Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Hypokalemic hypochloremic metabolic alkalosis is a well-known and typical manifestation of Cystic fibrosis. A case series is a small, but worth addition to the current literature. The manuscript does not add additional new informations to justify a publication in a high impact journal and has serious flaws. 

Comments:

Without a comparison with your total cohort judgement about risk factors is incomplete.

Sweat chloride at diagnosis and pancreatic insufficiency status are important basal characteristics

The differential diagnosis and the diagnostic workup should be reported in more detail as HHMA is sometimes the first symptom leading to the diagnosis of CF.

CFTR-Modulator therapy is not even mentioned. To my best knowledge this therapy was not accessible in Brazil during the time period. But this fact should be mentioned and discussed.

The microbiological characteristic and the individual description in tables 1 and 2 do not add value to the description.

Preventive strategies like increased salt intake are not even mentioned. 

It is important to highlight that the prevalence rate has to be interpreted in context to the reporting country in Brazil and that the climate plays an important role. A comparison with the literature/registry is lacking.

The literature is incomplete; I missed: 

• DOI: 10.1016/j.jcf.2025.12.006
• doi: 10.1136/bcr-2023-257348.
• doi: 10.1179/146532805X45719
• doi.org/10.1136/archdischild-2014-307384.744

 

 

Author Response

Comments and Suggestions for Authors

Hypokalemic hypochloremic metabolic alkalosis is a well-known and typical manifestation of Cystic fibrosis. A case series is a small, but worth addition to the current literature. The manuscript does not add additional new informations to justify a publication in a high impact journal and has serious flaws.

Reply: We sincerely thank the Reviewer for the careful evaluation of our manuscript and for the valuable comments and suggestions regarding potential improvements. We greatly appreciate the opportunity to revise the manuscript and to further strengthen its scientific contribution.

In response to the Reviewer’s concerns, we have substantially revised and expanded the manuscript. The new version was specifically improved with the aim of providing a more comprehensive characterization of patients with HHMA in the context of CF, as well as a more detailed assessment of their genetic profile.

Importantly, we have incorporated an analysis of CFTR gene variants in patients with HHMA and compared their genetic profile with that of other patients followed at our reference center in Brazil. We believe that this comparison adds relevant information, particularly considering the marked genetic diversity of the Brazilian population and the limited availability of data addressing the genetic characteristics of patients with HHMA in this population.

In addition, we have expanded the discussion and incorporated findings from the existing literature to place our results within a broader international context. These additions provide a more comprehensive overview of HHMA in CF and allow our findings to be discussed in relation to observations reported in different populations worldwide.

Therefore, we believe that the revised manuscript goes beyond a simple case series by providing clinical and genetic characterization of patients with HHMA, comparison with the broader CF population followed at our reference center, and integration of our findings with the international literature. We hope that these substantial revisions address the Reviewer’s concerns and enhance the relevance and contribution of the manuscript to the field.

We would be very grateful for the opportunity to have the revised manuscript reconsidered. We remain fully available to make any further corrections or modifications that may be necessary to improve the manuscript.

 

Comments:

Without a comparison with your total cohort judgement about risk factors is incomplete.

Reply: Thank you for this important comment. We agree that assessing potential risk factors requires an appropriate comparison group. In the revised manuscript, we have therefore refined the focus of the study to place greater emphasis on the genetic profile of patients with HHMA. Specifically, we performed a comparative analysis of CFTR variants between individuals with HHMA and the broader cohort of individuals with cystic fibrosis followed at our reference center. This comparison focused specifically on pathogenic CFTR variants, allowing us to better assess whether particular genetic variants were differentially represented among patients with HHMA. Accordingly, we have revised the manuscript to avoid broader inferences regarding clinical risk factors and to clarify that the comparative analysis is specifically related to the distribution of pathogenic CFTR variants.

 

We aimed to characterize the epidemiological and clinical features of HHMA in individuals with CF followed at a tertiary referral center, with emphasis on its frequency, biochemical profile, seasonality, recurrence, clinical presentation, and management, and to characterize the CFTR genetic profile of affected individuals and compare it with that of the broader CF population followed at the same center.

 

Sweat chloride at diagnosis and pancreatic insufficiency status are important basal characteristics

Reply: Thank you for this important comment. We agree that sweat chloride concentration at CF diagnosis and pancreatic insufficiency status are relevant baseline characteristics when clinically characterizing patients with HHMA. In our database, however, sweat chloride testing was recorded as a criterion for confirmation of the CF diagnosis, with at least two sweat chloride measurements above 60 mEq/L, but the exact chloride concentrations were not available. Therefore, although all patients had documented confirmation of the CF diagnosis based on the available diagnostic criteria, we were unable to assess the relationship between the quantitative sweat chloride levels at diagnosis and the occurrence of HHMA. Similarly, baseline information regarding pancreatic insufficiency was not systematically available in the database and therefore could not be reliably included in the analysis. We have now acknowledged these issues as limitations of the study and have avoided drawing conclusions regarding their potential association with HHMA.

 

The differential diagnosis and the diagnostic workup should be reported in more detail as HHMA is sometimes the first symptom leading to the diagnosis of CF.

