Beyond Foodborne HAV: Sexual Transmission Drives a New Wave of Cases in Romania

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments:

This study is highly relevant for advancing knowledge on hepatitis A virus (HAV) transmission in the adult population. However, although the statistical analysis subsection is clearly and well written, the manuscript does not adequately clarify how the exposure groups were defined and constructed.

 

Methodology:

Beyond the eligibility criterion of age ≥18 years, were there any additional restrictions for inclusion in the study? This question arises from the statement: “Fecal–oral transmission was considered for all patients, either by sexual practices (MSM) or by contaminated food”, as MSM refers to a predominantly male population. Additionally, the analysis included 191 unvaccinated individuals; however, it is unclear whether lack of vaccination was an observed characteristic of the study population or an explicit inclusion criterion.

The study population is defined on the basis of a hospital diagnosis of acute HAV infection, rather than on sexual orientation or sexual behavior, and this distinction should be made explicit from the beginning of the manuscript.

Regarding case definition, it is not clear whether individuals were included if they met at least one of the diagnostic criteria or if fulfillment of all criteria was required.

Conceptually, the exposure groups (MSM vs. FBT) do not appear to be mutually exclusive. All patients were assumed to share the same biological mechanism of transmission (fecal–oral), and the distinction lies in the context of exposure. Thus, the groups more appropriately represent different fecal–oral exposure contexts: sexual exposure, foodborne exposure, and a third category that is not addressed in the manuscript—mixed or indeterminate exposure.

Furthermore, it is not clearly described how each exposure category was operationally defined. The manuscript suggests that only two groups were considered (FBT and MSM), and that individuals identified as MSM were automatically classified into the MSM group, as if sexual behavior alone determined the transmission route. This approach raises concerns about potential exposure misclassification and oversimplification of transmission dynamics.

Author Response

Dear Reviewer,

Thank you for evaluating our work and for your constructive comments. We did our best to improve our manuscript!

Reviewer Overall Comment: This study is highly relevant for advancing knowledge on hepatitis A virus (HAV) transmission in the adult population. However, although the statistical analysis subsection is clearly and well written, the manuscript does not adequately clarify how the exposure groups were defined and constructed.

Authors' Response: Thank you very much for your positive assessment of the study’s relevance and for acknowledging the clarity of the statistical analysis. We appreciate your careful reading and agree that additional clarification regarding the definition and construction of the exposure groups was needed. We have revised the Methods section accordingly to ensure that these aspects are now clearly and transparently described.

Comments and answers:

Methodology:

Comment #1

Beyond the eligibility criterion of age ≥18 years, were there any additional restrictions for inclusion in the study? This question arises from the statement: “Fecal–oral transmission was considered for all patients, either by sexual practices (MSM) or by contaminated food”, as MSM refers to a predominantly male population. Additionally, the analysis included 191 unvaccinated individuals; however, it is unclear whether lack of vaccination was an observed characteristic of the study population or an explicit inclusion criterion.

Response #1 Inclusion criteria were age of at least 18 years and diagnosis of HAV, based on the case definition described in the Methods section. In all these patients diagnosed with HAV based on this definition, we identified the fecal-oral mode of transmission, either by sexual practices or by contaminated food. Males from the group with fecal-oral transmission due to sexual practices recognized high-risk sexual behavior (oral sex), and some of them had an epidemiological link. In this period, we noticed a high incidence of hepatitis A in MSM, in a shorter period of time, which was not the case in previous years. Some cases were also diagnosed with HIV or with STIs when admitted for hepatitis A, which confirmed their high-risk behaviors again. Males from the food-borne transmission were also asked about sexual practices, but they denied high-risk behavior. Similarly, a lack of vaccination was observed among the study population.

Thank you for this question. The only inclusion criteria for the study were age ≥18 years and a diagnosis of acute hepatitis A, as defined in the Methods section. No additional restrictions were applied. All patients meeting this definition were included, regardless of sex, gender, or presumed exposure context.

