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Journal of Clinical Microbiology, December 2001, p. 4404-4406, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4404-4406.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Evaluation of the Mycobacterium bovis Restriction Fragment Length Polymorphism Probe pUCD, in Combination with the Direct Repeat Probe, for Molecular Typing of Mycobacterium tuberculosis Strains in Ireland

Department of Microbiology, The Mater Misericordiae Hospital, Dublin,¹ and Department of Zoology² and Conway Institute of Biomedical Science,⁴ University College Dublin, Belfield, Dublin 4, and Department of Microbiology, University Hospital Cork, Wilton,³ Ireland

Received 23 April 2001/Returned for modification 23 July 2001/Accepted 8 October 2001

	ABSTRACT

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A mycobacterial restriction fragment length polymorphism probe, pUCD, has recently been described which represents an effective tool for the strain typing of Mycobacterium bovis. The present study evaluated this probe, in combination with the direct repeat probe (DR), for the molecular typing of 90 strains of Mycobacterium tuberculosis from 87 patients, looking at a group (62 isolates) of nonselected samples to assess pUCD combined with DR as a general tool and a subset of 32 isolates with a common specific IS6110 strain type in Ireland. Within the group of 62 isolates, pUCD-DR identified 42 strains and was comparable to both IS6110 (41 strains) and polymorphic guanine-cytosine-rich sequence (PGRS) (37 strains) analysis. pUCD-DR was found to be comparable to IS6110 and PGRS in identifying four separate clusters of isolates which were confirmed to be clinically related. pUCD-DR divided the common IS6110 isolates into six distinct types and was comparable to PGRS (seven strain types). The usefulness of this probe as an epidemiological tool is discussed.

	INTRODUCTION

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The reported incidence of tuberculosis in humans has increased dramatically in industrialized countries in recent years (7), prompting the development of improved methods for diagnosis and epidemiological tracing of Mycobacterium tuberculosis infection. Techniques commonly used for epidemiological typing include spoligotyping (2, 10), pulsed-field gel electrophoresis (17), PCR-based techniques such as variable-number tandem repeat analysis (6), heminested inverse PCR (15) or ligation-mediated PCR (16), and restriction fragment length polymorphism (RFLP) analysis (11, 14, 19). RFLP analysis has been demonstrated to be a robust and highly discriminatory tool for strain typing of M. tuberculosis in epidemiological studies (11). The tuberculosis complex-specific insertion sequence IS6110 is often used as a first-line tool to distinguish between M. tuberculosis isolates for epidemiological investigations due to its inherently high copy number and relatively high degree of strain polymorphism (18). Occasionally, some isolates which contain fewer copies of this element require supplementary typing with additional RFLP probes or alternative techniques. About 16% of Irish M. tuberculosis isolates exhibit a common banding pattern for IS6110 and require supplementary typing; this is commonly performed by RFLP analysis using a polymorphic guanine-cytosine rich sequence (PGRS).

Recently, a novel RFLP probe for the typing of isolates of Mycobacterium bovis, pUCD, was described (13). RFLP typing of M. bovis using pUCD generates a highly polymorphic yet simple-to-analyze banding pattern (12). It was reported that the pUCD banding pattern may be reproduced by a combination of three oligonucleotide probes to recreate the polymorphic bands of interest without compromising the discriminatory sensitivity of the probe. The application of this probe for typing M. bovis was reported to have a discriminatory power equivalent to that of IS6110, PGRS, and a direct repeat sequence (DR) in combination.

As pUCD is an effective tool for strain typing M. bovis, it was decided to investigate its applicability to typing of M. tuberculosis isolates. In this study, the pUCD oligonucleotide probe set was used in a mixed hybridization with DR and compared to IS6110 and PGRS to determine whether the discriminatory ability of pUCD was comparable to that of IS6110 and PGRS for typing M. tuberculosis and to investigate its usefulness as an alternative to PGRS where IS6110 is present in low copy numbers.

	MATERIALS AND METHODS

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RFLP analysis. Ninety M. tuberculosis strains from 87 patients, the type strain M. tuberculosis 14323, and three M. bovis isolates were cultured on Lowenstein-Jensen slopes with glycerol for 3 to 4 weeks at 37°C, and RFLP analysis was carried out as previously described (4) with probes IS6110 (9), PGRS (5), and pUCD (12) in combination with DR (8). PGRS, DR, and pUCD were obtained as commercially synthesized oligonucleotides with a digoxigenin label incorporated during synthesis.