Reply: We thank the reviewer for this important comment. We agree that HHMA may occasionally be an initial manifestation leading to the diagnosis of CF. In our cohort, all patients who underwent neonatal screening had already undergone neonatal screening before the occurrence of HHMA, and therefore HHMA was not the initial finding leading to the diagnosis of CF in these individuals. Among the remaining participants who did not have documented neonatal screening, the available clinical and laboratory information was insufficient to retrospectively validate whether HHMA preceded the diagnosis of CF or contributed to its identification. We have clarified this point in the Methods and Results sections to avoid overinterpretation of the temporal relationship between HHMA and CF diagnosis.

 

Results:

Among patients with documented neonatal screening, all had undergone screening before the occurrence of HHMA. For the remaining participants, the available data were insufficient to establish whether HHMA preceded CF diagnosis.

Methods:

Regarding the temporal relationship between HHMA and CF diagnosis, all patients who had undergone neonatal screening had received this screening before the occurrence of HHMA. Therefore, in these patients, HHMA was not the clinical finding that led to the diagnosis of CF. For participants without documented neonatal screening, the available records did not allow reliable retrospective determination of whether HHMA preceded the diagnosis of CF.

 

 

CFTR-Modulator therapy is not even mentioned. To my best knowledge this therapy was not accessible in Brazil during the time period. But this fact should be mentioned and discussed.

Reply: Thank you for this important comment. We agree that CFTR-modulator therapy is relevant to the interpretation of HHMA in cystic fibrosis and should be addressed in the manuscript.

The availability of CFTR-modulator therapy in Brazil evolved progressively during the study period (2014–2024) and remained restricted to specific genetic and clinical indications. Ivacaftor was incorporated into the Brazilian Unified Health System (SUS) in 2021 for patients with specific responsive CFTR variants, whereas lumacaftor/ivacaftor and tezacaftor/ivacaftor were not incorporated into the SUS. More recently, elexacaftor/tezacaftor/ivacaftor (ETI) was incorporated into the SUS in September 2023 for eligible patients aged ≥6 years carrying at least one F508del variant.

Importantly, none of the 41 patients included in our HHMA cohort was receiving CFTR-modulator therapy at the time of the HHMA episode. Therefore, the HHMA episodes analyzed in this study occurred without concomitant CFTR-modulator exposure. We have added this information to the revised manuscript and discussed the evolving therapeutic landscape in Brazil.

Because the retrospective database did not systematically capture longitudinal CFTR-modulator exposure, we could not evaluate whether subsequent treatment with CFTR modulators influenced the recurrence or clinical course of HHMA. Nevertheless, the absence of concomitant modulator therapy at the time of all HHMA episodes provides an important context for interpreting our findings.

 

Discussion:

An additional consideration is the progressive availability of CFTR-modulator therapies in Brazil during the study period. Access to these therapies was initially restricted to specific genetic and clinical indications. Ivacaftor was incorporated into the Brazilian Unified Health System (SUS – Sistema Único de Saúde) in 2021 for patients with specific responsive CFTR variants, whereas lumacaftor/ivacaftor and tezacaftor/ivacaftor were not incorporated into the SUS. Elexacaftor/tezacaftor/ivacaftor (ETI) was subsequently incorporated into the SUS in September 2023 for eligible patients aged ≥6 years carrying at least one F508del variant.

Importantly, none of the 41 patients in our cohort was receiving CFTR-modulator therapy at the time of the HHMA episode. Thus, all HHMA episodes analyzed in the present study occurred without concomitant CFTR-modulator exposure. This context is relevant when interpreting our findings, as the cohort predominantly reflects the clinical characteristics of HHMA before the widespread availability of highly effective CFTR-modulator therapy in Brazil. Future studies should assess whether broader access to CFTR modulators modifies the incidence, recurrence, or severity of HHMA.

 

Limitations:

None of the patients was receiving CFTR-modulator therapy at the time of the HHMA episode; however, because longitudinal treatment exposure was not systematically recorded, we could not assess whether subsequent CFTR-modulator therapy influenced the risk of HHMA recurrence or its clinical course.

 

The microbiological characteristic and the individual description in tables 1 and 2 do not add value to the description.

Reply: Thank you for this comment. We agree that an extensive individual description of the patients may not substantially contribute to the main interpretation of the study. However, we believe that the microbiological information provides relevant clinical context, as respiratory infections and airway colonization may contribute to dehydration, increased insensible fluid losses, reduced oral intake, and clinical deterioration, potentially acting as additional triggers for HHMA in susceptible patients. Therefore, we decided to retain the microbiological information but substantially reduce its narrative emphasis in the revised manuscript.

Regarding Tables 1 and 2, we agree that a detailed individual description of every patient may be excessive. Nevertheless, we believe that retaining the individual-level data provides transparency regarding the heterogeneity of age at presentation, clinical manifestations, seasonality, biochemical abnormalities, recurrence, and management, particularly because this is a case series and several patients presented with atypical or minimally symptomatic HHMA. To address the concern, we have revised the text to avoid repeating the information already presented in the tables and have limited the main text to the clinically relevant aggregate findings.