For all included cases, the mode of transmission was classified as fecal–oral based on epidemiological and behavioral information obtained during clinical interviews. Individuals reporting sexual practices with male partners were classified in the MSM group. During the study period, we observed a marked increase in hepatitis A cases among MSM, occurring over a relatively short interval compared with previous years. Several MSM patients reported high‑risk sexual behaviors (including oral–anal contact), and some had identifiable epidemiological links. A number of these individuals were also diagnosed with HIV or other sexually transmitted infections at admission, further supporting the classification of sexual exposure as the likely transmission context.

Patients in the non‑MSM group were also systematically asked about sexual practices, and all denied behaviors associated with sexual transmission. As detailed in the revised Methods section, their classification was based on non‑sexual fecal–oral exposure contexts.

Regarding vaccination status, lack of hepatitis A vaccination was not an inclusion criterion. Instead, it was an observed characteristic of the study population: none of the patients meeting the case definition had been previously vaccinated against HAV.

The Methods section has been updated to clarify these points (see lines 77-93).

Comment #2

The study population is defined on the basis of a hospital diagnosis of acute HAV infection, rather than on sexual orientation or sexual behavior, and this distinction should be made explicit from the beginning of the manuscript.

Response #2

We collected data from the patients' hospital records. We searched for all patients diagnosed with HAV, and each of them was asked about socio-demographical characteristics, including sexual orientation/behavior and mode of HAV transmission. When we had all the information about them, we could split the groups and analyse the data.

Comment #3

Regarding case definition, it is not clear whether individuals were included if they met at least one of the diagnostic criteria or if fulfillment of all criteria was required.

Response #3

Thank you for this observation. We agree that the original description of the case definition could be interpreted as ambiguous. To address this, we have revised the Methods section to clearly state that inclusion required fulfillment of the full diagnostic definition for acute hepatitis A: a compatible clinical presentation, elevated aminotransferases, and confirmation by positive anti‑HAV IgM serology. Individuals were included only when all components of the case definition were met. The methods section has been accordingly rewritten to improve clarity (see lines 79-81).

Comment #4

Conceptually, the exposure groups (MSM vs. FBT) do not appear to be mutually exclusive. All patients were assumed to share the same biological mechanism of transmission (fecal–oral), and the distinction lies in the context of exposure. Thus, the groups more appropriately represent different fecal–oral exposure contexts: sexual exposure, foodborne exposure, and a third category that is not addressed in the manuscript—mixed or indeterminate exposure.

Response #4

Thank you for this thoughtful comment. We agree that all patients shared the same biological mechanism of transmission (fecal–oral), and that the distinction between groups reflects different exposure contexts rather than different transmission pathways.

Exposure classification was based on detailed clinical interviews. Individuals reporting sexual risk behavior with male partners were classified as MSM. All individuals who explicitly denied MSM behavior or any sexual exposure risk were classified as non‑MSM. Within this non‑MSM group, several patients belonged to small epidemiological clusters—either intrafamilial or workplace‑related. These clusters were heterogeneous in age and gender and did not correspond to MSM sexual networks. In other non‑MSM cases, no such cluster could be identified. Importantly, none of the non‑MSM individuals reported sexual exposure, and no case in this group had evidence suggestive of sexual transmission.

Although a food‑borne HAV outbreak was reported in the mass media during the study period, a direct food‑exposure source could not be clearly established for individual patients. Nevertheless, all non‑MSM individuals reported behaviors or contexts consistent with non‑sexual fecal–oral transmission: they either purchased food from supermarkets (including the salads suspected in the outbreak), or originated from regions where similar cases were reported, including areas near the capital with documented water‑supply problems. Taken together, these patterns support the classification of all non‑MSM individuals as having acquired infection through non‑sexual fecal–oral routes.

For these reasons—absence of sexual risk behavior, heterogeneous but plausible non‑sexual epidemiological links, and no evidence of mixed exposure—a separate mixed or indeterminate exposure category was not applicable in our dataset.

