Dear Reviewer,

We would like to sincerely thank you for your careful and constructive review of our manuscript. We are grateful for your positive assessment that the study is a helpful addition to the literature on hematological and biochemical biomarkers and comorbidities in individuals with AUD and chronic viral illnesses in Uzbekistan. We also appreciate your detailed suggestions, which helped us improve the clarity, methodological transparency, terminology, interpretation, supplementary materials, and dataset anonymization.

We have revised the manuscript carefully in response to all comments. In addition, the supplementary materials and dataset deposited in Zenodo were revised for clarity and further anonymization. No changes were made to the study groups, statistical analyses, numerical results, or reported conclusions.

Below we provide a point-by-point response.

General comment: Terminology needs to be clear and more consistent when describing cases throughout the paper. The reviewer noted inconsistent use of “stage II chronic alcoholism,” “stage 2 alcohol use disorder,” “mild AUD,” and the term “addicts” in Figure 1.

Thank you for this important comment. We revised the terminology throughout the manuscript to improve consistency and to avoid stigmatizing language. The term “addicts” was removed and replaced with person-first terminology. We now consistently refer to the patient groups as “patients with AUD” or “AUD cohorts.”

We also clarified that the diagnosis was alcohol use disorder according to ICD-10 code F10.2. The wording suggesting that ICD-10 defines AUD by “stages” was removed or revised. In the Methods section, we now specify that inclusion criteria were male sex, age 20-65 years, inpatient status, and a clinical diagnosis of AUD (ICD-10 F10.2), established by an addiction psychiatrist and supported by AUDIT.

We also clarified the exclusion of individuals with mild alcohol-related conditions or clinically mild withdrawal presentations. The revised Methods state that individuals with mild alcohol-related conditions (ICD-10 F10.1) or clinically mild withdrawal presentations were excluded because their number was very small and because their short alcohol-use history and clinical profile were not comparable with patients with established long-term AUD.

Abstract: The abstract could better orientate the reader by clarifying that the viral infections studied refer to chronic viral infections, such as hepatitis B, hepatitis C, and HIV.

Thank you. We revised the Abstract to clarify this point. The Background now states that the study examined biomarker signatures in men with AUD in Uzbekistan, with and without chronic viral infections, specifically hepatitis B, hepatitis C, and HIV.

This clarification was also applied throughout the manuscript, including the Methods, Results, Discussion, figure/table legends, and data descriptions.

Methodology: Please include the specific dates of the 3-month study period, whether participants were inpatients or outpatients, and how biomarker values were selected if more than one value was available per participant.

Thank you. We expanded the Methods section substantially. We now specify that clinical and laboratory data were collected from 19 March to 22 June 2025. We also clarify that patients were recruited from inpatient units of the Republican Specialized Scientific and Practical Medical Center for Mental Health.

We added that laboratory measurements were obtained at admission, before treatment initiation, and were considered baseline values. Only admission values were included in the analysis, and follow-up measurements during treatment were excluded. This was added to clarify how biomarker values were selected.

Methodology: It is not clear whether controls were selected from patients at the centre or from another group.

Thank you for noting this. We clarified the control group source. The revised Methods state that the control group consisted of male volunteers of approximately similar age, including employees of the same institution and relatives of employees. Controls had no history of alcohol use disorder or psychoactive substance use, as confirmed by questionnaire-based screening and clinical assessment. We also clarified that one initially screened control participant was excluded because of a self-reported history of chronic hepatitis B, as the control group was defined to include individuals without known chronic viral infections.

Methodology: A brief sentence explaining the De Ritis ratio needs to be included in the Methods section rather than in the Discussion.

Thank you. We added a methodological explanation in the Data Collection section. The revised manuscript now states that the De Ritis ratio was calculated as the ratio of aspartate aminotransferase (AST) to alanine aminotransferase (ALT) and was used to characterize patterns of liver injury.

Methodology: Please explain how data on BMI, ethnicity, comorbidities, and alcohol withdrawal symptoms were obtained, and how withdrawal was categorized as mild, moderate, or severe.

Thank you. We expanded the Data Collection and Inclusion/Exclusion Criteria sections. The revised Methods now state that data on age, ethnicity, comorbidities, alcohol-use history, alcohol withdrawal symptoms, and clinical status were obtained from medical records, physician assessments, patient interviews, available collateral history, ultrasound reports, and documented diagnoses. Anthropometric data, including height and weight, were collected from medical records and questionnaires, and BMI was calculated as weight in kilograms divided by height in meters squared.

We also added a detailed explanation of alcohol withdrawal severity classification. Withdrawal severity was assessed clinically by the treating addiction psychiatrist and classified as mild, moderate, or severe according to the intensity of autonomic, somatic, and neuropsychiatric symptoms, including tremor, agitation, anxiety, sleep disturbance, nausea/vomiting, disorientation, hallucinations, delusional symptoms, and the need for inpatient monitoring or pharmacological management. Mild withdrawal was defined as limited autonomic or anxiety-related symptoms without clinically significant neuropsychiatric manifestations or somatic instability. Moderate withdrawal included more pronounced symptoms requiring inpatient observation and treatment, whereas severe withdrawal was characterized by delirium, hallucinations, delusional symptoms, marked agitation, or clinically significant somatic instability.