 

Results:

Airway microbiology findings were available for patients with documented cultures or previous colonization. Staphylococcus aureus alone was the most frequently identified microorganism, either alone or in combination with P. aeruginosa (Figure 1F, Table 2). Although microbiological findings were not a primary outcome of this study, they provide clinical context regarding concurrent or previous respiratory colonization among patients experiencing HHMA.

 

Preventive strategies like increased salt intake are not even mentioned. 

Reply: Thank you for this important comment. We agree that preventive strategies deserve greater emphasis, particularly because HHMA is closely related to excessive sodium and chloride loss through sweat. We have therefore expanded the Discussion to explicitly address preventive measures, including adequate salt and fluid intake, particularly during hot weather, periods of increased sweating, and intercurrent illness. We also emphasize the importance of caregiver education and early recognition of symptoms that may precede significant electrolyte disturbances. These measures may be particularly relevant during infancy and in patients with a previous episode of HHMA or recurrent episodes.

 

Discussion:

Preventive strategies should also be incorporated into the routine care of individuals with CF who are at risk of HHMA. Adequate sodium and fluid intake is particularly important during hot weather, periods of increased sweating, fever, respiratory or gastrointestinal illness, and reduced oral intake, when salt and water losses may increase substantially. Caregivers should receive clear guidance regarding adequate salt supplementation according to age and clinical circumstances, as well as appropriate hydration and early recognition of warning signs such as reduced oral intake, vomiting, lethargy, weight loss, or dehydration. Patients with a previous episode of HHMA may benefit from closer electrolyte monitoring during high-risk periods, given the recurrence observed in our cohort. These preventive measures may help reduce the risk of severe electrolyte disturbances and facilitate earlier intervention.

 

It is important to highlight that the prevalence rate has to be interpreted in context to the reporting country in Brazil and that the climate plays an important role. A comparison with the literature/registry is lacking.

Reply: We thank the reviewer for this important comment. We agree that the prevalence of HHMA should be interpreted within the specific epidemiological and geographic context of Brazil and that climatic conditions may contribute to the occurrence of electrolyte disturbances in individuals with CF. We have therefore expanded the Discussion to include three Brazilian publications addressing pseudo-Bartter syndrome in CF. One cohort from Ribeirão Preto, São Paulo, reported a prevalence of 41.6% among 12 infants with CF diagnosed through newborn screening. A Brazilian case series from Bahia reported pseudo-Bartter syndrome in 50% of 14 children with delayed CF diagnosis following false-negative or unsuccessful newborn screening. In addition, a Brazilian case report described severe pseudo-Bartter syndrome in a 5-year-old child with CF during the summer, in association with gastrointestinal fluid losses.

We have also expanded the discussion of climatic conditions. Rather than interpreting climate as a direct cause of HHMA, we now describe high ambient temperature as a potential contributing factor that may increase sweat-mediated sodium and chloride losses and thereby increase susceptibility to electrolyte disturbances. This interpretation is consistent with the observations discussed in the Brazilian studies, while acknowledging that HHMA is multifactorial and may also be influenced by age, nutritional status, pancreatic insufficiency, intercurrent illness, sodium intake, CFTR genotype, newborn screening, and access to specialized care.

Finally, we clarified that direct comparisons among the available Brazilian studies should be made cautiously because of differences in study design, sample size, age distribution, diagnostic strategies, and clinical characteristics. We also explicitly acknowledged that our single-center cohort from a specialized CF reference center cannot be considered representative of the Brazilian CF population as a whole. These changes have been incorporated into the Discussion.

 

The literature is incomplete; I missed: 

• DOI: 10.1016/j.jcf.2025.12.006
• doi: 10.1136/bcr-2023-257348.
• doi: 10.1179/146532805X45719
• doi.org/10.1136/archdischild-2014-307384.744

Reply: Thank you for pointing this out. We agree that these relevant studies were not adequately represented in the original version of the manuscript. We have now incorporated all four references into the revised manuscript and expanded the Discussion accordingly.

Specifically, we added the study by Berg et al. (DOI: 10.1016/j.jcf.2025.12.006), which provides recent evidence that elexacaftor/tezacaftor/ivacaftor improves sodium chloride and fluid conservation in individuals with cystic fibrosis and may reduce salt-wasting. We also incorporated the case report by Lopes et al. (DOI: 10.1136/bcr-2023-257348), which highlights pseudo-Bartter syndrome as an important initial manifestation of cystic fibrosis in infancy, particularly during periods of increased sweating and additional fluid and electrolyte losses. In addition, we included the study by Yalçin et al. (DOI: 10.1179/146532805X45719), which provides important data on the prevalence, clinical presentation, and recurrence of pseudo-Bartter syndrome in children with cystic fibrosis. Finally, we incorporated the conference report (DOI: 10.1136/archdischild-2014-307384.744), which further supports the occurrence of pseudo-Bartter syndrome as an early manifestation of cystic fibrosis in infancy. These references have been integrated into the relevant sections of the Discussion, and the corresponding citations have been added to the reference list.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript reports hypokalemic hypochloremic metabolic alkalosis in 41 patients with cystic fibrosis from a single referral center. The clinical dataset is useful, especially the information on age, recurrence, seasonality, genotype, electrolyte changes, and treatment. However, several methodological and interpretive issues should be addressed before publication. The manuscript currently relies mainly on descriptive results, while some conclusions are stronger than the data support.

