This approach can identify single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) in genes involved in insecticide resistance in Aedes aegypti mosquitoes. We designed and tested eleven amplicons, which included segments of the ace-1 (carbamate target), the Voltage-Gated Sodium Channel (vgsc; pyrethroids, DDT and organochlorines), and rdl (dieldrin) genes; thereby covering established knockdown resistance (kdr) mutations (e.g., S989P, I1011M/V, V1016G/I and F1534C), with the potential to identify novel ones. The amplicon assays were designed with internal barcodes, to facilitate multiplexing of large numbers of mosquitoes at low cost, and were sequenced using an Illumina platform. Our approach was evaluated on 152 Ae. aegypti mosquitoes collected in Cabo Verde, an archipelago with a history of arbovirus outbreaks. The amplicon sequence data revealed 146 SNPs, including four non-synonymous polymorphisms in the vgsc gene, one in ace-1 and the 296S rdl mutation previously associated with resistance to organochlorines. The 296S rdl mutation was identified in 98% of mosquitoes screened, consistent with the past use of an organochlorine compound (e.g., DDT). Overall, our work shows that targeted amplicon sequencing is a rapid, robust, and cost-effective tool that can be used to perform high throughput monitoring of insecticide resistance. Author summary Many viruses, such as dengue and Zika, are transmitted by Aedes aegypti mosquitoes. These vector-borne diseases are a major public health problem in the tropics and sub-tropics worldwide. The primary strategy to reduce their burden is vector control, including through the application of insecticides. However, many mosquito populations have developed resistance to insecticides. Here, we present a rapid, robust, and cost-effective tool that allows the screening of genes associated with insecticide resistance across many Aedes aegypti mosquitoes, identifying known resistance and novel mutations. This assay will support vector control strategies by informing on the emergence and spread of insecticide resistance mutations across Aedes aegypti populations.

Multi-resistant pathogenic strains of non-lactose fermenting Escherichia coli (NLF E. coli) are responsible for various intestinal and extraintestinal infections. Although several studies have characterised such strains using conventional methods, they have not been comprehensively studied at the genomic level. To address this gap, we used whole-genome sequencing (WGS) coupled with detailed microbiological and biochemical testing to investigate 17 NLF E. coli from a diagnostic centre (icddr,b) in Dhaka, Bangladesh. The prevalence of NLF E. coli was 10%, of which 47% (8/17) exhibited multi-drug resistant (MDR) phenotypes. All isolates (17/17) were confirmed as E. coli and could not ferment lactose sugar. WGS data analysis revealed international high-risk clonal lineages. The most prevalent sequence types (STs) were ST131 (23%), ST1193 (18%), ST12 (18%), ST501 (12%), ST167 (6%), ST73 (6%) and ST12 (6%). Phylogenetic analysis corroborated a striking clonal population amongst the studied NLF E. coli isolates. The predominant phylogroup detected was B2 (65%). The blaCTX-M-15 extended-spectrum beta-lactamase gene was present in 53% of isolates (9/17), whilst 64.7% (11/17) isolates were affiliated with pathogenic pathotypes. All extraintestinal pathogenic E. coli pathotypes demonstrated β-hemolysis. Our study underscores the presence of critical pathogens and MDR clones amongst non-lactose fermenting E. coli. We suggest that non-lactose fermenting E. coli be considered equally capable as lactose fermenting forms in causing intestinal and extraintestinal infections. Further, there is a need to undertake systematic, unbiased monitoring of predominant lineages amongst non-lactose fermenting E. coli that would help in better treatment and prevention strategies.

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the deadliest infectious diseases worldwide. Multidrug and extensively drug-resistant strains are making disease control difficult, and exhausting treatment options. New anti-TB drugs bedaquiline (BDQ), delamanid (DLM) and pretomanid (PTM) have been approved for the treatment of multi-drug resistant TB, but there is increasing resistance to them. Nine genetic loci strongly linked to resistance have been identified (mmpR5, atpE, and pepQ for BDQ; ddn, fgd1, fbiA, fbiB, fbiC, and fbiD for DLM/PTM). Here we investigated the genetic diversity of these loci across >33,000 M . tuberculosis isolates. In addition, epistatic mutations in mmpL5-mmpS5 as well as variants in ndh, implicated for DLM/PTM resistance in M. smegmatis, were explored. Our analysis revealed 1,227 variants across the nine genes, with the majority (78%) present in isolates collected prior to the roll-out of BDQ and DLM/PTM. We identified phylogenetically-related mutations, which are unlikely to be resistance associated, but also high-impact variants such as frameshifts (e.g. in mmpR5, ddn) with likely functional effects, as well as non-synonymous mutations predominantly in MDR-/XDR-TB strains with predicted protein destabilising effects. Overall, our work provides a comprehensive mutational catalogue for BDQ and DLM/PTM associated genes, which will assist with establishing associations with phenotypic resistance; thereby, improving the understanding of the causative mechanisms of resistance for these drugs, leading to better treatment outcomes.

Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis, is endemic in Pakistan. Resistance to both firstline rifampicin and isoniazid drugs (multidrug-resistant TB; MDR-TB) is hampering disease control. Rifampicin resistance is attributed to rpoB gene mutations, but rpoA and rpoC loci may also be involved. To characterise underlying rifampicin resistance mutations in the TB endemic province of Khyber Pakhtunkhwa, we sequenced 51 M. tuberculosis isolates collected between 2016 and 2019; predominantly, MDR-TB (n = 44; 86.3%) and lineage 3 (n = 30, 58.8%) strains. We found that known mutations in rpoB (e.g. S405L), katG (e.g. S315T), or inhA promoter loci explain the MDR-TB. There were 24 unique mutations in rpoA, rpoB, and rpoC genes, including four previously unreported. Five instances of within-host resistance diversity were observed, where two were a mixture of MDR-TB strains containing mutations in rpoB, katG, and the inhA promoter region, as well as compensatory mutations in rpoC. Heteroresistance was observed in two isolates with a single lineage. Such complexity may reflect the high transmission nature of the Khyber Pakhtunkhwa setting. Our study reinforces the need to apply sequencing approaches to capture the full-extent of MDR-TB genetic diversity, to understand transmission, and to inform TB control activities in the highly endemic setting of Pakistan.

Abstract Mosquitoes of the genus Aedes are the main vectors of many viruses, e.g. dengue and Zika, which affect millions of people each year and for which there are limited treatment options. Understanding how Aedes mosquitoes tolerate high viral loads may lead to better disease control strategies. Elucidating endogenous viral elements (EVEs) within vector genomes may give exploitable biological insights. Previous studies have reported the presence of a large number of EVEs in Aedes genomes. Here we investigated if flavivirus EVEs are conserved across populations and different Aedes species by using ~ 500 whole genome sequence libraries from Aedes aegypti and Aedes albopictus, sourced from colonies and field mosquitoes across continents. We found that nearly all flavivirus EVEs in the Ae. aegypti reference genome originate from four separate putative viral integration events, and that they are highly conserved across geographically diverse samples. By contrast, flavivirus EVEs in the Ae. albopictus reference genome originate from up to nine distinct integration events and show low levels of conservation, even within samples from narrow geographical ranges. Our analysis suggests that flaviviruses integrated as long sequences and were subsequently fragmented and shuffled by transposable elements. Given that EVEs of Ae. aegypti and Ae. albopictus belong to different phylogenetic clades and have very differing levels of conservation, they may have different evolutionary origins and potentially different functional roles.

Multidrug-resistant tuberculosis (MDR TB), pre-extensively drug-resistant tuberculosis (pre-XDR TB), and extensively drug-resistant tuberculosis (XDR TB) complicate disease control. We analyzed whole-genome sequence data for 579 phenotypically drug-resistant M. tuberculosis isolates (28% of available MDR/pre-XDR and all culturable XDR TB isolates collected in Thailand during 2014–2017). Most isolates were from lineage 2 (n = 482; 83.2%). Cluster analysis revealed that 281/579 isolates (48.5%) formed 89 clusters, including 205 MDR TB, 46 pre-XDR TB, 19 XDR TB, and 11 poly–drug-resistant TB isolates based on genotypic drug resistance. Members of most clusters had the same subset of drug resistance-associated mutations, supporting potential primary resistance in MDR TB (n = 176/205; 85.9%), pre-XDR TB (n = 29/46; 63.0%), and XDR TB (n = 14/19; 73.7%). Thirteen major clades were significantly associated with geography (p

Abstract During COVID-19, diagnostic serological tools and vaccines have been developed. To inform control activities in a post-vaccine surveillance setting, we have developed an online “immuno-analytics” resource that combines epitope, sequence, protein and SARS-CoV-2 mutation analysis. SARS-CoV-2 spike and nucleocapsid proteins are both vaccine and serological diagnostic targets. Using the tool, the nucleocapsid protein appears to be a sub-optimal target for use in serological platforms. Spike D614G (and nsp12 L314P) mutations were most frequent (> 86%), whilst spike A222V/L18F have recently increased. Also, Orf3a proteins may be a suitable target for serology. The tool can accessed from: http://genomics.lshtm.ac.uk/immuno (online); https://github.com/dan-ward-bio/COVID-immunoanalytics (source code).

