PREreview of Evolution of Pandemic Cholera at its Global Source

Summary and overall impression

This fascinating and data-rich paper tackles a long-held assumption: Bangladesh, particularly the Ganges Delta, is the epicenter of global cholera outbreaks. With support from an impressive dataset of over 2,300 samples from Bangladesh and Northern India, the authors argue that we may need to shift our understanding that the upper Ganges basin, especially parts of India, might be playing a bigger role in seeding global cholera strains.

The sections dealing with the evolution of sub-lineages, the gain/loss of phage defence elements, and their potential links to disease severity are all exciting. However, some claims feel a bit stronger than the data alone can carry, and the writing is dense or hard to follow in places. This can be an impactful piece of work with clearer framing and a bit more caution in the interpretations.

Abstract

•  Minor Flaw: The abstract lacks a focused framing around the key research question.

Suggestion: Reframe the abstract around the central hypothesis: “Does the upper Ganges basin in India play a larger role than Bangladesh in global cholera spread?”

•  Minor Flaw: Phrases like “mirrored gain/loss of anti-defence systems” are difficult for non-specialist readers.

Suggestion: Use simpler, clearer wording. Example: “We found repeating patterns in the presence or absence of genes that help cholera bacteria evade viruses, which may affect how strains spread and evolve.”

Introduction

• Major flaw: The introduction does not clearly state the paper’s key argument that India’s upper Ganges basin may be more central to global cholera spread than Bangladesh. This weakens the framing of the study.

Suggestion: Add a clear sentence early in the introduction, such as: “This study explores whether India’s upper Ganges basin, rather than the Ganges delta in Bangladesh, may be the primary source of pandemic cholera lineages.”

• Major Flaw: The terms Ganges delta and Ganges basin are used without explanation, though they are crucial to the paper’s geographic argument. Readers unfamiliar with the region may not grasp the difference.

Suggestion: Briefly define both terms early in the introduction. For example: “The Ganges delta refers to the coastal region in Bangladesh and West Bengal, while the Ganges basin spans a broader area upstream in northern India.”

• Minor Flaw: The introduction is packed with background information in long paragraphs, which can overwhelm the reader and dilute the paper’s focus.

Suggestion: Break the introduction into shorter paragraphs, each focused on one theme:

-Global burden of cholera

- Importance of genomic surveillance

-The Ganges region as a historical hotspot

-Hypothesis about India’s upstream role

Methods

Strength:

• The authors use an impressive number of genomes (~2,300), which provides strong

statistical power and broad representation across regions.

• Tools like Snippy, Gubbins, IQ-TREE, and BEAST are widely accepted and standard

for microbial genomic epidemiology.

• The authors go beyond phylogenies to look at functional genes and structural elements,

adding depth to the evolutionary interpretation.

Major Flaw: The methods are described in long, text-heavy blocks that are hard to follow. This reduces clarity, especially for readers who want to understand or replicate the study.

Suggestion: Use a visual flowchart or a clear step-by-step breakdown to show the analytical process. This will help readers quickly grasp the sequencing, filtering, alignment, and phylogenetic methods.

Minor Flaw: The paper mentions “high-quality genomes” but does not define the criteria used to include or exclude them. This limits transparency.

Suggestion: Briefly explain what filtering thresholds or quality control steps were applied (e.g., coverage depth, assembly metrics, contamination filtering).

Results

Major Flaw: Some claims in the Results appear to draw causal inferences from associational data.

For example, links between gene deletions (e.g., phage defense islands) and disease severity or spread are discussed as they drive these outcomes. However, the paper does not present statistical models (e.g., regression analysis, adjusted comparisons) to support causality, only temporal and genomic associations.

Suggestion: Reframe these claims as hypotheses rather than conclusions unless statistical tests directly support causation. For instance, clarify that observed patterns are correlations, and highlight the need for functional studies or adjusted models to test causal mechanisms.

Minor Flaw: While the genomic analysis is impressive, the section contains too much information without enough structure. Key findings get lost in the detail.

Suggestion: Break the results into smaller, clearly labelled sections. Add summary sentences at the end of each key point (e.g., “This suggests X sub-lineage may have emerged independently in Y region”). This will help readers follow the significance of each result.

Discussion

Major Flaw: The paper strongly frames India as the main source of global cholera spread, but the data do not fully support this. It’s a problem as there has been no adjustment for sampling bias or differences in surveillance systems across regions.

Suggestion: Soften the language and add nuance. Example edit: “These findings suggest the upper Ganges basin may play a significant role in cholera spread, though differences in sampling and surveillance must be considered.”

Minor flaw: The discussion overlooks key social and ecological drivers such as human mobility, trade, water systems, and healthcare access.

Suggestion: Add a short paragraph acknowledging these influences.

Example: “Cholera spread is also shaped by human behaviour and infrastructure, which should be considered in future analyses.”

Minor flaw: The discussion jumps between ideas without a clear structure.

Suggestion: Use short sub-sections or clearer paragraph breaks by topic:

-Phage pressure

-Regional comparisons

- Spread/export

-Limitations

End each theme with a summary sentence. Example: “Cholera spread is also shaped by human behaviour and infrastructure, which should be considered in future analyses.”

Conclusion

Minor Flaw: The conclusion summarises the paper well, but doesn’t clearly state what should happen next.

Suggestion: Add 2–3 specific priorities. Example: “To improve future cholera preparedness, countries should expand local sequencing infrastructure, invest in phage surveillance and prioritise regional data sharing networks”

Minor Flaw: The ending feels soft. A sharper final statement could reinforce the paper’s significance.

Suggestion: End with a strong call to action or takeaway message. Example: “Cholera’s future spread may hinge not only on pathogens but also on how quickly we act to track, share, and respond.”

Final Note: This is an important study with the potential to shift how we think about the global spread of cholera. The data is impressive, the methods are strong, and the big-picture questions are worth asking. However, the paper would benefit from pulling back a bit in its conclusions, simplifying some dense sections, and sharpening its main message.

Competing interests

The authors declare that they have no competing interests.