Assessment of Mycobacterial Interspersed Repetitive Unit-QUB Markers To Further Discriminate the Beijing Genotype in a Population-Based Study of the Genetic Diversity of Mycobacterium tuberculosis Clinical Isolates from Okinawa, Ryukyu Islands, Japan

Abstract

The present investigation focused on genetic diversity and drug resistance of 101 Mycobacterium tuberculosis strains isolated between July 2003 and February 2005 in the Okinawa prefecture, Ryukyu Islands, Japan. A high rate of clustering (87%, eight clusters, 2 to 69 strains/cluster) was observed upon spoligotyping; most of it was due to the lower discriminatory power of this method for the Beijing lineage (n ‫؍‬ 72; 71.3% of the isolates). The remaining diversity was limited to seven clusters (two to five isolates/cluster), with the following distribution of major lineages: ill-defined T (n ‫؍‬ 13; 12.8%), ancestral East African-Indian (n ‫؍‬ 6; 5.9%), Haarlem (n ‫؍‬ 4; 4%), Latin American-Mediterranean (n ‫؍‬ 2; 2%), X1 (n ‫؍‬ 1; 1%), and a total absence of the central Asian clade. Three remaining strains could not be classified on the basis of their spoligotype pattern and were labeled "unknown." Subtyping with mycobacterial interspersed repetitive units (MIRUs) in association with additional QUB minisatellites was performed to discriminate among the Beijing strains. Based on an "in-house" spoligotyping/MIRU database (n ‫؍‬ 694 Beijing strains), eight highly discriminative MIRU loci for Beijing strains were selected (loci numbered 10, 16, 23, 26, 27, 31, 39, and 40). The highest discriminatory power (h) observed in our sample (n ‫؍‬ 72; M-26, 0.385; M-10, 0.38; M-31, 0.255; M-16, 0.238) was too low, and 73.6% of the Beijing strains from Okinawa remained clustered. Typing of Beijing strains with additional QUB loci (with the exception of "one-copy" QUB-1451) resulted in higher discriminatory powers: QUB-11b, 0.68; QUB-11a, 0.656; QUB-26, 0.644; QUB-18, 0.553; QUB-4156, 0.5; and QUB-1895, 0.453. A definitive algorithm on the use of QUB markers to subtype Beijing isolates in expanded studies would shed light on their hypervariability, which may sometimes blur recognition between epidemiologically linked Beijing isolates. The total absence of multiple drug resistance among Beijing isolates from Okinawa, as well as the relatively older ages of the patients (majority above 60 years), shows that tuberculosis (TB) is a declining disease in Okinawa, and an adequate TB control program has successfully avoided both the emergence and the spread of multidrug-resistant TB in this insular setting.Understanding the population structure and transmission dynamics of circulating Mycobacterium tuberculosis clades provides a unique insight into crucial public health issues, such as the appearance and persistence of variants escaping human immune response or the emergence of resistance to antibiotics. PCR-based methods, such as spoligotyping (17), may be a good indicator of strain identity by providing information about epidemiologically important clones, particularly when used in a new setting (17,30,40). Although spoligotyping may correctly identify "outbreak" episodes as well as reflect the spread of the disease due to human migratory movements, it is not suitable when used alone for purely epidemiological investigations as it ...

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