The Methods section has been revised accordingly to reflect these clarifications (see lines 92-108).

Comment #5

Furthermore, it is not clearly described how each exposure category was operationally defined. The manuscript suggests that only two groups were considered (FBT and MSM), and that individuals identified as MSM were automatically classified into the MSM group, as if sexual behavior alone determined the transmission route. This approach raises concerns about potential exposure misclassification and oversimplification of transmission dynamics.

Response #5

We agree that the initial version of the manuscript did not sufficiently detail how the exposure categories were operationally defined. As noted in our previous responses, the revised Methods section clearly describes the criteria used to assign individuals to the MSM and FBT groups, including the behavioral, epidemiological, and contextual elements considered, as well as the potential for misclassification. We have also acknowledged the lack of molecular sequencing as a limitation. These clarifications have now been incorporated into the updated Methods section (see lines 94-111).

P.S.: A revised manuscript has been submitted, with all changes clearly marked in grey shading for ease of evaluation.

Thank you, once again!

On behalf of all authors, 

Sincerely,

Ruxandra Moroti and Irina Ianache

01 Feb 2026

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Overall comments: This manuscript presents the clinical and laboratory findings of patients with acute hepatitis A virus infection in two tertiary hospitals in Bucharest over a one-year period (March 2022-2023). Approximately half of the 191 patients are MSM, half of whom are also HIV positive (n=48). The authors refer to this study as “….one of the first documented hepatitis A virus (HAV) outbreaks
in Romania involving dual transmission pathways: sexual transmission among MSM and foodborne
transmission within the general population.” Unfortunately, this claim is significantly overstated; the study lacks basic epidemiological investigation typical of an outbreak. It is unclear how and when the outbreak started, when it peaked, and when it subsided, if it did at all. The epidemiologic linkages between the outbreak cases are poorly, if at all, characterized, especially among the foodborne transmission cases, as no clusters seem to have been identified. A major drawback of this work is the lack of molecular sequencing of different HAV isolates, which is crucial in identifying individual outbreak clusters and transmission networks among the MSM population, which might help guide health response measures to contain the outbreak.

Specific comments:

1. Introduction, line 70; not sure of the proper terminology, but “fisting” may be more appropriate than “..screw you withand”.
2. Material and Methods, line 79; the selected cut-off for ALT >10x normal values as one of the case definitions is problematic, as both European (ECDC) and North American guidelines recommend ALT>5x values. As a result, some of the milder HAV cases may have been missed. In addition, the selected age eligibility (over 18 years of age) is inappropriate regarding the foodborne transmission pathway, as this artificially limits the range of affected cases.
3. Results, line 105; please clarify text.
4. Results overall: epidemiological data is either incomplete or missing regarding cluster links for both MSM and FBT.
5. Results, lines 122-23; Jaundice is the main symptom triggering patients to seek medical help from infectious disease specialists, as the other ones listed by the authors are too nonspecific; how were the 10% of the patients without jaundice diagnosed?
6. Discussion; the authors should spend more time discussing their finding that MSM patients “experienced significantly greater hepatocellular injury”. Despite the undetectable viral load, their CD4 counts ( 609 cells/μL (IQR 464–865) are lower than the average among the general population (800-1000), and therefore, based on the mechanism of liver injury, which is immune-mediated, one might expect a milder disease course. As for the other factors, potentially involved, such as STIs the authors might compare the latter in their MSM cohort with and w/o HIV to rule out or include this as a proper risk factor of greater hepatocellular injury.

Author Response

Dear Reviewer,
Thank you for evaluating our work and for your constructive comments. We did our best to improve our manuscript!

