Direct sequencing technologies for bacterial sexually transmitted infections

Background

Over the last 10 years, genomics, particularly next-generation sequencing (NGS), has become a crucial approach for the monitoring and diagnosing of infectious diseases. It plays a vital role in enabling high-resolution surveillance, assisting with outbreak identification and investigation, facilitating contact tracing through the identification of transmission pathways, and enabling antimicrobial resistance (AMR) target detection.^1,2

However traditional microbial genomics is not a perfect fit for all bacterial pathogens, as it has typically been based on sequencing from a cultured isolate. In some cases, culturing of patient samples can prove time consuming and technically demanding, especially for sexually transmitted pathogens that might be fastidious or not readily culturable. Moreover, as diagnostic testing methods are increasingly shifting away from culture-based approaches to molecular testing direct on specimen and rapid point-of-care testing, there is a noticeable decline in the production of culture isolates.³

Although innovative culture-independent diagnostic approaches offer many advantages to patients and clinicians, dropping culture rates or an inability to culture can create gaps in disease surveillance including typing and AMR monitoring. Culturing for bacterial sexually transmitted infections (STIs) can be particularly challenging in remote areas meaning treatment surveillance data can be heavily skewed by urban cases. Problematically, some of the most overrepresented populations in Australian STI notifications are located in regional and remote areas.⁴ Therefore, it is increasingly important to develop surveillance approaches that are suitable for testing directly on patient specimens.

Metagenomic (direct sequencing) approaches have the potential to provide clinical and public health information directly from bacterial STI-positive patient samples. Although several challenges remain in integrating these methods into the routine testing of STIs in laboratories, particularly regarding cost, turnaround time and the need for suitable workflows and bioinformatic solutions, there are a number of promising approaches already developed for a range of pathogens.⁵

Neisseria gonorrhoeae

Neisseria gonorrhoeae is a major public health concern, with some lineages developing resistance to the last line of antibiotics used empirically to treat gonorrhoea.^6–10 Despite global and Australian reports of N. gonorrhoeae isolates with reduced susceptibility to ceftriaxone and resistance to azithromycin, the standard treatment for gonorrhea in Australia remains a combination of injectable ceftriaxone and oral azithromycin.¹¹ In response to increasing AMR infections, the absence of an effective vaccine and the lack of newly developed antibiotics classes, the World Health Organization (WHO) added N. gonorrhoeae to the list of high-priority pathogens.¹²

Phenotypic methods such as culture are still used for the diagnosis and AMR profiling of this pathogen, enabling genomics and population-level studies of N. gonorrhoeae diversity to occur. However, due to the high notification rate, most public health laboratories cannot afford to sequence all cultures, making routine comprehensive molecular surveillance uncommon. Molecular-based techniques on patient samples are the most widely used for diagnosing gonorrhoea (80% of notifications in Australia). However, these techniques require additional, often bespoke, molecular assays to detect AMR-associated mutations and associated genes or sequencing based typing schemes, which are costly and labour intensive.

Advanced, culture-independent methods like metagenomic approaches offer significant advantages for simultaneous detection of AMR genes and mutations, virulence genes, and strain typing in real time. This approach has the potential to revolutionise the treatment of gonorrhoea by guiding targeted therapies more effectively even in regions where culture is too challenging.¹³ Several studies have successfully yielded AMR and typing information from N. gonorrhoeae-positive samples without the need for culture by using metagenomic approaches such as shotgun sequencing or capture hybridisation methods (Fig. 1).^14,15 Additionally, amplicon-based techniques might also prove useful and has the potential to be particularly cost effective.¹⁶

Fig. 1.

Flowchart demonstrating the different workflows available for direct sequencing of STI-positive specimens. Common workflow is shown by grey arrows. Amplicon based sequencing steps have green arrows. Shotgun WGS has red arrows and enrichment-based sequencing is shown with purple arrows. This figure was created with Biorender.com.

Treponema pallidum

Syphilis, caused by Treponema pallidum, has re-emerged as a significant threat to public health. In 2020, there were 7.1 million new reported cases worldwide.¹⁷ Notably, in high-income regions such as Australia, heterosexual populations are now being affected after decades of low infection rates. Concerningly, congenital syphilis cases are rising across many countries, with a 937% rise in congenital syphilis cases in the USA and 358% increase in Australia over the past decade.^17,18 Groups particularly vulnerable to syphilis include men who have sex with men (MSM) and Aboriginal and Torres Strait Islander communities.^17,19

Syphilis offers significant diagnostic challenges as it is extremely difficult to culture. Diagnosis primarily relies on serological testing, with multiple tests needed to differentiate between active and past infections, a process that can be particularly challenging in remote areas. Serological testing for syphilis is negative in ~20% of primary syphilis cases. Nucleic-acid amplification tests (NAATs) such as quantitative polymerase chain reaction (qPCR) are uncommon but increasingly encouraged to improve the diagnosis of primary syphilis.²⁰

