Date Published: October 4, 2016
Publisher: Public Library of Science
Author(s): Nicola Casali, Agnieszka Broda, Simon R. Harris, Julian Parkhill, Timothy Brown, Francis Drobniewski, John Z Metcalfe
Abstract: BackgroundA large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster.Methods and FindingsWe sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998–22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation.ConclusionsOur study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases.
Partial Text: A cluster of isoniazid-monoresistant tuberculosis cases centred on North London was initially detected in 2000 . Molecular typing based on IS6110 restriction fragment length polymorphism (RFLP) analysis confirmed the isolates comprised a putative outbreak, and retrospective investigation revealed that the first cluster case probably occurred in 1995. RFLP-based typing of isoniazid-monoresistant isolates and, later, routine 24-loci mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing showed that by 2008, there were 343 culture-proven cases, 299 (87%) of which were diagnosed in London . By the end of 2013, the cluster comprised 501 individuals nationally . Compared to other tuberculosis cases in London, outbreak cases were more likely to be United Kingdom born (53%) and of white or black Caribbean ethnicity. Patients were from a wide social background with foci in high risk groups, including the homeless, injection-drug users, and prisoners [1,4,5].
To our knowledge, this is the largest WGS-based study of an outbreak of M. tuberculosis reported to date. Analysis of 344 isolates, sharing an identical 24-loci VNTR profile molecular type, collected from individual patients over 14 y, revealed that 96 (38%) were indistinguishable, and only one differed by more than five SNPs from this clone. Contrary to that observed in some recent studies, WGS was able to reveal the direction of transmission of tuberculosis in only a small proportion (16/344) of cases within the outbreak. We observed the acquisition and spread of a rare RpoB mutation conferring rifampicin resistance that supported the conclusions of a public health investigation into this MDR cluster . Despite the overall lack of variation in isolates between patients, intra-patient isolate diversity was detected in longitudinal isolates (up to 4 SNPs in 4 y), across specimen sites (up to 1 SNP in contemporary isolates), and within a single specimen (10 SNPs in 27 individual colonies).