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Whole-Genome Sequencing Allows for More Precise Tuberculosis Tracking During an Outbreak

NEW YORK (GenomeWeb News) – Whole-genome sequencing appears to be better than standard strain typing for tracing tuberculosis outbreaks, according to a paper published yesterday in PLOS Medicine.

The researchers, led by Stefan Niemann from Forschungszentrum Borstel, used both whole-genome sequencing and genotyping to analyze 86 cases from a long-term tuberculosis outbreak in Germany, finding that whole-genome sequencing analysis more closely aligned with contact tracing data and the known spread of the disease through the city of Hamburg and neighboring Schleswig-Holstein during that time. Further, whole-genome sequencing allowed the researchers to more specifically identify the strains of Mycobacterium tuberculosis involved in the outbreak.

"Only genome-based investigations allowed us to trace the spread of M. tuberculosis with the resolution needed to visualize transmission patterns correctly," Andreas Rötzer, from Forschungszentrum Borstel and the first author of the study, said in a statement.

The researchers noted that to develop better ways to control tuberculosis outbreaks, improved and more refined surveillance of the disease is needed to track its spread, and the spread of specific strains, through human populations.

In this study, using classical strain typing, Niemann, Rötzer, and their colleagues first homed in on a cluster of 86 tuberculosis cases, caused by a particular strain of the bacterium, that cropped up in Hamburg and Schleswig-Holstein during the 1997 to 2010 tuberculosis outbreak there. The researchers analyzed those cases using DNA fingerprinting, spoligotyping, and 24-locus MIRU-VNTR genotyping. They also sequenced the samples using an Illumina platform.

Contact tracing data, which was collected by public health officials using standard questionnaires, indicated that the cases in this cluster arose due to eight multiple transmission chains, which, the researchers reported, was not reflected in the genotyping data.

However, using whole-genome sequencing, the researchers identified 85 SNPs that could be used to carve the cases up into seven sub-clusters and 36 unique cases. A majority of those SNPs, the researchers noted, were non-synonymous.

Most of the isolates grouped together into one clade, which the researchers dubbed the Hamburg clone. Further, they found that the Hamburg clone was first isolated in 1998 and remained part of the outbreak through the end of the study, while other isolated clones had a smaller expansion and led to smaller clusters of cases.

"These data revealed that [whole-genome sequencing]-based analysis resolved the 86 isolates at a much higher discriminatory level than classical genotyping, and was, in addition, in accordance with the temporal and spatial expansion pattern," the researchers wrote.

The contact tracing data further indicated that there were links among 31 patients, forming eight different transmission chains. Of those the isolates from those 31 patients, 19 had no SNP differences between them. The other 12 differed by, at most, three SNPs.

"[W]hile whole-genome variability detected among the outbreak isolates was high enough to resolve the outbreak, the level of genome-wide variation in definite transmission chains was very low, limited to no more than three SNPs," the researchers added.

Drawing on a mutation rate for M. tuberculosis determined using a macaque model of the disease, the researchers traced the Hamburg clone to its most recent common ancestor, which they pinpointed to 1995, a few years prior to the onset of the outbreak.

Since the Hamburg clone appeared to preferentially expand over that span of time in the patient population, the researchers examined whether there were any social or environmental factors, such as HIV status or alcohol abuse, that distinguished the Hamburg clone patient populations from other tuberculosis patients, but they found no differences between the patient populations. They also found no evidence of a "super-spreader" of the disease, though they could not fully exclude that possibility.

The researchers then selected one isolate of the Hamburg tuberculosis clone for further analysis. The isolate, called 71199/99, was subjected to whole-genome sequencing using the 454 platform, with both standard and paired-end reads, and its sequence was manually annotated. As compared to the reference, this isolate had a larger genome and it contained a number of genomic rearrangements and a few polymorphisms affecting coding regions.

"We hypothesize that the increasing number of cases caused by the Hamburg clone might have resulted from some of the small genetic changes that accumulated before the initial spread … which possibly enhanced the fitness of this particular clone," the researchers said. "However, this hypothesis would be difficult to prove, even using available experimental models of infection."

Overall, the researchers said that their study underscores the potential for whole-genome sequencing to aid in tuberculosis surveillance and control, as well as for other infectious diseases.