Mpox diagnostics: a swift and integrated approach to outbreak control

2022 mpox outbreaks reveal diagnostic gaps

Prior to 2022, monkeypox (mpox) was largely considered a zoonotic pathogen. Infections were predominantly described from the African continent with occasional overspill events as a result of contact between humans and small rodents.¹ The global transmissions of mpox in 2022, manifested predominately through close or sexual contacts among men who have sex with men was unanticipated.² Owing to the lack of commercial assays and accessibility to control materials (containment requirements), diagnoses of mpox at the early stage of the outbreak were restricted to specialised laboratories.

As part of Australia’s outbreak response, literature review and mapping of existing diagnostic capabilities were conducted.³ It highlighted several challenges in the control of mpox, for example, paucity of evidence on viral shedding dynamics and infectivity, the extent of serological cross-reactivity, absence of rapid diagnostics and a lack of genomic characterisation to inform transmission linkages.

Diagnostics strategies for mpox control

For early diagnosis and control of mpox, a centralised high-throughput testing in a reference laboratory was considered the preferred approach initially. This was carefully balanced against biosafety considerations, readiness of multi-targeted mpox assays to minimise diagnostic escape, capabilities to conduct viral culture and genomic characterisation and better coordinated responses. This approach was effective in the eradication of mpox from Australia across multiple states. As mpox continues to spread across the globe in 2023–24, diagnostic strategies evolved to focus on early detection of symptomatic returned travellers to prevent local spread. As eradication remains the priority of the disease control strategy in Australia, assays with high sensitivity, specificity and rapid turnaround time are critical.

Evaluation of viral dynamics and shedding

As mpox shedding dynamics were poorly described during the early stage of the outbreak, studies conducted to evaluate the viral shedding patterns were particularly important in informing testing and control strategies.^4–8 Multiple body site sampling tests were conducted in the Victorian Infectious Diseases Reference Laboratory (VIDRL) in partnership with Melbourne Sexual Health Centre (MSHC) to evaluate monkeypox virus (MPXV) shedding patterns, viral loads and infectivity of different samples types.⁹ Virus shedding was observed from multiple body sites; in symptomatic individuals, swabs obtained from involved sites (vesicles, anorectal) have the highest yield whereas oropharyngeal swabs were more useful for pre-symptomatic individuals.^9,10 This information was important in guiding public health policy in testing high exposure risk individuals. In parallel infectivity studies demonstrated viability of MPXV in oral and anal sites, indicating that abstinence from close physical and sexual contact for a longer period of time is required to prevent transmission.¹⁰

Rapid multi-target assay in vesicular diseases diagnosis

Early confirmation of mpox and exclusion of other causes of vesicular diseases are both important in facilitating clinical assessment of suspicious cases. Evaluation of QIAStat-Dx viral vesicular panel (MPXV clades I & II, HSV1, HSV2, HHV-6, VZV, EV), a rapid 1-h multiplexed assay developed by Qiagen, was evaluated in parallel with the reference PCR at VIDRL. The assays demonstrated high level of concordance without cross-reactivity with other viruses from the Poxviridae family (vaccinia, orf, molluscum contagiosum viruses).¹¹ This is one of the earliest studies to inform rapid assay performance and provided an option for rapid diagnosis at primary clinician testing centres. The assessment could serve as an independent review of assay performance to facilitate regulatory approvals.

Evaluation of high-throughput PCR assays

Most of the currently available molecular assays for mpox are either real-time PCR or loop mediated isothermal amplification (LAMP)-based assays, targeting conserved central coding regions (e.g. E9L_NVAR, B6R, F3L).^12,13 The National High Security Quarantine Laboratory at VIDRL serves as a reference laboratory for smallpox and orthopoxvirus by performing culture, distributing inactivated control materials and evaluating the analytical performance of high-throughput molecular assay to support diagnostic laboratories. A study comparing published primers and probes from VIDRL, the US Centers for Disease Control and Prevention (CDC) and US Army Medical Research Institute of Infectious Diseases (USAMRIID) with 13 other commercial PCR kits demonstrated good concordance between PCR assays, although significant variability in limits of detection (LODs) was observed for a few assays (LODs between 57 and 14,495 copies mL^–1).¹⁴ Interestingly, a few MPXV-specific assays non-specifically detected vaccinia virus. These data suggest that variabilities in assay sensitivity and specificity should be taken into consideration in testing strategies, in particular for the detection of MPXV in non-vesicular samples (e.g. urine, semen, blood), where viral loads can be low or variable. The study findings help support the development of WHO mpox laboratory testing policy.¹⁵