Abstract Background Tuberculosis (TB), particularly multi- and or extensive drug resistant TB, is still a global medical emergency. Whole genome sequencing (WGS) is a current alternative to the WHO-approved probe-based methods for TB diagnosis and detection of drug resistance, genetic diversity and transmission dynamics of Mycobacterium tuberculosis complex (MTBC). This study compared WGS and clinical data in participants with TB. Results This cohort study performed WGS on 87 from MTBC DNA isolates, 57 (66%) and 30 (34%) patients with drug resistant and susceptible TB, respectively. Drug resistance was determined by Xpert® MTB/RIF assay and phenotypic culture-based drug-susceptibility-testing (DST). WGS and bioinformatics data that predict phenotypic resistance to anti-TB drugs were compared with participant’s clinical outcomes. They were 47 female participants (54%) and the median age was 35 years (IQR): 29–44). Twenty (23%) and 26 (30%) of participants had TB/HIV co-infection BMI

ABSTRACTTuberculosis disease (TB), caused by Mycobacterium tuberculosis, is a major public health issue in Thailand. The high prevalence of modern Beijing (Lineage 2.2.1) strains has been associated with multi- and extensively drug-resistant infections (MDR-, XDR-TB), complicating disease control. The impact of rarer proto-Beijing (L2.1) strains is less clear. In our study of thirty-seven L2.1 clinical isolates spanning thirteen years, we found a high prevalence of XDR-TB cases (32.4%). With ≤ 12 pairwise SNP distances, 43.2% of L2.1 patients belong to MDR-TB or XDR-TB transmission clusters suggesting a high level of clonal expansion across four Thai provinces. All XDR-TB (100%) were likely due to transmission rather than inadequate treatment. We found a 47 mutation signature and a partial deletion of the fadD14 gene in the circulating XDR-TB cluster, which can be used for surveillance of this rare and resilient M. tuberculosis strain-type that is causing increasing health burden. We also detected three novel deletion positions, a deletion of 1285 bp within desA3 (Rv3230c), large deletions in the plcB, plcA, and ppe38 gene which may play a role in the virulence, pathogenesis or evolution of the L2.1 strain-type.

Abstract Background Mycobacterium tuberculosis resistance to anti-tuberculosis drugs is a major threat to global public health. Whole genome sequencing (WGS) is rapidly gaining traction as a diagnostic tool for clinical tuberculosis settings. To support this informatically, previous work led to the development of the widely used TBProfiler webtool, which predicts resistance to 14 drugs from WGS data. However, for accurate and rapid high throughput of samples in clinical or epidemiological settings, there is a need for a stand-alone tool and the ability to analyse data across multiple WGS platforms, including Oxford Nanopore MinION. Results We present a new command line version of the TBProfiler webserver, which includes hetero-resistance calling and will facilitate the batch processing of samples. The TBProfiler database has been expanded to incorporate 178 new markers across 16 anti-tuberculosis drugs. The predictive performance of the mutation library has been assessed using > 17,000 clinical isolates with WGS and laboratory-based drug susceptibility testing (DST) data. An integrated MinION analysis pipeline was assessed by performing WGS on 34 replicates across 3 multi-drug resistant isolates with known resistance mutations. TBProfiler accuracy varied by individual drug. Assuming DST as the gold standard, sensitivities for detecting multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) were 94% (95%CI 93–95%) and 83% (95%CI 79–87%) with specificities of 98% (95%CI 98–99%) and 96% (95%CI 95–97%) respectively. Using MinION data, only one resistance mutation was missed by TBProfiler, involving an insertion in the tlyA gene coding for capreomycin resistance. When compared to alternative platforms (e.g. Mykrobe predictor TB, the CRyPTIC library), TBProfiler demonstrated superior predictive performance across first- and second-line drugs. Conclusions The new version of TBProfiler can rapidly and accurately predict anti-TB drug resistance profiles across large numbers of samples with WGS data. The computing architecture allows for the ability to modify the core bioinformatic pipelines and outputs, including the analysis of WGS data sourced from portable technologies. TBProfiler has the potential to be integrated into the point of care and WGS diagnostic environments, including in resource-poor settings.

