Case-based and wastewater surveillance for mpox

Authors: Modjadji, WARN-ID, and SEARCH; Email: data@modjadji.info

Funded, in part, by: The Bill & Melinda Gates Foundation and the U.S. National Institutes of Health

Version: 1.0

Created: 29 Aug 2024

Updated: 19 Sep 2024

License: Creative Commons Attribution 4.0 International

Purpose of surveillance

Case- and environmental/wastewater-based surveillance play distinct, but overlapping and complementary roles. Case-based surveillance at clinics works at the level of individuals and is typically focused on diagnosing patients and, via reporting mechanisms, can serve as a passive surveillance tool to understand the presence or absence of infectious diseases in a community. Wastewater-based surveillance works at the population level and can serve several roles, including the (1) detection of pathogens, (2) measurement of disease prevalence, and the (3) study of disease dynamics in the community over time.

For mpox surveillance, both case- and wastewater-based surveillance should be considered. Molecular tools, such as quantitative PCR (qPCR) and droplet digital PCR (ddPCR) can be used for both surveillance modalities, including for diagnostics in a case-based (clinical) setting and detection, and for viral load estimation (burden) and longitudinal studies of mpox dynamics in a wastewater setting. Given the multiple “clades” of mpox virus (MPXV; primarily “Ia” and “Ib”, prevalent in e.g., Central Africa, and “IIb”, prevalent in West Africa and responsible for the 2022 global outbreak), specific qPCR and ddPCR assays have been developed to aide in the “typing” of MPXV into its respective clades. However, this requires multiple independent assays, is poorly scalable, and vulnerable to drop-out, as we have recently seen with Clade Ib. Therefore, whenever possible and as described below, we propose to perform typing and more thorough characterization using sequencing-based assays.

The importance of MPXV sequencing

In addition to qPCR and ddPCR, as described above, sequencing of MPXV should be considered for mpox surveillance. Obtaining MPXV genomic sequences over time will enrich the utility of any surveillance-based network, including giving us information about (1) the presence, absence, and emergence of potential viral deletions, mutations of interest, including SNPs that may “tag” potential clades, sub-clades, and lineages of interest, (2) typing of MPXV into clades, sub-clades, and lineages, which will be especially important in a situation where multiple clades may be co-circulating, and (3) genomic epidemiological studies to investigate the evolution, spread, and emergence of MPXV and it’s clades, sub-clades, and lineages over time.

Given the relatively large size (~200kb) of the DNA-based genome of MPXV, we recommend organizations and labs engaged in mpox surveillance consider separate strategies for case- and wastewater-based sequencing. For case-based sequencing, we propose that organizations consider full-genome sequencing of MPXV using targeted amplicon-based sequencing covering the whole MPXV DNA genome (see, guide from ARTIC Network). Metagenomic (untargeted) or probe-based enrichment (semi-targeted) methods can also be used, but are generally cost-intensive. By obtaining full-length genomic sequences, we can (1) gain a rich understanding of the evolution, spread and dynamics of MPXV over time, (2) detect novel mutations, insertions, and deletions of interest, (3) distinguish zoonotic vs human-to-human transmission chains, and (4) provide fine-grained insights into transmission networks of multiple MPXV clades, sub-clades, and lineages, whether co-circulating in the same community or across multiple different communities.

For wastewater-based sequencing, we propose that organizations and labs consider using a focused (single-tube) panel for targeted amplicon-based sequencing that can be performed on commonly available platforms, including Illumina and Nanopore/MinION. This assay only captures a fraction of the MPXV genome, however, given the limited genetic diversity, slower rate of evolution, and lower levels of the virus in wastewater compared to e.g., SARS-CoV-2, we believe this is most sensitive and cost-effective way to type MPXV clades and obtain better data on the spread and evolutionary dynamics of MPXV from wastewater sources, especially in areas where co-circulation of clades may occur. By performing focused sequencing of MPXV material present in wastewater, it is possible to (1) type clades, sub-clades, and potential lineages of interest in the community and (2) obtain in-depth information on the co-circulation and dynamics of MPXV, including insights into transmission networks in the community. Given the flexibility of the platform, should a novel mutation of interest (e.g., a “tagging” SNP identifying a potential lineage or key adaptive mutation) be identified via case-based sequencing, the focused amplicon panel can be rapidly updated to target new parts of the MPXV genome over time. Compared to e.g., the usage of qPCR and/or ddPCR platforms for “tagging” SNPs of interest, focused amplicon-based sequencing will provide a much more flexible, adaptable, and extendable platform once introduced as part of a surveillance workflow.