Methodology: Please comment on whether delusions/hallucinations were related to alcohol withdrawal or could be related to primary mental illness, and how these data were obtained.

Thank you. We added a clarification in the Data Collection section. The revised manuscript states that delirium, hallucinations, and delusional symptoms were recorded only when documented by the treating addiction psychiatrist during the withdrawal episode. These data were obtained from clinical examination, patient interview, medical history, available collateral history, and medical record review.

We also clarified that symptoms were considered withdrawal-related when they occurred in the context of alcohol withdrawal and were not documented as part of a primary psychiatric disorder. According to the medical records, these patients had no previous registration or documented history of primary psychotic disorders.

Methodology: The Methods section states that patients with mild withdrawal symptoms were excluded. Please explain why this decision was made.

Thank you. We clarified this point in the Study Design and Population section and in the Inclusion/Exclusion Criteria section. Individuals with mild alcohol-related conditions (ICD-10 F10.1) or clinically mild withdrawal presentations were excluded because their number was very small and because their short alcohol-use history and clinical profile were not comparable with patients with established long-term AUD. This was intended to maintain clinical comparability within the analytical cohort.

Results: The statement regarding “right shift” in the differential count and its interpretation as suggestive of megaloblastic anemia or hepatic/renal impairment requires further support.

Thank you for this very helpful comment. We revised this section to avoid unsupported interpretation. The term “right shift” was removed. We now state that an increased proportion of segmented neutrophils was observed in 7.6–9.8% of patients; however, because direct morphological assessment of neutrophil hypersegmentation was not performed, this finding should be interpreted cautiously and cannot be used to infer megaloblastic anemia or hepatic/renal impairment.

This revision ensures that the interpretation remains aligned with the data actually available in the study.

Discussion: Please review the Discussion to ensure that claims do not go beyond the data presented or cited.

Thank you. We carefully revised the Discussion to make the interpretation more cautious and better aligned with the available data. Several statements were softened or clarified. For example, causal or overly strong wording was revised to more cautious language such as “may reflect,” “may aggravate,” “suggesting a greater likelihood,” and “should be interpreted cautiously.”

We also revised the interpretation of neuropsychiatric symptoms, platelet counts, creatinine/MELD, ROC findings, bilirubin findings, and biomarker diagnostic utility to avoid overinterpretation.

Discussion: The term “granulocytopenia” is used, but data on eosinophils and basophils are not provided. Please clarify whether this refers specifically to low neutrophils or to low granulocytes.

Thank you. We revised this terminology. Because eosinophil and basophil data were not included, we removed the broader term “granulocytopenia” where it could be misleading and clarified the finding as neutropenia/neutrophilic granulocyte compartment suppression.

The revised Discussion now states that alcohol-related disruption of granulopoiesis has been described as a mechanism contributing to impaired innate immune defense and increased susceptibility to infection. However, in the present AUD cohorts, neutropenia was rare and comparable between virus-positive and virus-negative patients, suggesting that suppression of the neutrophilic granulocyte compartment was not a prominent hematological feature in these patients.

Discussion: The reference to previous research on Candida albicans-specific Th17 cells would benefit from further explanation of its relevance to this work.

Thank you. We expanded this part of the Discussion to better explain the relevance of the cited work. The revised text now explains that inflammatory marker abnormalities may reflect not only viral-associated inflammatory burden but also alcohol-related disruption of gut-liver immune signaling. We added that Zeng et al. showed that chronic alcohol exposure is associated with intestinal fungal dysbiosis and increased Candida albicans-specific Th17 responses, with these cells detected in the circulation and liver of patients with alcohol-associated liver disease. We also noted that experimental models showed ethanol-associated migration of Candida albicans-specific Th17 cells from the intestine to the liver, where IL-17-mediated signaling contributed to hepatic inflammation and injury.

This explanation was added to clarify why gut-derived microbial/fungal immune pathways may be relevant to systemic and hepatic inflammation in patients with AUD and chronic viral comorbidity.

Discussion: The statement that the findings support including creatinine in the MELD score is not adequately supported, as mortality was not explored.

Thank you. We agree and revised this section substantially. The unsupported statement was removed. The revised Discussion now states that creatinine was significantly lower in patients with AUD, consistent with prior reports. We explain that, in the context of chronic alcohol exposure, reduced creatinine may reflect lower skeletal muscle mass, malnutrition, or altered protein metabolism rather than preserved renal function.

We also clarified that creatinine remains an important component of liver disease severity assessment, including the MELD score, where it is considered together with bilirubin and INR to support risk stratification in advanced liver disease. We no longer imply that our findings validate mortality prediction. Instead, we state that lower creatinine in the present cohorts should be interpreted as part of the broader metabolic and somatic profile of patients with AUD rather than as an isolated renal marker.