Major comments

1. The study design should be described correctly. The manuscript calls the work a “serial case report,” but it includes 257 patients and analyzes 41 HHMA cases over ten years. This is more appropriately described as a retrospective case series or retrospective observational study. Please use one consistent term throughout the title, abstract, Results, and Methods.
2. The diagnostic criteria require clarification. The authors define HHMA using pH, bicarbonate, sodium, chloride, and potassium, but bicarbonate values are not shown in the main results or tables. Please provide bicarbonate measurements for each patient or explain why these data are unavailable. The method used to exclude respiratory compensation should also be stated.
3. The statistical analysis is too limited. Only descriptive statistics are presented. The authors should compare recurrent and non-recurrent cases, particularly for age, sex, genotype, season, and electrolyte values. The proposed association between F508del and recurrence should not be presented strongly without statistical testing.
4. The conclusion that HHMA is associated with male sex is not sufficiently supported. There is no comparison with the sex distribution of the full cystic fibrosis cohort. The higher number of male HHMA cases may simply reflect the underlying cohort composition. Please provide the sex distribution of all 257 patients and perform an appropriate comparison.
5. The genotype analysis needs improvement. The manuscript combines several different genotype categories, including patients with missing or unidentified alleles. Please clearly report the number of fully genotyped patients and separate confirmed genotypes from incomplete genetic information. Claims regarding “severe phenotypes” should be moderated unless supported by genotype–phenotype analysis.
6. The seasonality analysis should account for repeated episodes. It is unclear whether the percentages refer to patients or total HHMA episodes. Because some patients had multiple episodes, each episode should be analyzed separately, or the authors should clearly explain how recurrent events were counted.
7. The microbiology section appears disconnected from the main study question. Airway colonization is described, but no analysis shows whether colonization was associated with HHMA severity, recurrence, or treatment. The authors should either perform such analyses or shorten this section and avoid suggesting a causal relationship.
8. The manuscript contains conflicting statements about informed consent. The Methods state that written informed consent was obtained, while the ethics section states that consent was waived. This must be corrected.
9. Figure 3 should be revised. The figure is visually attractive but includes conclusions such as genotype linkage and diagnostic implications that are not fully demonstrated by this study. It also appears different in style from the other figures. The authors should simplify the figure, remove unsupported statements, and ensure that all graphical elements are scientifically accurate.
10. The Discussion should better distinguish the present findings from previous knowledge. The authors should explain what this cohort adds beyond earlier reports of pseudo-Bartter syndrome in cystic fibrosis. The possible roles of host responses, infection, and epithelial regulation may also be discussed more carefully. Relevant examples include studies showing that host RNA-binding proteins can regulate infection-associated epithelial apoptosis and epithelial–mesenchymal changes:Frontiers in Immunology. 2024;15:1431207. eLife. 2025;14:e107132.

Minor comments

1. Correct “Cystic Fibrosis Transmembrane Regulator” to “Cystic Fibrosis Transmembrane Conductance Regulator” throughout.
2. Report continuous variables using median and interquartile range when distributions are not normal.
3. Define whether electrolyte measurements were collected before treatment.
4. Check the units for sodium, potassium, chloride, and bicarbonate and use them consistently.
5. Simplify Table 1, which is difficult to read because of the large number of abbreviations.
6. Avoid repeating the same findings in the Abstract, Results, Discussion, Figure 3, and Conclusion.
7. Revise the English for grammar, punctuation, and sentence length.

Recommendation

The study contains clinically useful observations, but the study classification, diagnostic reporting, statistical analysis, ethics statements, and interpretation require substantial revision. I recommend major revision.

Author Response

Comments and Suggestions for Authors

This manuscript reports hypokalemic hypochloremic metabolic alkalosis in 41 patients with cystic fibrosis from a single referral center. The clinical dataset is useful, especially the information on age, recurrence, seasonality, genotype, electrolyte changes, and treatment. However, several methodological and interpretive issues should be addressed before publication. The manuscript currently relies mainly on descriptive results, while some conclusions are stronger than the data support.

Reply: We sincerely thank the Reviewer for the careful and constructive assessment of our manuscript. We appreciate the recognition of the clinical relevance of our dataset, particularly the information regarding age at presentation, recurrence, seasonality, CFTR genotype, electrolyte abnormalities, and treatment.

We agree that the original manuscript relied predominantly on descriptive analyses and that some interpretations were stronger than could be directly supported by the study design and available data. In response to the Reviewer's comments, we have substantially revised the manuscript to strengthen the methodological description, clarify the scope and limitations of the analyses, and ensure that the interpretation of the findings remains appropriately cautious.

In particular, we have revised the Discussion to avoid causal interpretations, especially regarding the relationship between environmental temperature and HHMA. We now describe seasonality and warm environmental conditions as potential contributing or modifying factors rather than as direct causes. We have also expanded the discussion of potential confounding factors and the limitations associated with the retrospective design, single-center setting, descriptive nature of the study, and heterogeneity of the clinical population.