Assays for wastewater surveillance

Both “molecular” and “sequencing” based tools should be considered for wastewater-based surveillance of mpox. Molecular tools, like qPCR- and ddPCR-based assays, should be used for early detection of mpox soon after it enters a community, as well as to quantify the burden (load) of disease in a population. While qPCR and ddPCR assays can be used for“typing” of MPXV clades, focused amplicon-based sequencing, allows for more fine-grained information and provides a more flexible and adaptable framework that can be expanded and modified over time, if necessary. This becomes especially important in situations in which different ‘types’ of MPXVs may be circulating in the same community (e.g., Clades Ib and IIb).

Molecular assays for detection and burden of mpox in the community

Adding ddPCR- and/or qPCR-based assays for the detection of MPXV in wastewater should be possible as a relatively simple extension of existing COVID-19 (or other) environmental surveillance programs (Table 1). For example, in San Diego, we added the general MPXV G2R_G qPCR assay developed by Li et al. to our existing COVID-19 wastewater surveillance program. This work, in response to the 2022 multi-country outbreak of Clade IIb MPXV, allowed for detection of mpox shortly after it was present in the community and tracking of burden and growth dynamics over time. In South Africa, NICD has optimized a ddPCR method described by Fan et al in their routine wastewater surveillance for SARS-CoV-2 and other pathogens.

Assays for pan-MPXV detection and burden estimation

To detect MPXV in wastewater samples, we utilize specific primers and probes (listed here) that target conserved and distinctive regions of the MPXV genome. Generic qPCR and ddPCR assays are employed to detect MPXV and to monitor the viral load over time (Table 1). We include crAssphage and Pepper Mild Mottle Virus (PMMoV), viral markers prevalent in human feces, to enable normalization between samples collected from the same wastewater treatment plant and to account for variations in the amount of human fecal matter present in the wastewater, thereby improving the accuracy of viral quantification over time. We are currently successfully using PMMoV as a fecal marker in San Diego and in South Africa, but due to differences in food consumption this might vary across locations. A detailed qPCR protocol is available here.

Assays for typing of MPXV clades Ia, Ib, and IIb

Differentiating between MPXV clades Ia, Ib, and IIb requires additional clade-specific ddPCR or qPCR assays (Table 2), as generic assays, as described above, will detect all clades and cannot effectively distinguish between them. Clade-specific assays target insertions/deletions that are unique for a particular clade, thereby providing high specificity for a particular clade. This specificity is especially important in wastewater surveillance in locations where multiple clades may co-circulate, or in regions where mpox was introduced for the first time, or re-merged after a long time of absence. PCR-based assays have a quick turn-around time, are affordable, and require relatively simple lab equipment. However, they are unable to differentiate between multiple introductions or distinct transmission chains belonging to the same clade. In such cases, focused sequencing is required to resolve finer-scale epidemiological questions and track virus evolution.

As with the generic qPCR assays, markers like crAssPhage and PMMoV are used for normalization to account for variations in viral concentration across samples and time. This normalization ensures that trends in MPXV detection reflect true changes in viral prevalence rather than technical or sampling differences. For those seeking to implement this approach, detailed protocols, including primer sequences for clade-specific assays, can be found here.

Sequencing assay for typing, co-circulation, and network dynamics of mpox in the community

Viruses tend to be present at far lower concentrations in wastewater than in clinical specimens, and genomes are often highly fragmented. As such, sequencing approaches requiring long amplicons will generally fail, and smaller amplicons are needed to ensure reliable amplification. Limiting both the number of amplicons and amplicon size (225-275 bp) ensures that the sequencing assay is optimized for higher sensitivity (Table 3). Such a focused sequencing approach allows for the detection of genomic regions that are essential for clade, subclade, and lineage identification, which is crucial for understanding the emergence and spread of MPXV.

By using focused amplicon sequencing it is possible to differentiate between MPXV Clades I and II, identify sub-clades (e.g. Clade Ib vs Clade IIb), and even lineages within Clade IIb. The use of tagging SNPs, specific to different clades or lineages, allows for the detection of co-circulating lineages and mutations of interest that might emerge during outbreaks. Furthermore, as case-based sequencing identifies additional mutations of interest, e.g., associated with increased disease severity or transmission advantages, these markers can easily be incorporated into the amplicon schemes, enhancing the method’s utility over time.

