Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study

Abstract

In an outbreak investigation of Mycobacterium tuberculosis comparing whole genome sequencing (WGS) with traditional genotyping, Stefan Niemann and colleagues found that classical genotyping falsely clustered some strains, and WGS better reflected contact tracing.

Abstract

Background

Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains.

Methods and Findings

During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed “ Hamburg clone”) started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance.

Conclusions

Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context.

Please see later in the article for the Editors' Summary

Editors' Summary

Background

Tuberculosis—a contagious bacterial disease that usually infects the lungs—is a major public health problem, particularly in low- and middle-income countries. In 2011, an estimated 8.7 million people developed tuberculosis globally, and 1.4 million people died from the disease. Tuberculosis is second only to HIV/AIDS in terms of global deaths from a single infectious agent. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is readily spread in airborne droplets when people with active disease cough or sneeze. The characteristic symptoms of tuberculosis include persistent cough, weight loss, fever, and night sweats. Diagnostic tests for the disease include sputum smear analysis (examination of mucus coughed up from the lungs for the presence of M. tuberculosis), mycobacterial culture (growth of M. tuberculosis from sputum), and chest X-rays. Tuberculosis can be cured by taking several antibiotics daily for at least six months, although the recent emergence of multidrug-resistant M. tuberculosis is making tuberculosis harder to treat.

Why Was This Study Done?

Although efforts to reduce the global burden of tuberculosis are showing some improvements, the annual decline in the number of people developing tuberculosis continues to be slow. To develop optimized control strategies, experts need to be able to accurately track M. tuberculosis transmission within human populations. Because M. tuberculosis, like all bacteria, accumulates genetic changes over time, there are many different strains (genetic variants) of M. tuberculosis. Genotyping methods have been developed that identify different bacterial strains by examining specific regions of the bacterial genome (blueprint), but because these methods examine only a small part of the genome, they may not distinguish between related transmission chains. That is, traditional strain genotyping methods may not be able to determine accurately where a tuberculosis outbreak started or how it spread through a population. In this longitudinal cohort study, the researchers compare the ability of whole genome sequencing (WGS), which is rapidly becoming widely available, and traditional genotyping to provide information about a recent German tuberculosis outbreak. In a longitudinal cohort study, a population is followed over time to analyze the occurrence of a specific disease.

What Did the Researchers Do and Find?

During long-term (1997–2010) population-based molecular epidemiological surveillance (disease surveillance that uses molecular techniques rather than reports of illness) in Hamburg and Schleswig-Holstein, the researchers identified a large tuberculosis outbreak caused by M. tuberculosis isolates of the Haarlem lineage using classical strain typing. The researchers examined each of the 86 isolates from this outbreak using WGS and classical genotyping and asked whether the results of these two approaches correlated with contact tracing data (information is routinely collected about the people a patient with tuberculosis has recently met so that these contacts can be tested for tuberculosis and treated if necessary) and with the spatio-temporal distribution of outbreak cases. WGS of the isolates identified 85 single nucleotide polymorphisms (SNPs; genomic sequence variants in which single building blocks, or nucleotides, are altered) that subdivided the outbreak into seven clusters of isolates and 36 unique isolates. The WGS results showed that the first isolates of the outbreak were incorrectly clustered by classical genotyping and that one strain—the “ Hamburg clone”—started expanding in 1998. Notably, the genome-based clustering patterns were in better accordance with contact tracing data and with the geographical distribution of cases than clustering patterns based on classical genotyping, and they identified eight confirmed human-to-human transmission chains that involved 31 patients and a maximum of three SNPs. Finally, the researchers used their WGS results to estimate that the Hamburg clone emerged between 1993 and 1997, shortly before the discovery of the tuberculosis outbreak through epidemiological surveillance.

What Do These Findings Mean?

These findings show that WGS can be used to identify specific strains within large tuberculosis outbreaks more accurately than classical genotyping. They also provide new information about the evolution of M. tuberculosis during outbreaks and indicate how WGS data should be interpreted in future genome-based molecular epidemiology studies. WGS has the potential to improve the molecular epidemiological surveillance and control of tuberculosis and of other infectious diseases. Importantly, note the researchers, ongoing reductions in the cost of WGS, the increased availability of “bench top” genome sequencers, and bioinformatics developments should all accelerate the implementation of WGS as a standard method for the identification of transmission chains in infectious disease outbreaks.

Additional Information

Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001387.

• The World Health Organization provides information (in several languages) on all aspects of tuberculosis, including the Global Tuberculosis Report 2012

• The Stop TB Partnership is working towards tuberculosis elimination; patient stories about tuberculosis are available (in English and Spanish)

• The US Centers for Disease Control and Prevention has information about tuberculosis, including information on tuberculosis genotyping (some information in English and Spanish)

• The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis

• The Tuberculosis Survival Project, which aims to raise awareness of tuberculosis and provide support for people with tuberculosis, provides personal stories about treatment for tuberculosis; the Tuberculosis Vaccine Initiative also provides personal stories about dealing with tuberculosis

• MedlinePlus has links to further information about tuberculosis (in English and Spanish)

• Wikipedia has a page on whole-genome sequencing (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)