Furthermore, we have incorporated additional relevant literature suggested by the Reviewer and clarified how our findings compare with previous Brazilian and international studies. The revised manuscript also provides a more cautious interpretation of the observed associations with CFTRgenotype, recurrence, age, and seasonality.

We believe that these revisions have improved the methodological rigor and interpretive balance of the manuscript while preserving the clinical relevance of the findings.

 

Major comments

1. The study design should be described correctly. The manuscript calls the work a “serial case report,” but it includes 257 patients and analyzes 41 HHMA cases over ten years. This is more appropriately described as a retrospective case series or retrospective observational study. Please use one consistent term throughout the title, abstract, Results, and Methods.

Reply: Thank you for this important methodological comment. We agree that the term “serial case report” was inappropriate for the design and scope of our study. The study includes 257 individuals with CF followed at a single referral center over a ten-year period, of whom 41 developed HHMA. Therefore, we have revised the manuscript to consistently describe the study as a retrospective case series.

Accordingly, the term “serial case report” has been replaced throughout the Title, Abstract, Methods, Results, and Discussion with “retrospective case series,” as appropriate. We also revised the title to more accurately reflect the study design and its main components.

The revised title is:

 

Hypokalemic hypochloremic metabolic alkalosis in cystic fibrosis: A retrospective observational study with clinical characterization, CFTR genetic profiling, and comparative genetic analysis with patients from a Brazilian reference center.

 

These changes were made to ensure methodological accuracy and consistency throughout the manuscript.

 

1. The diagnostic criteria require clarification. The authors define HHMA using pH, bicarbonate, sodium, chloride, and potassium, but bicarbonate values are not shown in the main results or tables. Please provide bicarbonate measurements for each patient or explain why these data are unavailable. The method used to exclude respiratory compensation should also be stated.

Reply: Thank you for this important comment. We agree that the method used to address respiratory compensation should be stated more explicitly.

We have revised the Methods section to clarify that HHMA was defined by the concomitant presence of hypokalemia, hypochloremia, and primary metabolic alkalosis, based on elevated pH and bicarbonate, in the appropriate clinical context of salt and fluid loss. Respiratory compensation was considered a physiological response to the metabolic alkalosis and therefore was not used as a criterion to exclude HHMA. Rather, a primary respiratory acid–base disorder was considered unlikely in the absence of clinical or laboratory evidence suggesting an independent respiratory disturbance.

We have also clarified that, when blood gas measurements were available, the acid–base profile was interpreted considering the expected respiratory compensation for metabolic alkalosis. Thus, a compensatory increase in pCO₂ was not interpreted as evidence against the diagnosis of HHMA. This clarification has now been incorporated into the Methods section.

 

CF diagnosis was established based on compatible clinical features and/or family history, associated with two sweat chloride tests ≥ 60 mEq/L and/or identification of two pathogenic CFTR variants [49–51]. HHMA was defined by the presence of: (i) primary metabolic alkalosis (pH ≥ 7.45 and bicarbonate ≥ 24 mmol/L); (ii) hyponatremia (≤ 135 mmol/L); (iii) hypochloremia (≤ 99 mmol/L); and (iv) hypokalemia (≤ 3.5 mmol/L) [13]. The classification of metabolic alkalosis was based on the concomitant elevation of pH and bicarbonate, together with the characteristic clinical context of salt and fluid loss. Respiratory compensation was considered an expected physiological response to metabolic alkalosis and was not considered evidence against the diagnosis of HHMA. A primary respiratory acid–base disorder was not considered when there was no clinical or laboratory evidence suggesting an independent respiratory disturbance. When blood gas measurements were available, the acid–base profile was interpreted in the context of the expected respiratory compensation for metabolic alkalosis rather than as a criterion for excluding HHMA.

 

1. The statistical analysis is too limited. Only descriptive statistics are presented. The authors should compare recurrent and non-recurrent cases, particularly for age, sex, genotype, season, and electrolyte values. The proposed association between F508del and recurrence should not be presented strongly without statistical testing.

Reply: Thank you for this important comment. We agree that the use of descriptive statistics limits the extent to which associations between clinical characteristics and recurrence can be inferred.

In our cohort, recurrent HHMA episodes occurred in only a relatively small number of patients. The limited number of recurrent cases substantially reduces the statistical power and makes formal comparative analyses between recurrent and non-recurrent cases potentially unstable and susceptible to type II error. For this reason, we have avoided presenting the association between the F508del genotype and recurrence as a definitive statistical association and have revised the corresponding statements throughout the manuscript to reflect this limitation. The association is now presented as an observed pattern that warrants confirmation in larger cohorts rather than as an established relationship.

We have also clarified the primary focus of the present version of the manuscript, which is the genetic characterization of patients with HHMA. In response to the Reviewer's suggestion, we expanded the genetic analysis to provide a detailed comparison of the CFTRgenotypic profile between patients with HHMA and those without HHMA within the same reference-center population. This comparative analysis provides additional context for the observed predominance of F508del among patients with HHMA and allows the genetic findings to be interpreted in relation to the broader CF population treated at our center.

