The Significance of Spoligotyping Method in Epidemiological Investigations of Tuberculosis

Introduction: The control of tuberculosis (TB) requires methods for rapid detection and tracing sources of infection, so that further transmission can be arrested. Recent developments in molecular biology have resulted in techniques that allow prompt identification and tracking specific strains of M. tuberculosis as they spread through the population. Most of these techniques take advantage of M. tuberculosis DNA polymorphism and are based on various repetitive DNA elements as genetic markers. Each method yields strain-specific genetic profiles (fingerprints). Strains showing identical fingerprints are referred to as clustered and are usually associated with recent transmission, whereas strains whose fingerprints are unique are presumed to represent remote transmission, a reactivation of infection acquired in the distant past. Material and methods: In recent years, spoligotyping has become one of the most widely used genotyping method for epidemiological studies of TB. Spoligotyping is a PCR-based method allowing to analyze strain-dependent polymorphisms observed in spacer sequences present within the direct repeat (DR) genomic region of M. tuberculosis complex strains. Spoligotyping provides some important advantages over other genotyping techniques. These are simplicity, rapidity, high reproducibility and stability of the results, with the latter being expressed in a simple digital pattern, readily named and databased, and the ability to perform spoligotyping directly on clinical samples, without the need for prior culture. However, spoligotyping has relatively low discriminatory capacity, which makes it necessary to use secondary fingerprinting methods to prove clonality between isolates. Results: The aim of this study was to evaluate the usefulness of spoligotyping in epidemiological investigations of TB by analyzing 16 isoniazid-resistant M. tuberculosis strains isolated from patients with pulmonary TB in the central region of Poland. A total of 11 distinct spoligopatterns were obtained. 9 isolates were represented by a unique pattern, whereas 7 were clustered in 2 groups of 5 and 2 isolates, respectively. When compared with an international spoligodatabase SpolDB4, 13 isolates shared already described spoligotypes, whereas 3 did not match any existing spoligopattern in database and were defined as orphans. Spoligotyping overestimated the number of clustered isolates in one of its two clusters when compared to IS6110 Mtb1/ /Mtb2 PCR. Strains clustered using the latter method were assumed to be closely epidemiologically related. Conclusions: This report demonstrates the utility of spoligotyping as an initial screening technique, to be supplemented by another typing method of greater discriminatory power, such as the IS6110 Mtb1/Mtb2 PCR in order to better recognize the epidemiological links between TB patients.