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Genotypic changes in DNA fingerprinting patterns of Mycobacterium tuberculosis strains from HIV‐positive persons in Sardinia

Sechi, L A.1; Zanetti, S1; Dupré, I1; Aceti, A2; Sanguinetti, M3; Fadda, G3


1Dipartimento di Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica

2Istituto di Malattie Infettive e Parassitarie, Facoltà di Medicina e Chirurgia, Università degli studi di Sassari, Viale S. Pietro 43/b, 07100 Sassari

3Istituto di Microbiologia, Facoltà di Medicina e Chirurgia ‘Agostino Gemelli’, Università Cattolica del Sacro Cuore, 00168 Roma, Italy.

Sponsorship: This work was supported by the first national project ‘Tubercolosi’ of the Istituto Superiore di Sanità, Rome and 60% MURST.

Date of receipt: 3 June 1998; accepted: 10 June 1998.

Amongst infectious diseases, tuberculosis is still the major killer in the world [1]. In some developing countries tuberculosis infection has reached epidemic proportions and is increasing worldwide [2]. This rapid growth is thought to be correlated in part to the movement of people from developing countries [3], where tuberculosis largely occurs, to industrialized countries. It is also due in part to AIDS, as Mycobacterium tuberculosis infection appears to be one of the first indications of a loss of immune system function, and those infected are susceptible to a number of opportunistic infections [3], including infections by other mycobacteria, and finally because the expectation of life is increased. In the present study, we applied a computer-assisted method to analyse DNA fingerprints [4] and construct dendrograms of M. tuberculosis strains isolated from HIV-positive patients.

IS6110 DNA fingerprinting is a useful tool for typing M. tuberculosis strains isolated in any geographic region because it allows the definition and characterization of the transmission of the infection [2,4–7]. There are no meaningful differences in the patterns of strains isolated in Northern and Southern Sardinia, suggesting that there is no segregation of a particular strain in these areas. Fifty-one strains of M. tuberculosis were isolated from different specimens of HIV-positive patients in the Clinical Infectious Disease Department of Sassari Hospital. The samples were collected from 1992 until 1996. Twenty-six strains were collected in the Clinical Department of Infectious Disease of Cagliari Hospital (Southern Sardinia). The M. tuberculosis H37R v strain, purchased from American Type Culture Collection, was used as a control strain.

The dendrograms (Fig. 1) indicated that amongst the strains of M. tuberculosis isolated in Sardinia there were high genetic distances, and therefore the strains were not epidemiological related. These results support the hypothesis that, in Sardinia, tuberculosis is more frequently the result of an endogenous reactivation process rather than an exogenous (re-)infection process. The 51 strains isolated in Sassari (Northern Sardinia) gave 45 different patterns of hybridization, whereas the 26 strains from Southern Sardinia showed 13 different patterns. In the epidemiological study summarized in the dendrograms, there was no evidence of any correlation amongst HIV infection and particular strains of M. tuberculosis

Different strains isolated from different patients showed an identical pattern, and are represented in the dendrogram with an Sab (Similarity ratio value) coefficient of 1.0 (Fig. 1). The strains CA1 and CA9 had an identical pattern with IS6110 DNA fingerprinting (Fig. 1A). Moreover, CA4, CA5 and CA6 also showed an identical pattern. All patients from which these strains were isolated were being treated in the same hospital. Fig. 1B shows the homology between the strains SS1, SS3, SS5 and SS8. These strains were isolated from HIV-infected patients in the same hospital. SS4 strain has a high degree of homology with this cluster of strains, and was isolated from a patient in the same hospital during the same period. Strains SS57 and SS69 also have the same fingerprint, and furthermore, SS65 has a high degree of homology (95%) with them. We did not have clinical data on the patients from which the strains were isolated. Five sequential isolates from a patient in a 1 year period (SS71, SS72, SS73, SS74 and SS75) were analysed by IS6110 DNA fingerprinting. Strains SS71, SS72, SS73 and SS74 produced identical fingerprinting patterns, whereas SS75 was lacking one band (Fig. 2). SS75 was isolated at the end of the 12 months.

It is unlikely that the extra band was the result of an incomplete digestion or degradation of DNA. The same pattern and band intensity was obtained in different experiments. It has been reported that IS6110 is very stable in the genome [8]. Moreover, van Soolingen et al. [9] described a microepidemic that occurred in a Czechoslovakian family, where an isolate from a girl aged 14 years differed by one band from the strains isolated from the other members of the family (six people living in the same house). Here, we have reported a change in the pattern of a serial of isolates from the same person after 1 year since the first strain was isolated. These observations may be helpful in understanding the transmission of M. tuberculosis within different communities.

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