Accordingly, the revised manuscript places greater emphasis on the comparative CFTR genetic profile of patients with and without HHMA, while interpreting recurrence-related findings cautiously because of the limited number of recurrent cases.

 

1. The conclusion that HHMA is associated with male sex is not sufficiently supported. There is no comparison with the sex distribution of the full cystic fibrosis cohort. The higher number of male HHMA cases may simply reflect the underlying cohort composition. Please provide the sex distribution of all 257 patients and perform an appropriate comparison.

Reply: Thank you for this important comment. In accordance with the reviewer’s suggestion, we have added the sex distribution of the entire CF cohort (n = 257), comprising 135 males and 122 females, and performed a comparative analysis between patients with and without HHMA. The corresponding results have been added to the revised manuscript and presented in the comparative table. Importantly, we have also revised the interpretation of the association with male sex to avoid overstating the finding. The higher proportion of males among patients with HHMA is now interpreted in the context of the sex distribution of the overall CF cohort, and we have avoided implying a causal relationship. This comparative analysis provides a more appropriate assessment of whether the observed sex distribution among HHMA cases differs from that expected based on the underlying CF population.

 

1. The genotype analysis needs improvement. The manuscript combines several different genotype categories, including patients with missing or unidentified alleles. Please clearly report the number of fully genotyped patients and separate confirmed genotypes from incomplete genetic information. Claims regarding “severe phenotypes” should be moderated unless supported by genotype–phenotype analysis.

Reply: Thank you for this important comment. The genotype analysis has been revised and expanded in the current version of the manuscript. We have included the available CFTR genotypes for all patients in the cohort, both those with HHMA and those without HHMA, allowing a direct comparison of the genetic profiles between the two groups.

In addition, we performed two complementary approaches to genotype classification. First, patients were categorized according to the presence of the F508del variant, including F508del/F508del, F508del/other variant, and other genotypes. Second, the identified CFTR variants were grouped according to the functional classes I–VI, as described in the literature, with classes I–III generally representing variants associated with more severe CFTR dysfunction. Genotypes with incomplete, unidentified, or unavailable allelic information were explicitly identified and were not treated as confirmed specific genotypes.

Accordingly, the revised manuscript now clearly distinguishes fully characterized genotypes from incomplete genetic information and provides the detailed genotype distribution for both patients with and without HHMA. We have also moderated the interpretation of genotype severity and now describe the findings in terms of the distribution of F508del and CFTR functional classes rather than assuming a severe phenotype solely on the basis of genotype. This approach provides a more appropriate genotype–phenotype assessment while avoiding conclusions that are not directly supported by the available data.

 

1. The seasonality analysis should account for repeated episodes. It is unclear whether the percentages refer to patients or total HHMA episodes. Because some patients had multiple episodes, each episode should be analyzed separately, or the authors should clearly explain how recurrent events were counted.

Reply: Thank you for this important comment. The seasonality analysis has been clarified in the revised manuscript. The information is now presented in greater detail in the corresponding table, with the denominator and unit of analysis clearly specified. In addition, the figure legend has been revised to explicitly describe how recurrent HHMA episodes were accounted for in the seasonal analysis. Thus, the reader can distinguish the distribution of patients from the distribution of HHMA episodes and understand how recurrent events contributed to the reported seasonal pattern.

 

1. The microbiology section appears disconnected from the main study question. Airway colonization is described, but no analysis shows whether colonization was associated with HHMA severity, recurrence, or treatment. The authors should either perform such analyses or shorten this section and avoid suggesting a causal relationship.

Reply: Thank you for this valuable comment. We agree that the microbiological findings were not directly related to the primary objectives of the study. Therefore, rather than performing additional analyses that would not be adequately supported by the available sample size, we have shortened and streamlined this section in the revised manuscript. The microbiological information is now presented only as a descriptive characteristic of the cohort, and any wording that could imply a causal or clinically significant association between airway colonization and HHMA severity, recurrence, or treatment has been removed.

 

1. The manuscript contains conflicting statements about informed consent. The Methods state that written informed consent was obtained, while the ethics section states that consent was waived. This must be corrected.

Reply: We sincerely apologize for this inconsistency in the manuscript. We confirm that written informed consent was obtained from all participants or their legal guardians, as applicable. The statement indicating that informed consent was waived was included inadvertently and was incorrect. We have corrected the ethics and informed consent statements throughout the revised manuscript to ensure consistency.

 

Informed Consent Statement: Written informed consent was obtained from all individual participants included in the study or from their legal guardians in the case of minors or individuals unable to provide consent.

 

1. Figure 3 should be revised. The figure is visually attractive but includes conclusions such as genotype linkage and diagnostic implications that are not fully demonstrated by this study. It also appears different in style from the other figures. The authors should simplify the figure, remove unsupported statements, and ensure that all graphical elements are scientifically accurate.

Reply: Thank you for this valuable comment. We agree that Figure 3 included graphical elements and statements that could be interpreted as extending beyond the evidence directly supported by our data. To avoid potential misinterpretation and ensure that the manuscript remains focused on the findings supported by the study, we have removed Figure 3 from the revised manuscript.