Reviewer Overall comments:

This manuscript presents the clinical and laboratory findings of patients with acute hepatitis A virus infection in two tertiary hospitals in Bucharest over a one-year period (March 2022-2023). Approximately half of the 191 patients are MSM, half of whom are also HIV positive (n=48). The authors refer to this study as “….one of the first documented hepatitis A virus (HAV) outbreaks in Romania involving dual transmission pathways: sexual transmission among MSM and foodborne transmission within the general population.” Unfortunately, this claim is significantly overstated; the study lacks basic epidemiological investigation typical of an outbreak. It is unclear how and when the outbreak started, when it peaked, and when it subsided, if it did at all. The epidemiologic linkages between the outbreak cases are poorly, if at all, characterized, especially among the foodborne transmission cases, as no clusters seem to have been identified. A major drawback of this work is the lack of molecular sequencing of different HAV isolates, which is crucial in identifying individual outbreak clusters and transmission networks among the MSM population, which might help guide health response measures to contain the outbreak.

Authors response to the general comment:

Thank you for your thoughtful overall assessment of our manuscript. We appreciate your recognition of the clinical and laboratory value of the dataset and agree that several aspects of the initial version required clearer epidemiological framing.

We acknowledge that the original wording may have overstated the strength of the outbreak characterization. As you correctly note, the absence of molecular sequencing and the limited availability of formal public‑health outbreak investigations restrict our ability to delineate precise transmission chains, identify definitive clusters, or describe the temporal dynamics of a confirmed outbreak. We have therefore revised the manuscript to avoid overinterpretation and to more accurately reflect the observational nature of our data.

In the revised Methods and Results sections, we now provide a clearer description of how exposure groups were defined, how epidemiological information was collected, and what types of linkages could and could not be established. We also explicitly acknowledge the limitations related to the lack of molecular sequencing and the incomplete characterization of epidemiological links, particularly among individuals classified as having food‑borne transmission.

Our intention was to document an unusual temporal aggregation of HAV cases in our region, with a marked increase among MSM and a concurrent rise in cases without sexual exposure, rather than to claim a fully characterized outbreak investigation. The revised text now reflects this more cautiously and transparently.

We thank you again for highlighting this important point, which has helped us improve the accuracy and clarity of the manuscript.

Reviewer Specific comments:

Comment #1

Introduction, line 70; not sure of the proper terminology, but “fisting” may be more appropriate than “..screw you withand”.

Response #1

Thank you for this observation. We agree that the original wording was inappropriate and resulted from an unintended phrasing error. The revised text now reads: “The study aimed to describe the clinical and epidemiological features, laboratory findings, and outcomes in individuals diagnosed with HAV in two tertiary care centers in XX…” (see lines 69 - 70).

Comment #2

Material and Methods, line 79; the selected cut-off for ALT >10x normal values as one of the case definitions is problematic, as both European (ECDC) and North American guidelines recommend ALT>5x values. As a result, some of the milder HAV cases may have been missed. In addition, the selected age eligibility (over 18 years of age) is inappropriate regarding the foodborne transmission pathway, as this artificially limits the range of affected cases.

Response #2

Thank you for this comment. We agree that the initial wording in the Methods section may have suggested that ALT >10× the upper limit of normal was used as an inclusion criterion. This was not the case. As clarified in the revised text, the case definition required all the following: compatible clinical symptoms, elevated aminotransferases, and positive anti‑HAV IgM serology. We did not apply a specific ALT cut-off for inclusion (see the corrected text in the Methods section, lines 79-91). In our cohort, however, all patients who met the full diagnostic criteria (clinical presentation + elevated aminotransferases + positive IgM anti‑HAV) had ALT values ≥10× normal at presentation. We did not encounter patients with suggestive symptoms and confirmed HAV infection whose ALT values were only 5× normal. Therefore, no milder cases were excluded based on ALT levels.