	RESULTS AND DISCUSSION

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The 90 isolates were divided into two groups. The first set of 62 samples was not selected but represented routine samples available in the laboratory. The second set was a subselected group of isolates with the most common IS6110 strain type in Ireland. Nomenclature for IS6110 and PGRS analysis was designated with a letter and a number, while pUCD nomenclature was represented by a number only.

The first set of 62 samples, recovered from 60 patients, analyzed by IS6110 identified 41 different types, while pUCD-DR distinguished 42 types and PGRS identified 37 different strain types. Within this set, four clusters of identical isolates were identified, and clinical data confirmed these to be related to specific outbreaks (Table 1). The largest group, nine isolates (numbers 1, 2, 3, 7, 9, 11, 12, 14, and 17), were from children attending a large secondary school where an outbreak of M. tuberculosis was investigated by public health specialists. IS6110 or PGRS analysis was able to identify eight isolates as identical, while pUCD-DR analysis maintained epidemiological concordance among all nine isolates. An extra copy of IS6110 was observed in one isolate within the cluster, while PGRS results showed minor differences in the banding pattern associated with a different isolate within this group. In this instance, pUCD-DR was better than IS6110 or PGRS at maintaining epidemiological concordance from this outbreak. The results for isolates from the remaining three outbreaks (isolates 4 and 8 comprised a small outbreak in a prison, isolates 59, 60, and 61 were from a family outbreak, and isolates 39, 40, 41, 42, and 43 were from an outbreak among the crew of a passenger ferry) were identical with all three probes. pUCD-DR on one occasion discriminated between two isolates from the same patient (isolates 32 and 33). This has been seen before with IS6110 (1).

View this table: [in this window] [in a new window]   TABLE 1.   Epidemiological concordance data processed for 62 M. tuberculosis isolates from 61 patientsa

Although IS6110 is the most commonly applied RFLP probe for preliminary screening of M. tuberculosis isolates and has been proven effective for discrimination between isolates that have a high copy number, when the copy number is low it is less discriminating, suggesting the need for secondary markers (3).

The most common IS6110 strain type in Ireland, which encompasses 16% of isolates, contains only two copies of IS6110. To examine the ability of pUCD to discriminate within this IS6110 strain type, a second set of 32 isolates which were preselected for this specific IS6110 banding pattern were chosen, including four isolates from the original group. pUCD-DR was able to identify six different types within this group, while PGRS identified seven different types (Table 2). However, both PGRS and pUCD predominantly assigned these IS6110 isolates to a single strain type; PGRS identified 20 isolates as type A1 and 7 as A2, while pUCD identified 26 of the 32 isolates as type 31. Neither probe was particularly useful in discriminating within the prevalent IS6110 strain type B1. Two epidemiologically linked cases from the same family (isolates 73 and 89) were found by all three typing methods to be indistinguishable from other unrelated isolates. No evidence of epidemiological links between other isolates with the B1 (IS6110), A1 (PGRS), and 31 (pUCD) combination has been identified.

View this table: [in this window] [in a new window]   TABLE 2.   Epidemiological data for 32 different M. tuberculosis isolates, from 30 patients, which have two copies of IS6110 and the most common banding pattern found in Irelanda

pUCD-DR analysis was comparable to PGRS analysis where isolates had three or more copies of IS6110. pUCD-DR generates a simplistic banding pattern containing six to eight distinct bands which was more amenable to band analysis than PGRS, where the bands are often close together and are difficult to resolve. These results suggest that pUCD-DR-based RFLP typing is comparable to PGRS typing and generates a useful degree of M. tuberculosis strain discrimination while maintaining known epidemiological concordance relating to the samples. This probe combination represents a useful secondary tool for future epidemiological studies of M. tuberculosis.

	ACKNOWLEDGMENTS

We are grateful to Mary O'Meara and Margaret O'Sullivan, specialists in public health medicine, for epidemiological information.

This work was supported by a grant from The Mater College of Postgraduate Education and Research and the Department of Agriculture Food and Rural Development.

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Microbiology, The Mater Misericordiae Hospital, Eccles St., Dublin 7. Phone: (353) 1 8032379. Fax: (353) 1 8034781. E-mail: rhone{at}mater.ie.

	REFERENCES

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