In addition, we have refined the overall focus of the study to emphasize the clinical characterization of HHMA and, particularly, the CFTR genetic profile of affected patients and its comparison with the pathogenic CFTR variant profile of the broader cystic fibrosis cohort followed at our Brazilian reference center. Accordingly, the revised manuscript avoids graphical or interpretative statements regarding genotype–phenotype relationships that are not directly demonstrated by the available data.

 

1. The Discussion should better distinguish the present findings from previous knowledge. The authors should explain what this cohort adds beyond earlier reports of pseudo-Bartter syndrome in cystic fibrosis. The possible roles of host responses, infection, and epithelial regulation may also be discussed more carefully. Relevant examples include studies showing that host RNA-binding proteins can regulate infection-associated epithelial apoptosis and epithelial–mesenchymal changes:Frontiers in Immunology. 2024;15:1431207. eLife. 2025;14:e107132.

Reply: We thank the Reviewer for this important comment. We agree that the Discussion should more clearly distinguish the findings of the present cohort from previously published reports of pseudo-Bartter syndrome in cystic fibrosis. Accordingly, the Discussion has been revised to better emphasize the contribution of our study, particularly the characterization of 41 HHMA cases identified over a 10-year period in a Brazilian CF referral center, including the description of recurrence, seasonal distribution, electrolyte abnormalities, clinical characteristics, and, importantly, the detailed comparison of the CFTR genotype profile between patients with and without HHMA. The revised manuscript also includes a broader analysis of F508del status and CFTR variant classes, providing additional information on the genetic profile of patients with HHMA within the context of the broader CF population followed at our center.

We also expanded the Discussion to acknowledge that HHMA is likely influenced by multiple interacting factors, including salt and fluid losses, environmental temperature, age, nutritional status, pancreatic function, intercurrent respiratory or gastrointestinal conditions, and individual epithelial ion-transport regulation. In response to the Reviewer’s suggestion, the cited studies regarding host RNA-binding proteins, infection-associated epithelial apoptosis, and epithelial–mesenchymal changes (Frontiers in Immunology. 2024;15:1431207; eLife. 2025;14) were considered in the revised discussion as potential mechanistic context. However, we have carefully avoided implying a direct association between these mechanisms and HHMA, as host responses, RNA-binding proteins, epithelial apoptosis, and epithelial–mesenchymal changes were not directly evaluated in our study.

Thus, these mechanisms are presented as potential areas for future investigation rather than as explanations demonstrated by the present dataset. We believe this revision better defines the novel contribution of our cohort while placing the findings within the broader biological context of CF epithelial dysfunction and infection-related responses.

 

Importantly, the present study extends previous reports by providing a longitudinal characterization of HHMA in a relatively large cohort of individuals with CF followed at a single Brazilian referral center over a 10-year period. Beyond estimating the prevalence of HHMA, our study describes its age distribution, recurrence, seasonal pattern, clinical and biochemical characteristics, treatment, and, particularly, its genetic profile. The revised analysis includes a detailed comparison of CFTR genotypes between individuals with and without HHMA, including F508del status and the distribution of variants according to functional classes. This approach provides additional information on the genetic background of HHMA within the broader CF population and may help clarify potential genotype-related susceptibility to salt-wasting episodes. Nevertheless, the multifactorial nature of HHMA suggests that genetic background alone is unlikely to fully explain its occurrence. Host factors, epithelial ion transport, environmental exposure, nutritional status, and intercurrent infections or gastrointestinal losses may interact to determine the clinical expression of salt and fluid depletion.  

Emerging evidence also suggests that epithelial responses to infection and inflammation may involve complex regulatory mechanisms that extend beyond CFTR dysfunction itself. Studies investigating host RNA-binding proteins have demonstrated their potential role in regulating infection-associated epithelial apoptosis and epithelial–mesenchymal changes [35]. Although these mechanisms were not directly evaluated in the present study and no causal relationship with HHMA can be established from our data, they provide a broader biological context in which host responses, epithelial integrity, and infection-related processes may potentially influence the susceptibility or clinical expression of electrolyte disturbances in CF. Future studies integrating CFTR genotype, epithelial function, inflammatory responses, infection status, and environmental exposure may help determine whether these pathways contribute to the heterogeneity of HHMA.

 

Minor comments

1. Correct “Cystic Fibrosis Transmembrane Regulator” to “Cystic Fibrosis Transmembrane Conductance Regulator” throughout.

Reply: We sincerely apologize for this terminology error. The name of the gene/protein has been corrected from “Cystic Fibrosis Transmembrane Regulator” to “Cystic Fibrosis Transmembrane Conductance Regulator” in the two occurrences identified in the manuscript. We thank the reviewer for bringing this to our attention.

 

1. Report continuous variables using median and interquartile range when distributions are not normal.

Reply: We thank the reviewer for this important comment. In accordance with the recommendation, continuous variables with non-normal distributions are now reported as median and interquartile range (IQR). Accordingly, the laboratory findings in the manuscript and the corresponding tables have been revised to present pH, sodium, potassium, chloride, and bicarbonate as median (IQR) values rather than means.