Regarding age eligibility, we agree that the initial text did not fully explain why only adults were included. Although access to pediatric data would have been possible, our study specifically targeted the adult population, as we observed a sudden and marked increase in HAV cases among adult males—many identifying as MSM—beginning in early 2022, shortly after the relaxation of pandemic‑related restrictions. This pattern suggested an emerging adult‑focused transmission context, distinct from the usual pediatric epidemiology of HAV in our region. For this reason, the investigation was designed to characterize the clinical and epidemiological features of this adult cluster rather than to describe the broader pediatric burden.

We have clarified this rationale in the Introduction section, lines 65-68.

Comment #3

Results, line 105; please clarify the text.

Response #3

It was an error – an interposition of several words – we corrected this – actual line no 127.

Comment #4

Results overall: epidemiological data is either incomplete or missing regarding cluster links for both MSM and FBT.

Response #4

We agree that the epidemiological data available to us did not allow detailed reconstruction of cluster linkages for either the MSM or the food‑borne transmission groups. As noted, the absence of molecular sequencing represents a major limitation, and we have explicitly acknowledged this in the manuscript.

In addition, the revised Methods section now provides a clearer description of how the two exposure groups were defined and the rationale for this classification (please see lines 95-112). These clarifications outline the epidemiological and behavioral information used for group assignments and transparently describe the constraints that limited more granular cluster analysis.

A limitation of the study is the lack of access to molecular testing, which prevented us from obtaining information on the cluster links for MSM and FBT. We mentioned that as a limitation of our study in the manuscript

Comment #5

Results, lines 122-23; Jaundice is the main symptom triggering patients to seek medical help from infectious disease specialists, as the other ones listed by the authors are too nonspecific; how were the 10% of the patients without jaundice diagnosed?

Response #5

The patients without jaundice (approximately 10%) presented to the emergency department with other gastrointestinal symptoms such as nausea, vomiting, abdominal discomfort, or fatigue. Routine laboratory testing performed at presentation revealed markedly elevated aminotransferases, raising suspicion for acute hepatitis. These patients were subsequently referred to our infectious diseases departments, where HAV infection was confirmed by positive anti‑HAV IgM serology.

Comment #6

 Discussion: the authors should spend more time discussing their finding that MSM patients “experienced significantly greater hepatocellular injury”. Despite the undetectable viral load, their CD4 counts ( 609 cells/μL (IQR 464–865) are lower than the average among the general population (800-1000), and therefore, based on the mechanism of liver injury, which is immune-mediated, one might expect a milder disease course. As for the other factors, potentially involved, such as STIs, the authors might compare the latter in their MSM cohort with and w/o HIV to rule out or include this as a proper risk factor of greater hepatocellular injury.

Response #6

Thank you for this insightful comment. We agree that the relationship between HIV status, immune function, and the degree of hepatocellular injury warrants clarification. In our cohort, people living with HIV were on antiretroviral therapy, had undetectable viral loads, and demonstrated good immunological status, with a median CD4 count above 500 cells/μL. Therefore, significant immunosuppression was unlikely to explain the observed differences in liver injury.

Importantly, not all MSM in our study lived with HIV, and the greater hepatocellular injury observed in the MSM group was not necessarily attributable to HIV infection itself. Other factors may have contributed, including the possibility of a higher infectious inoculum associated with specific sexual practices that facilitate fecal–oral HAV transmission. This hypothesis is consistent with observations from other HAV outbreaks among MSM.

We have addressed this point in the Discussion (lines 177-186) and in the Conclusion (lines 255-259).  

Regarding the potential role of concurrent STIs, we acknowledge that a more detailed comparison between MSM with and without HIV could provide additional insight (we added commentaries in the Discussion section (lines 187-191) and in the Conclusions (lines 259-262)). However, STI screening was not uniformly available to all patients, limiting our ability to perform a robust subgroup analysis and clarifying the potential mechanisms that may explain the greater hepatocellular injury observed in MSM.

Additional response regarding the Tables in the manuscript:

Thank you for your feedback regarding the tables. Although no specific changes were suggested, we carefully reviewed the structure and content of the tables and agreed that two of them (Tables 2 and 3) contained overlapping information. To improve clarity and readability, we have merged these into a single table and streamlined the data presentation. We believe this revision enhances the coherence and usability of the results.