Discussion: Please clarify the comment regarding the small number of female participants, as females were explicitly excluded.

Thank you. We revised the Limitations section to clarify this point. The revised text states that the analysis was restricted to male patients because only eight female patients met the initial screening criteria, which was insufficient for meaningful sex-stratified analysis. Given known sex-related differences in alcohol metabolism, body composition, and biochemical and hematological profiles, women were therefore excluded from the final analytical dataset. We also added that the findings should be interpreted as primarily applicable to male patients with AUD, and that future studies should include adequately powered female cohorts.

Discussion: Please clarify the statement regarding low PLT and withdrawal-related neuropsychiatric complications, as the cited paper refers to delirium tremens and withdrawal seizures, and the current paper did not appear to collect these outcomes. The current paper also analyzed blood results categorically and has limited ability to comment on severity of thrombocytopenia.

Thank you for this important clarification. We revised this section to avoid overinterpretation. We no longer refer to severe thrombocytopenia as an outcome category. Instead, we provide the platelet findings in relation to the reference range and explicitly state the available clinical data.

The revised Discussion now states that thrombocytopenia was observed in patients with AUD. We added median PLT values across cohorts and the proportions of participants with PLT values below the reference range. We also clarified that, although platelet counts below 119,000/μL have been associated with increased risk of withdrawal seizures and delirium tremens in previous research, clinically documented delirium and hallucinations during withdrawal were recorded in only 16 of 292 patients with AUD in our cohort; among them, only one had PLT below the reference range, one had PLT at the lower reference limit, and 14 had values within the reference range.

We therefore revised the conclusion to state that reduced PLT was an important hematological feature of AUD but showed no clear correspondence with recorded withdrawal-related delirium or hallucinations in this cohort.

Discussion: The statement about combining biochemical, hematological, and physiological markers to enhance diagnostic accuracy needs further context on what is being diagnosed, why diagnostic accuracy may be difficult, and the clinical importance of accurate diagnosis.

Thank you. We revised the ROC interpretation in both the Results and Discussion sections. We clarified that two diagnostic comparisons were assessed: first, discrimination between all patients with AUD and alcohol-free controls; second, differentiation between patients with AUD and chronic viral infection and patients with AUD without chronic viral infection.

We also clarified that AUROC was used to quantify discriminatory performance and that AUROC values below 0.5 reflected inverse discrimination according to the predefined class coding, rather than necessarily indicating absence of discriminatory value.

In the Discussion, we revised the statement to clarify that combined biomarker panels may be more informative than isolated markers for distinguishing AUD from alcohol-free controls and for identifying viral comorbidity within AUD cohorts. We also added clinical context: chronic alcohol exposure and viral infections can produce overlapping hepatic, inflammatory, and metabolic abnormalities, making interpretation of single laboratory parameters difficult. We further clarified that markers with limited discriminatory performance, including platelets, hemoglobin, and BMI, should be interpreted as supportive clinical features rather than standalone diagnostic indicators.

Supplementary tables: Within Table S1, please provide a legend with abbreviations written out in full and provide the thresholds defined as normal range.

Thank you. Supplementary Table S1 was revised. We added a legend defining hematological abbreviations, including Hb, LYM, MCV, MON, MPV, NEUT, PLT, ESR, and WBC. We also added the specific reference intervals used to define low, normal, and high values.

Supplementary tables: Within Table S2, please clarify whether glucose is random or fasted.

Thank you. Supplementary Table S2 was revised to clarify glucose sampling. We added that glucose values in the patient group represent random/non-fasting blood glucose measured at admission, whereas blood samples from controls were obtained under fasting conditions. This clarification was also added to the Methods section.

Supplementary tables: Consistency in bilirubin labeling would be helpful.

Thank you. We harmonized bilirubin terminology throughout the manuscript and supplementary materials. We now use direct (conjugated) bilirubin and indirect (unconjugated) bilirubin consistently where relevant.

Dataset: Participant ID and “encrypted name, nation” may contain identifying information. Please consider whether further anonymization is needed.

Thank you. We carefully reviewed and revised the dataset. The dataset file deposited in Zenodo was additionally anonymized. Potentially identifying or non-essential fields, including the encrypted name and nationality-related field, were removed. The dataset now retains anonymized participant IDs and the variables required for reproducibility of the analysis.

Dataset: The “note” column contains comorbidity information not required for replication and may be identifying if specific dates are included. Please consider removing this column.

Thank you. We removed the free-text note column from the dataset to reduce re-identification risk. Comorbidity-related information necessary for the analysis is retained in structured, analyzable variables, while potentially identifying free-text information was removed.

Additional data and references

During the revision, we also checked the reference list and corrected/replaced several references where needed to ensure that all cited sources accurately support the corresponding statements. The Zenodo repository was updated to include the additionally de-identified underlying dataset and clarified extended data files. No changes were made to study groups, statistical analyses, numerical results, or reported conclusions.

We are grateful to the reviewer for the detailed and constructive comments. We believe that the revised version is clearer, more transparent, more cautious in interpretation, and better aligned with the available data and reporting standards.