 

1. Define whether electrolyte measurements were collected before treatment.

Reply: Thank you for this important comment. We confirm that all electrolyte measurements reported in this study were obtained before the initiation of treatment for HHMA. We have clarified this information in the Materials and Methods and in the description of the biochemical profile in the Results section.

 

Methods:

All electrolyte and acid–base measurements included in the analysis were obtained at the time of HHMA diagnosis and before the initiation of intravenous or oral fluid, electrolyte, or other specific treatment for the episode.

 

1. Check the units for sodium, potassium, chloride, and bicarbonate and use them consistently.

Reply: We thank the reviewer for this comment. We carefully checked the units for sodium, potassium, chloride, and bicarbonate and standardized their presentation throughout the manuscript. All values are now consistently reported in mmol/L in the text and corresponding tables.

 

1. Simplify Table 1, which is difficult to read because of the large number of abbreviations.

Reply: Thank you for this helpful comment. We agree that the original Table 1 was difficult to read because it combined a large number of clinical, treatment, and recurrence variables and relied extensively on abbreviations. To improve clarity and facilitate interpretation, we have reorganized the individual-level information into separate tables according to clinical domain. The revised tables use full terms whenever possible and substantially reduce the use of abbreviations. Clinical characteristics and presentation are now presented separately from treatment and recurrence data, while biochemical measurements are presented in a dedicated table. We believe this restructuring provides a more accessible and didactic presentation of the individual patient-level data while preserving the information relevant to this case series.

 

1. Avoid repeating the same findings in the Abstract, Results, Discussion, Figure 3, and Conclusion.

Reply: We thank the reviewer for this comment. To reduce redundancy and improve the overall presentation of the manuscript, Figure 3 has been removed. We also revised the Abstract, Results, Discussion, and Conclusion to minimize repetition and ensure that each section presents the findings according to its specific purpose.

 

1. Revise the English for grammar, punctuation, and sentence length.

Reply: We thank the reviewer for this comment. The manuscript has been carefully revised for English grammar, punctuation, clarity, and sentence structure. Long and complex sentences were also revised and, where appropriate, divided into shorter sentences to improve readability and overall clarity.

 

Recommendation

The study contains clinically useful observations, but the study classification, diagnostic reporting, statistical analysis, ethics statements, and interpretation require substantial revision. I recommend major revision

Reply: We sincerely thank the Reviewer for the comprehensive and constructive assessment of our manuscript. We appreciate the recognition of the clinical relevance of our observations and the opportunity to substantially revise the manuscript.

In response to the Reviewer’s recommendation, we have undertaken a major revision addressing the study classification, diagnostic criteria, statistical analysis, ethics statements, presentation of laboratory findings, interpretation of the results, and overall clarity of the manuscript.

Specifically, the study is now consistently described as a retrospective case series, replacing the previous terminology of “serial case report.” We have clarified the diagnostic criteria for HHMA, including the definition of metabolic alkalosis and the interpretation of respiratory compensation. Laboratory findings are now consistently reported using median and interquartile range (IQR) when appropriate, and the units for sodium, potassium, chloride, and bicarbonate have been standardized to mmol/L.

We have also strengthened the comparative genetic analysis by including the broader CF cohort followed at our reference center and have revised the interpretation of CFTR genotype findings, recurrence, sex distribution, and other potential risk factors to avoid unsupported causal or associative conclusions. The sex distribution of the complete cohort was added, and the limitations imposed by the relatively small number of recurrent cases are now explicitly acknowledged.

The manuscript has also been revised to provide greater detail regarding the temporal relationship between HHMA and CF diagnosis, the availability of CFTR-modulator therapy in Brazil, preventive strategies, climatic and seasonal considerations, and the comparison of our findings with previous Brazilian and international reports. The relevant references suggested by the Reviewers have been incorporated into the revised manuscript.

Regarding the microbiological findings, we have shortened this section and now present these data only as descriptive clinical information, without implying associations with HHMA severity, recurrence, or treatment that were not directly evaluated. Similarly, unsupported genotype–phenotype interpretations have been moderated.

The ethics statements have been carefully reviewed and corrected to ensure consistency, including clarification that written informed consent was obtained from participants or their legal guardians, as applicable. We have also substantially revised the tables to improve readability and reduced redundancy throughout the manuscript.

Importantly, Figure 3 has been removed in response to the concerns regarding redundancy, graphical interpretation, and statements that could extend beyond the evidence provided by the study.

Finally, the entire manuscript has undergone extensive English-language revision, including grammar, punctuation, sentence structure, clarity, and sentence length.

We believe that these comprehensive revisions have substantially strengthened the methodological rigor, transparency, and scientific interpretation of the manuscript. We have carefully addressed all comments raised by the Reviewers and believe that the revised version provides a more balanced and clinically relevant contribution to the literature on HHMA in cystic fibrosis.

We sincerely thank the Reviewers and the Editorial Team for their valuable comments and consideration of our revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

good to publish

Author Response

Thank you for the positive assessment. We sincerely appreciate your evaluation and recommendation that the manuscript is suitable for publication.