Application of mpox genomics for surveillance

In recent years, public health authorities have begun relying heavily on genomic surveillance to assist with epidemiological linkages. However, it is often challenging to determine the threshold for genomic linkages in emerging pathogens without prior validation with epidemiological data. Whole-genome sequencing of MPXV was performed from multiple body sites using sample cohorts collected at MSHC. The presence of different subclades of MPXV in some individuals suggested potential complex transmission chains.¹⁶ Additionally, the presence of large deletions and elevated number of mutations in the MPXV genome (due to human APOBEC3 enzyme editing activity) provides evidence that MPXV is continuously evolving and diversifying during human-to-human transmissions. The lack of geographical representative genomic data poses a challenge in using genomics for case linkages. Despite this, there are a number of public health applications in mpox genomics, for example (i) global contextualisation to determine origin of case importation,¹⁷ (ii) assessment for diagnostic and potentially therapeutic or vaccine escape ¹⁸ and (iii) excluding case or cluster linkages. Most importantly, the early genomic characterisation of local clusters continues to guide public health actions based on scientific evidence.

Cutting-edge CRISPR technology for rapid PoCTs

Despite the availability of good commercial mpox PCR assays,¹⁴ because of the lack of endemicity in Australia, few of these tests have undergone regulatory approval. For an effective control of sexually transmitted infection (STI) outbreaks, investment in capability to develop in-house diagnostic assays, especially good quality point-of-care tests (PoCTs) is necessary. Advancement in CRISPR-based diagnostics have catalysed the development of DNA- or RNA-targeting assays that display high sensitivity and specificity, high portability, rapid turnaround, and require minimal manipulation (Fig. 1). Owing to the highly programmable nature of the CRISPR system, these assays can be designed in-house, with the flexibility to be rapidly modified in the event of diagnostic escape.^19–22 However, complexed computational capability is required for high-throughput primer and guide RNA design, essential for target amplification and the CRISPR assay (Fig. 1).

Fig. 1.

CRISPR Diagnostic Platforms – SHERLOCK and DETECTR. (a) Key steps in CRISPR diagnostic assay design using the pangenome approach in PathoGD. (b) Two CRISPR diagnostic systems. In the SHERLOCK system, target DNA or RNA are amplified with recombinase polymerase amplification (RPA) and RT-RPA (reverse transcription RPA) respectively. An in vitro transcription (IVT) step with T7 polymerase converts DNA into RNA for detection with Cas13. Binding of the gRNA specifically designed to complement the target sequence triggers the collateral cleavage of quenched fluorescent reporters by the Cas13 endonuclease. The reporter system can be adapted to different readouts including lateral flow assay (LFA), fluorescence, next-generation sequencing (NGS) or electrochemical detection. In the DETECTR system, a similar approach is applied using Cas12a endonuclease for the detection of DNA.

Recently, PathoGD, a bioinformatic pipeline for high-throughput recombinase polymerase amplification (RPA) primer and guide RNA design has been developed, which allows for rapid design of CRISPR–Cas12a-based diagnostic assays.²³ This automated, end-to-end computational tool is a major developmental milestone, as it enables the assay to be developed in diagnostic laboratories with minimal bioinformatic capabilities.²⁴

During the 2022 mpox outbreak, several assays incorporating CRISPR technology were developed for the rapid detection of MPXV, including one in Australia.²⁵ This CRISPR–Cas12a based assay was the first Australian assay, designed for applications in the field (lateral flow readout) and laboratory (fluorescent readout). This assay enabled the detection of MPXV within 45 min with 100% specificity and a LOD comparable to real-time PCR (1 copy μL^–1). This assay was subsequently streamlined to a portable heating instrument, allowing deployment outside of the laboratory (Fig. 2).

Fig. 2.

Application of CRISPR diagnostic assay in disease outbreak investigation.

Summary

As mpox continues to spread in 2024, repeated importation of cases is posing a significant challenge to disease control in Australia. Reference laboratories play a significant role in early characterisation of infectivity, viral dynamics, genomics and distribution of control materials. As the outbreaks continue to evolve, in-house capability to develop innovative rapid diagnostic assays becomes important in preventing endemicity and reducing reliance on commercial assays. Investing in computational infrastructure and CRISPR diagnostics not only provides flexibility in responding to outbreaks and tracking diagnostic escape in viruses, but could also serve as a cost-effective public health strategy for controlling STIs.