In the absence of culture, genomic methods for T. pallidum have had to focus on metagenomic approaches. Enrichment-based whole-genome sequencing (WGS) has been successful in generating molecular-typing information able to inform on the emergence and evolution of T. pallidum, revealing the co-circulation of two major lineages (Nichols and SS14) across continents.²¹ This expansion aligns with the global resurgence of syphilis. In Australia, targeted whole-genome enrichment sequencing has also been applied to clinical T. pallidum samples, able to provide insights into the contemporary re-emergence of syphilis in Australia, revealing not only that multiple circulating strains linked to globally disseminated lineages are responsible for the epidemic, but that particular sub-lineages were associated with MSM and heterosexual groups.¹⁸

Mycoplasma genitalium

There has been a rise in Mycoplasma genitalium cases over the past decade, particularly among high-risk demographics, with reported incidence ranging from 5 to 20%.²² This surge coincides with a rise in infections resistant to treatment, leading to M. genitalium named as an emerging concern in STI treatment guidelines by the US Centers for Disease Control and Prevention (CDC).²³ The incidence of macrolide resistance has surged to 50% and the efficacy of fluoroquinolones has declined across urban Australia.²⁴ Understanding the AMR profile of M. genitalium is essential for delivering effective patient care and curbing the emergence of highly resistant strains.

Mycoplasma genitalium is highly fastidious, presenting a major hurdle in obtaining cultured isolates. Consequently, only a small number of isolates have been sequenced and made publicly available, leading to a limited understanding of the diversity and mechanisms of AMR in this organism.²⁵ Presently, epidemiological studies mainly rely on individual molecular assays that target specific genes (such as resistance genes) or regions of the genome with high diversity to glean insights. Therefore, a method enabling genomic sequencing of M. genitalium directly from clinical samples would be ideal, offering a valuable tool to enhance our comprehension of the population structure of this organism.

Sequencing methods that incorporate enrichment strategies, where for example sequence specific baits are used to pull out M. genitalium-specific DNA over host and other DNA in the sample, present a promising approach to yield sufficient sequence information from M. genitalium-positive samples (Fig. 1). Preliminary data generated at the Doherty Institute, in collaboration with the Melbourne Sexual Health Centre, suggest that enrichment sequencing is a feasible approach to overcoming limited culturing capacity and provide information on prevalence and distribution of resistance mechanisms (F. Azzato, G. Taiaroa, J. Fernando, V. De Petra, M. Taouk, L. Vodstrcil, E. Plummer, S. Chea, S. Paricha, K. Raios, L. Caly, B. P. Howden, C. Bradshaw and D. A. Willamson, in prep.). Targeted amplicon-type approaches have also been shown to be successful in yielding sequences data on AMR-associated targets in M. genitalium-positive patient specimens.^26,27

Chlamydia trachomatis

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in females.²⁸ Although classified as a bacterium, C. trachomatis will only grow intracellularly, similar to viruses. For this reason, culture of C. trachomatis is difficult, requiring time-consuming cell-culture methods. Instead, diagnosis of infection must focus on alternative methods including antigen tests, NAATs and occasionally serology. In the absence of high numbers of isolates, typing and phylogenetic studies using genomics have been performed using metagenomic shotgun WGS.^29,30

Interestingly, shotgun metagenomic approaches offer additional benefits for C. trachomatis infections where sequencing all genetic material present in the patient sample has increased understanding of co-infections, whereby C. trachomatis may increase susceptibility to human papillomavirus (HPV) infection and play a role in viral persistence.³¹ Alternatively, studies utilising enrichment steps have been reported 10-fold higher sensitivity in generating C. trachomatis sequences, which, although more expensive, does increase the likelihood of yielding successful results.³² More recently, a primer-based amplification approach has been reported for C. trachomatis that has the potential to provide a cost-effective and sensitive approach to generating genomic sequences directly from clinical samples.³³

Microbiome

Shotgun metagenomics offers the ability to generate a comprehensive analysis of all bacterial species present in a sample, which will allow for insights into mixed infections involving either multiple strains of a single pathogen or multiple pathogen species. Such studies show promise in assisting in identifying the role of different bacteria in STIs, and will potentially leading to more sophisticated diagnostic and treatment strategies, and ultimately a deeper understanding of the genital microbiome through metagenomics may contribute to unravelling complex conditions such as bacterial vaginosis.^34,35

Summary

Direct sequencing presents a variety of exciting solutions to challenges in the STI public health space, including typing, AMR tracking and surveillance. However, several obstacles hinder routine adoption in public health and clinical microbiology laboratories. Cost remains a significant barrier, particularly for baiting approaches, necessitating ongoing efforts to reduce expenses. Additionally, bioinformatic tools must be optimised for accessibility and efficiency, and laboratory workflows need automation to handle large sample volumes efficiently, with reporting tailored to end-user needs for AMR and phylogenetic data. Ethical considerations must also be adequately addressed, especially regarding incidental findings and patient privacy and confidentiality raised by methods that sequence host DNA as well as pathogen DNA. Establishing robust protocols for patient de-identification, data security and handling incidental findings is imperative.

Despite these challenges to routine implementation, the future is promising for direct sequencing technologies addressing the current gaps in typing and AMR surveillance of under-cultured and unculturable STI pathogens.