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Sequencing Offers Look at Tuberculosis Diversity, Spread in HIV-Infected Individuals

Mycobacterium tuberculosis, scanning electron micrograph

NEW YORK (GenomeWeb) – Sequencing samples from different parts of the body can uncover new mutations in Mycobacterium tuberculosis bacteria that are useful for understanding the pathogen's diversity and spread in individuals infected with both M. tuberculosis and HIV, new research suggests.

Researchers from the US, South Africa, and Israel used whole-genome sequencing to profile M. tuberculosis isolates in thousands of post-mortem samples taken from the lung, liver, spleen, and other body sites in dozens of South African individuals with TB who had been treated for fewer than four days before their deaths. Most of the deceased individuals were also infected with HIV. The findings, published in Nature Medicine, suggest that M. tuberculosis can move about as easily between organs as it can inside each lung in HIV-infected individuals.

"Extensive sampling from multiple body sites and detection of minor alleles within each sample shows that even the notoriously slowly evolving M. tuberculosis presents substantial within-patient diversity," the authors wrote. "The distribution of this diversity across the body suggests that, at least in people with HIV, dissemination from the lungs follows a similar mechanism as dissemination from the lungs to extra-pulmonary organs."

The researchers reasoned that it should be possible to retrace M. tuberculosis movement within the lungs of individuals with TB and from the lungs to other parts of the body — information that could offer clues to the pathogen's ability to infect and evolve within infected individuals.

"In theory, the same principles that are used to track spread across the globe and between individuals can inform our understanding of how M. tuberculosis spreads within the lungs and throughout the body," they explained, noting that "[a]lthough M. tuberculosis primarily causes lung infections, it can disseminate to extra-pulmonary organs, particularly in subjects who are co-infected with HIV."

The team collected minimally invasive autopsy samples from 100 deceased individuals with TB in KwaZulu-Natal, South Africa who had been treated for three days or less. Consistent with patterns in the region, all but four of the deceased individuals were also infected with HIV.

Using samples from three sites per lung and pooled samples from the liver, spleen, and endotracheal aspirate, the researchers attempted to culture M. tuberculosis prior to sequencing, narrowing their analyses to 44 individuals for whom lung cultures and cultures from another body site could be produced, including 42 HIV-positive individuals.

All told, the team considered 2,693 samples from 329 body sites in these individuals — isolates sequenced at Rockville, Maryland-based Macrogen with Illumina's HiSeq 2000. There, samples were sequenced to between 59-fold and 178-fold coverage, on average.

When they analyzed these sequences, the researchers uncovered infections involving multiple M. tuberculosis strains in four of the individuals, though there was also evidence of sub-lineages formed due to intra-individual de novo M. tuberculosis mutations occurring over as many as 16 years.

Multiple M. tuberculosis genotypes did exist in the individuals, moving both within the lungs and to other body sites with comparable difficulty or ease, the authors explained. "These results support the hypothesis that both intra-lung and inter-organ spread are mediated by similar dissemination mechanisms."