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Rates of bacterial sexually transmitted infections (STIs) are rising, and accessing their genomes provides information on strain evolution, circulating strains and encoded antimicrobial resistance (AMR). Notable pathogens include Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP), globally the most common bacterial STIs. Mycoplasmoides (formerly Mycoplasma ) genitalium (MG) is also a bacterial STI that is of concern due to AMR development. These bacteria are also fastidious or hard to culture, and standard sampling methods lyse bacteria, completely preventing pathogen culture. Clinical samples contain large amounts of human and other microbiota DNA. These factors hinder the sequencing of bacterial STI genomes. We aimed to overcome these challenges in obtaining whole-genome sequences and evaluated four approaches using clinical samples from Argentina (39), and Switzerland (14), and cultured samples from Finland (2) and Argentina (1). First, direct genome sequencing from swab samples was attempted through Illumina deep metagenomic sequencing, showing extremely low levels of target DNA, with under 0.01% of the sequenced reads being from the target pathogens. Second, host DNA depletion followed by Illumina sequencing was not found to produce enrichment in these very low-load samples. Third, we tried a selective long-read approach with the new adaptive sequencing from Oxford Nanopore Technologies, which also did not improve enrichment sufficiently to provide genomic information. Finally, target enrichment using a novel pan-genome set of custom SureSelect probes targeting CT, NG, TP and MG followed by Illumina sequencing was successful. We produced whole genomes from 64% of CT-positive samples, from 36% of NG-positive samples and 60% of TP-positive samples. Additionally, we enriched MG DNA to gain partial genomes from 60% of samples. This is the first publication to date to utilize a pan-genome STI panel in target enrichment. Target enrichment, though costly, proved essential for obtaining genomic data from clinical samples. These data can be utilized to examine circulating strains and genotypic resistance and guide public health strategies.

Whole-genome analysis has provided insights into the evolution of Chlamydia trachomatis and, recently, into circulating strains that cause lymphogranuloma venereum (LGV). A large LGV outbreak of a new ompA -genotype, L2b, was first reported in Europe in the early 2000s, primarily affecting men who have sex with men (MSM), and then expanded globally. More recent work shows that this outbreak is diversifying into variants of described ompA -genotypes, with the same L2b genomic backbone. This study extends the investigation of LGV cases to Argentina and Finland. In 2017, an LGV outbreak was described in Argentina characterized by distinct genomic features shown by both ompA -genotyping and Multi-Locus Sequence Typing (MLST) analysis. We have obtained whole-genome sequences from cultured isolates and clinical samples via SureSelect (Agilent) target enrichment. Based on ompA and phylogenetic analyses, we describe further diversity within the ompA -genotype L2b clade, illustrating the transmission dynamics in Argentina and Finland. A key finding is that of a novel clade of Argentinian samples, characterized by a proposed new ompA -genotype L4. Additionally, we present the genome sequence of a non-LGV strain associated with anorectal proctitis. These findings contribute to the investigation of LGV evolution, particularly with the presence of the novel L4 lineage, and provide insights into genomic diversity and transmission dynamics of C. trachomatis .

During the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent-something that is unknown at the time of sequencing-and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses., Competing Interests: The authors declare no conflict of interest.