Review 2: "Mapping and Sequencing of Cases from an Ongoing Outbreak of Clade Ib Monkeypox Virus in South Kivu, Eastern Democratic Republic of the Congo between September 2023 to June 2024"

RR\ID Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review: This manuscript describes the most recent mpox outbreak in the eastern region of the Democratic Republic of Congo, from September 2023 – June 2024. The authors summarize the historical context and previously observed transmission patterns for the different mpox clades that have emerged. The focus of this manuscript is the outbreak of a Clade 1 sub-lineage in South Kivu, a mining town that lies adjacent to Rwanda, Burundi, and Tanzania. The manuscript provides details with regards to the sociodemographic characteristics of those affected, and distribution of disease amongst consenting persons admitted to a health facility after presenting with signs and symptoms consistent with mpox infection. The study population included 670 hospitalized persons, 96% of whom had PCR confirmed mpox infection. The authors provide important clinical outcomes for persons with severe disease who progressed to death, and though limited in scope as introduction of a standardized clinical case report form was only introduced in the latter part of the study period, provides important insights into the potential higher morbidity among pregnant women and the association of mpox with pregnancy loss. The team identifies potential behavioral factors associated with mpox, with a high proportion of persons with mpox identified to have spent time in a bar, and engaged in sexual activity in that context. The team provides a temporal timeline of the emergence of cases, including temporal mapping within the region. Whole genome sequencing was performed on a subset of participants and sequences with >85% genome coverage (N=58) that was distributed across space and time to allow for molecular characterization. The manuscript is well written and provides valuable insights into this dynamic and ongoing outbreak.

Below are specific recommendations and request for clarification:

1. It would be helpful to the reader to know the total number of persons who were hospitalized with mpox, total approached to enroll in the study, and total who declined to enroll would help to assess generalizability of the findings described. Are there certain subpopulations e.g. miners, that are more likely to be hospitalized? This is important to address as this may be strongly related to the mode of acquisition that is identified in this cohort.

2. The authors should clarify the basis for the decision to hospitalize persons presenting with signs and symptoms consistent with mpox. Are all persons with such a presentation admitted or only those with concern for severe disease?

3. Related to #1 above, it would be helpful to provide further details on the clinical presentation observed – summary statistics related to presence of the various known clinical characteristics would be helpful to include. This may include the proportion with fever, headache, myalgias, sore throat e.t.c.; the proportion of other indicators of disease severity – proportion with lesions >=25% of body surface, airway involvement/compromise, CNS disease, and ocular involvement would be helpful.

4. The authors identify sexual contact in bar settings as a prominent risk factor for disease, and separately provide data on the number of sex workers who work in those bars.

   What proportion of the cases were among professional sex workers? What other occupations were represented among the cases? Providing the actual occupation of mpox cases would be helpful to include especially if there are distinct patterns that emerge that may provide a basis for distribution of preventive services and health communication and education. This is very important to avoid stigmatizing specific sub-populations as this may have long term negative consequences and impede disease control efforts.

5. Are persons with mpox maintained in the health setting from the time of diagnosis until full recovery or are do persons return to the home environment? What infection prevention modalities are communicated to decrease transmission?

6. In the cluster description, there is reference to “Cluster A shows many unique mutations suggesting undetected circulation or circulation outside the Kamituga health zone”. However, the sequences are recovered from samples from detected affected cases. The language should be clarified, perhaps to indicate that there is evidence of shared common ancestor within this cluster linking to cases outside of Kamituga health zone area under study, including within the DRC (Kamanyola) in Cluster A, as well in Kenya, Sweden and Thailand in Cluster A1.

7. In the last paragraph of the results section, the authors present their findings related to the presence of APOBEC3 associated mutations. This reviewer’s understanding is that APOBEC3 mutations are suggestive of immune-mediated evolutionary selection pressure during infection within a host. In this case, these mutations have been maintained in ongoing transmissions. The authors refer to their identifying as “indicative of human-to-human transmission”. Is the suggestion here that there is increased viral fitness and transmissibility associated with these mutations? If that is the assertion, this interpretation is best suited in the discussion with any accompanying literature that supports this interpretation.

8. The authors should clarify the specific concern of potential for under diagnosis of cases related to Clade 1b mpox. The discussion point currently reads as “Genomic analysis shows evidence for considerable underdetection”. The point could be strengthened by  the authors explicitly stating that the presented genomic analysis identifies linkages and shared common ancestors outside of the immediate geographic area, and serves as notice of the importance of activating a public health response to ensure early identification, detection, prevention and treatment.