Objective:

To determine how tuberculosis (TB) cases diagnosed in Almería and Madrid were connected by transmission, using integrated genomic data, detailed epidemiological interviews, and targeted strain sequencing.

Approach:

• Sample Collection: Researchers combined isolates and sequencing data from TB surveillance in Almería with available data and stored isolates from Madrid.
• DNA Extraction and Purification: DNA was extracted from inactivated positive cultures using the QIAamp DNA Mini Kit. Stored Madrid cultures used for retrospective tracking were heat-inactivated for PCR.
• Genomic Analysis: Illumina and nanopore sequencing were used to analyze isolates and examine differences among related strains.
• Cluster Analysis: Researchers compared single-nucleotide polymorphism (SNP) differences and combined genomic findings with detailed interviews to investigate links among cases.
• Multiplex PCR for Targeted Sequencing: A strain-specific assay targeted marker SNPs in Madrid isolates; whole-genome sequencing confirmed newly identified cluster cases.

Key Findings:

• The integrated investigation identified a 13-case cluster spanning Almería and Madrid. Interviews revealed family connections, movement between the regions, and possible exposures at social gatherings that were not apparent from initial investigations.
• One patient had symptoms for at least a year before diagnosis and advanced TB at presentation. The authors identified this prolonged diagnostic delay as a likely contributor to extensive transmission.
• Targeted sequencing of 164 Madrid isolates identified four additional candidate cases, subsequently confirmed as part of the cluster by whole-genome sequencing.

Interpretation:

Combining data across regions with genomic analysis and more detailed interviews clarified connections that geographically separate investigations had missed.

Limitations:

• Madrid did not have population-wide genomic surveillance, so additional cases may have gone undetected.
• Detailed follow-up interviews were unavailable for some Madrid cases, leaving their specific exposure links unresolved.

Conclusion:

This cluster illustrates how mobility within one country and prolonged diagnostic delay can complicate TB transmission investigations. The authors call for genomic and epidemiological surveillance that extends across regional boundaries.