Use of Genome Level-Informed PCR as a New Investigational Approach for Analysis of Outbreak-Associated Mycobacterium tuberculosis Isolates

Abstract

Mycobacterium tuberculosis strain CH, the index isolate linked to a major tuberculosis outbreak associated with high levels of transmissibility and virulence, was characterized by microarray analysis by use of a PCR product array representative of the genome of M. tuberculosis strain H37Rv. Seven potential genomic deletions were identified in CH, five of which were confirmed by PCR analysis across the predicted deletion points. The panel of five PCRs required to individually interrogate these loci was collectively referred to as the genome level-informed PCR (GLIP) assay. GLIP analysis was performed with CH, 12 other epidemiologically linked isolates, and 43 recent, non-outbreak-associated isolates derived from patients within the local area. All 13 outbreak-linked isolates showed a profile corresponding to the presence of all five deletions. These 13 isolates were also found to share common variable-number tandem repeat and mycobacterial interspersed repetitive unit profiles. None of the 43 non-outbreak-associated isolates exhibited the five-deletion profile. Although three individual deletions were present in upwards of 44% of the non-outbreak-associated isolates, no single-deletion isolates were detected. Interestingly, none of these deletions had been previously recognized, and sequence analysis of the immediate flanking regions in CH failed to identify a likely mechanism of deletion for four of the five loci. The GLIP assay also proved valuable in ongoing surveillance of the outbreak, rapidly identifying a further two outbreak-associated cases months after the initial cluster and, importantly, dismissing a further 12 epidemiologically suspect cases, which allowed the optimum deployment of public health resources.Mycobacterium tuberculosis remains a major killer, accounting for more than 2 million deaths annually. Its success as a pathogen is highlighted by its remarkable ability to spread among human populations. It is estimated that more than 2 billion people, a third of the world's population, are infected with this bacterium (29). The major burden is borne by underdeveloped nations. However, even in settings with excellent public health and clinical facilities, sporadic outbreaks of tuberculosis remain a continuing threat (7,23).In 2001 the largest recognized outbreak of tuberculosis in a United Kingdom school was detected in Leicester. The index patient was a 14-year-old student who had been complaining of a chronic cough for 9 months prior to being diagnosed with sputum smear-positive cavitary pulmonary tuberculosis (9). Subsequent screening and investigation by the health authority of the entire school population led to the diagnosis of a further 77 cases of active disease and 254 cases of latent tuberculosis among students, staff, and family contacts of the index patient and secondary cases. This outbreak occurred in Leicester, a city with rates of tuberculosis exceeding four times the national average (24; P. Monk, unpublished data). Given the large scale of the outbreak and the elevated rates of tu...

A deletion defining a common Asian lineage ofMycobacterium tuberculosisassociates with immune subversion

“…Initially focusing on its outbreak potential, we hypothesized that the immunomodulatory phenotype of CH might be a consequence of interruption of one of the 10 ORFs affected by large sequence polymorphisms in this strain (10). We therefore complemented these lesions in trans by cloning the complete sequence of the deleted genes into pSMT3 to bring the genes under the control of the constitutive hsp60 promoter.…”

“…Rv1519 has been lost from CH as a result of a deletion that also involves Rv1520 (10). This large sequence polymorphism has recently been designated RD750, a relatively ancient single polymorphism that defines the East African-Indian lineage (also known as Delhi, CAS, or South Asian lineage) (3).…”

“…The growth rate, resistance to H 2 O 2 , NO, and decreased pH of CH, CAS2 (a second isolate from the outbreak, indistinguishable from CH by microarray and PCR genotyping; ref. 10), and H37Rv (the sequenced type strain) were investigated. CH and CAS2 grew more slowly and showed greater susceptibility to H 2 O 2 and decreased pH (Fig.…”

“…In 2001, a large school-associated outbreak of tuberculosis occurred in Leicester, U.K., and both the genotype of the outbreak strain, CH, and the acquired immune responses of the exposed schoolchildren were characterized in unusual detail (10)(11)(12). CH was transmitted extensively from a single index case, leading to 254 cases of latent infection by tuberculin skin testing (TST) amongst 1,128 other pupils tested.…”

“…Outbreaks of tuberculosis present natural ''experiments'' in which the genotype of the strain responsible may be related to its propensity to cause clinical disease (8)(9)(10). In 2001, a large school-associated outbreak of tuberculosis occurred in Leicester, U.K., and both the genotype of the outbreak strain, CH, and the acquired immune responses of the exposed schoolchildren were characterized in unusual detail (10)(11)(12).…”