Abstract Human tuberculosis disease (TB), caused by Mycobacterium tuberculosis (Mtb), is a complex disease, with a spectrum of outcomes. Genomic, transcriptomic and methylation studies have revealed differences between Mtb lineages, likely to impact on transmission, virulence and drug resistance. However, so far no studies have integrated sequence-based genomic, transcriptomic and methylation characterisation across a common set of samples, which is critical to understand how DNA sequence and methylation affect RNA expression and, ultimately, Mtb pathogenesis. Here we perform such an integrated analysis across 22 M. tuberculosis clinical isolates, representing ancient (lineage 1) and modern (lineages 2 and 4) strains. The results confirm the presence of lineage-specific differential gene expression, linked to specific SNP-based expression quantitative trait loci: with 10 eQTLs involving SNPs in promoter regions or transcriptional start sites; and 12 involving potential functional impairment of transcriptional regulators. Methylation status was also found to have a role in transcription, with evidence of differential expression in 50 genes across lineage 4 samples. Lack of methylation was associated with three novel variants in mamA, likely to cause loss of function of this enzyme. Overall, our work shows the relationship of DNA sequence and methylation to RNA expression, and differences between ancient and modern lineages. Further studies are needed to verify the functional consequences of the identified mechanisms of gene expression regulation.

Abstract Background Mixed, polyclonal Mycobacterium tuberculosis infection occurs in natural populations. Developing an effective method for detecting such cases is important in measuring the success of treatment and reconstruction of transmission between patients. Using whole genome sequence (WGS) data, we assess two methods for detecting mixed infection: (i) a combination of the number of heterozygous sites and the proportion of heterozygous sites to total SNPs, and (ii) Bayesian model-based clustering of allele frequencies from sequencing reads at heterozygous sites. Results In silico and in vitro artificially mixed and known pure M. tuberculosis samples were analysed to determine the specificity and sensitivity of each method. We found that both approaches were effective in distinguishing between pure strains and mixed infection where there was relatively high (> 10%) proportion of a minor strain in the mixture. A large dataset of clinical isolates (n = 1963) from the Karonga Prevention Study in Northern Malawi was tested to examine correlations with patient characteristics and outcomes with mixed infection. The frequency of mixed infection in the population was found to be around 10%, with an association with year of diagnosis, but no association with age, sex, HIV status or previous tuberculosis. Conclusions Mixed Mycobacterium tuberculosis infection was identified in silico using whole genome sequence data. The methods presented here can be applied to population-wide analyses of tuberculosis to estimate the frequency of mixed infection, and to identify individual cases of mixed infections. These cases are important when considering the evolution and transmission of the disease, and in patient treatment.

Background Increased tuberculosis disease burden arises as a result of low treatment success rates stemming from the emergence of second-line drug resistance. We aimed at determining the usefulness of second-line drug (SLD) resistance markers as proxy indicators of time to sputum culture conversion; a renowned predictor of Tuberculosis treatment outcome, among SLD-resistant tuberculosis (TB) patients tested at the Uganda National TB Reference Laboratory (NTRL). Methods A cross-sectional study was conducted on 72 bacteriologically confirmed SLD resistant TB patients with datasets including culture conversion time and second line probe assay mutation profiles between 01/06/2017 and 31/12/2019. The data were then imported into STATA v15 for descriptive statistical analysis, Univariate cox proportional hazard model analysis and Kaplan-Meier survival curves at a 5% level of significance; p-value ≤0.05. Results Results indicate the median time was achieved at 3 (0–12) months across the studied patients. The rrs G1484T mutation associated with conferring drug resistance to injectable agents was observed to have the shortest median conversion time of 1.5 months, longest by the gryB E540D at 5 months. A single mutation in the gryA gene locus showed higher converted proportions 70.8% (58.9–81.0) than those that had two 8.3% (3.1–17.3) or three 2.7% (0.3–10.0) mutations. Conclusions The studied second-line drug resistance markers had no statistically significant association with the time to sputum culture conversion, although increased drug resistance levels reduced the converted proportions and stressed the need to utilize molecular diagnostics data and other crucial variables to better comprehend proxy indicators of SLD resistant tuberculosis management.