Importantly, this approach is cost-effective and flexible, making it suitable for low-resource settings where extensive genomic surveillance may not be feasible. Although not as affordable as clade-based qPCR assays, focused amplicon sequencing provides much higher resolution and is highly scalable using commonly available sequencing instruments and resources.

Design and validation of a focused amplicon scheme for MPXV wastewater sequencing

To enable typing and tracking of MPXV directly from wastewater sources, we developed a focused amplicon-based sequencing using a minimal barcode set for MPXV, which provides an efficient method for identifying viral clades, subclades, and lineages (Table 3). To ensure that the full diversity of MPXV lineages is captured, we extracted a comprehensive set of barcode sequences from the Nextstrain phylogenetic tree. We selected 45 key non-homoplasic nucleotide sites to discriminate between all clades, subclades and lineages. This non-redundant set of 45 nucleotide positions forms the foundation of the amplicon design. A detailed overview of the process of how these sites were selected can be found here.

Next, we targeted conserved regions flanking these key nucleotide sites, resulting in a total of 40 amplicons that cover approximately 10% of the MPXV genome and all necessary genetic markers for clade, sub-clade, and lineage identification. The use of conserved regions allows the primers to remain effective across a wide variety of MPXV strains. In combination with a short amplicon design, ranging from 225 to 275 bp, this maximizes sensitivity, particularly in degraded or low-quality samples like those often encountered in wastewater sequencing. This focused amplicon scheme requires less than 100,000 reads per sample and greatly enhances both the cost-effectiveness and scalability of MPXV wastewater sequencing, making it accessible to a broader range of sequencers and public health laboratories. Full details of the protocol, including primer sequences and amplification conditions, can be found here.

As an initial validation step, we validated the non-redundant barcode set using in silico simulations, demonstrating that all known MPXV clades, subclades, and lineages can be reliably identified with Freyja (see below). Freyja MPXV barcodes to delineate mixed mpox samples are available here. By focusing on a minimal barcode set, our approach balances sensitivity with practicality, reducing the need for a complex and extensive primer library while maintaining high-resolution tracking of viral diversity. Further validations are currently on-going and protocols and repositories will be updated accordingly.

Clade typing and analyzed of MPXV sequencing data using Freyja

To analyze MPXV sequencing data from wastewater, our bioinformatics tool Freyja leverages unique clade, sub-clade (e.g., Ia, Ib, IIb), and lineage (e.g., B.1) mutational barcodes. These barcodes are automatically derived and updated in real-time via phylogenetic trees and annotations available through leading community resources, including Nextstrain. Using these barcodes, Freyja can infer clade and lineage prevalence from wastewater samples, tracking of virus evolution and spread/network dynamics within communities, along with estimation of associated clade and lineage growth parameters. This allows for integration of wastewater and case-based sequencing data for outbreak analyses. Prevalence estimates from wastewater can be spatially resolved and compared with case-based surveillance in matched regions, providing key genomic epidemiology information to identify and track outbreaks and guide public health responses.

Case-based surveillance using targeted amplicon-based sequencing

Case-based surveillance using targeted amplicon-based sequencing is an essential tool for tracking the evolution and spread of viruses, including MPXV. Unlike focused amplicon sequencing for wastewater, full-length genome sequencing requires larger amplicons to span the entire MPXV genome, typically around 2,000-3,000 bp (See detailed guide for case-based sequencing from the ARTIC Network here). Full-length genome sequencing is critically important to identify the emergence of novel mutations that may not be captured by focused amplicon schemes, and enables more detailed genomic-based investigations. When critical new mutations are identified during case-based surveillance, we can expand the focused amplicon scheme with additional amplicons, ensuring that novel mutations can be tracked in wastewater as well.

Compendium of relevant links and resources

Molecular Testing Guidance Document from Africa CDC

Protocol for MPXV detection by qPCR in wastewater

Protocol for MPXV sequencing using focused amplicon sequencing

MPXV primer sequences

MPXV focused amplicon scheme design

Freyja software

Freyja MPXV barcodes

NextStrain phylogenetic trees

Case-based amplicon sequencing guide from the ARTIC Network