On behalf of all authors, we thank you for your constructive comments and for the opportunity to improve our manuscript.

Sincerely,

Ruxandra Moroti and Irina Ianache

1 of Feb 2026

P.S.: A revised manuscript has been submitted, with all changes clearly marked in grey shading for ease of evaluation.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed the points raised in the review. They clarified the case definition, inclusion criteria, and analytical construction of exposure groups, explicitly distinguishing between the fecal-oral transmission mechanism and the different exposure contexts. Although exposure classification continues to be based on clinical and epidemiological information, rather than molecular confirmation, this limitation is now clearly recognized and justified. Overall, the revisions significantly improved the conceptual clarity and methodological transparency of the manuscript.

Author Response

Dear Reviewer, 

Thank you very much for your observation regarding the tables. Although no specific modifications were requested, we carefully re‑examined the structure and content of all tables in light of your comment. During this review, we noted that two of them (Tables 2 and 3) presented partially overlapping information. To improve clarity and overall readability, we have merged these into a single, consolidated table and streamlined the data presentation.

In addition, we have lightly shaded selected rows and columns to enhance visual guidance and facilitate easier navigation through the key results.

We believe that these revisions improve both the coherence of the manuscript and the usability of the results section.

Thank you,

On behalf of all authors,

Sincerely,

Ruxandra Moroti and Irina Ianache

3 February 2026

 

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript addresses the main reviewer’s concern regarding the insufficient epidemiological investigation of the current HAV outbreak by including all the additional information that could have been collected by the authors, especially regarding outbreak cases associated with foodborne transmission (FBT). Hence, the revised M&M section outlines in a better way the two different groups affected and incorporates a bit more epidemiological links within the FBT cases. The authors also clarified the outbreak case definition and age eligibility. Greater consideration was ascribed to the issue of hepatocellular injury in MSM compared to the FBT group in the Discussion section.

In their point-to-point response, the authors agreed that the lack of molecular sequencing of the outbreak HAV isolates was an important limitation of this study, however this was left out in the track changes of the revised manuscript. This reviewer believes that a one-to two-sentence paragraph should be included in the Discussion section.

Overall, the inclusion of more epidemiological data, discussion and limitations in the revised manuscript improved its quality.

Author Response

Reviewer Comments:

The revised manuscript addresses the main reviewer’s concern regarding the insufficient epidemiological investigation of the current HAV outbreak by including all the additional information that could have been collected by the authors, especially regarding outbreak cases associated with foodborne transmission (FBT). Hence, the revised M&M section outlines in a better way the two different groups affected and incorporates a bit more epidemiological links within the FBT cases. The authors also clarified the outbreak case definition and age eligibility. Greater consideration was ascribed to the issue of hepatocellular injury in MSM compared to the FBT group in the Discussion section.

In their point-to-point response, the authors agreed that the lack of molecular sequencing of the outbreak HAV isolates was an important limitation of this study, however this was left out in the track changes of the revised manuscript. This reviewer believes that a one-to two-sentence paragraph should be included in the Discussion section.

Overall, the inclusion of more epidemiological data, discussion and limitations in the revised manuscript improved its quality.

Authors' response:

Dear Reviewer,

Thank you for the thorough evaluation and for acknowledging the improvements made in the revised manuscript.

Regarding the comment on molecular sequencing, we agree that the lack of access to genotyping represents an important limitation of our study. Although this aspect was already mentioned in the Methods section, we have now added a dedicated statement in the Discussion to explicitly address its implications for interpreting the outbreak dynamics. The new text has been incorporated into the revised manuscript (lines 214-219).

We are grateful for your constructive input, which has improved the clarity and overall quality of the manuscript.

Thank you once again, 

On behalf of all authors, 

Ruxandra Moroti and Irina Ianache

3 February 2026