Molecular Evidence of Endogenous Reactivation of Mycobacterium tuberculosis after 33 Years of Latent Infection

Troels Lillebaek 2 3 Asger Dirksen 0 2 Inga Baess 2 3 Benedicte Strunge 2 4 Vibeke . Thomsen 2 3 A se B. Andersen 1 2 0 Department of Pulmonary Medicine, Gentofte University Hospital , Gentofte 1 Infectious Diseases Clinic, Rigshospitalet University Hospital , Copenhagen 2 Received 6 August 2001; revised 4 October 2001; electronically published 17 January 2002. Financial support: Grant from the Danish Lung Association; the European Community program Quality of Life and Management of Living Resources (grant 2000-0630). Laboratory for Mycobacteriology, Statens Serum Institut , Artillerivej 5, DK-2300 Copenhagen S , Denmark 3 International Reference Laboratory for Mycobacteriology, Statens Serum Institut (National Institute for Prevention and Control of Infectious Diseases and Congenital Disorders) 4 Department of Pulmonary Medicine, Holstebro Hospital , Holstebro , Denmark Since Robert Koch described the cause of tuberculosis in 1882, the natural history of the disease after primary infection has been subject to debate. Only 10% of infected individuals develop active disease, which may appear years to decades after infection. Late onset has been attributed to the endogenous reactivation of dormant bacteria. However, this has not been documented by molecular means for latencies of more than a few years. In Denmark, we have recently recultured 205 freeze-dried Mycobacterium tuberculosis strains obtained from 1961 through 1967. These historical strains are analyzed by DNA restriction fragment- length polymorphism testing, and their DNA patterns are compared with those of 4008 recently obtained clinical specimens. This has, surprisingly, yielded molecular evidence of M. tuberculosis reactivation after 33 years of latent infection. A father and son who developed tuberculosis in 1961 and in 1994, respectively, were the only patients infected with strains that share an identical DNA pattern. - Primary infection with Mycobacterium tuberculosis leads to clinical disease in only 10% of cases [1]. Most infected individuals are able to mount an effective immune response, which limits proliferation of the bacilli and produces a long-lasting partial immunity, both to new infections (often referred to as exogenous reinfections) and to the reactivation of latent bacilli (often referred to as endogenous reactivation) [2, 3]. Approximately one-half of infected individuals who develop clinical disease experience early progressive disease that occurs within 5 years of infection, and the rest have late disease, which is caused by reactivation as long as several decades after infection [2, 4]. The relative contributions of reactivation and reinfection, and even whether each occurs, have been the subject of considerable controversy [2, 3, 5 7]. The introduction of molecular subtyping methods has permitted characterization of specific strains of M. tuberculosis and determination of whether isolates from different patients might have a common origin [8]. Therefore, the potential exists for determining whether disease in a particular patient is Methods Data collection. In Denmark, all microbiological tests for mycobacteria have been carried out at the International Reference Laboratory for Mycobacteriology (IRLM) at Statens Serum Institut (SSI) in Copenhagen since 1922. This is the only laboratory that performs culture-based tuberculosis (TB) diagnosis for Denmark, Greenland, and the Faroe Islands. It also serves as an international reference laboratory for Iceland and Lithuania. Because all M. tuberculosis specimens from Denmark, Greenland, and the Faroe Islands are processed in a single laboratory and because of the long-standing mandatory centralized TB notification system in Denmark, we believe that our data are nearly complete and highly representative of culturepositive TB from the areas we cover. This is of major importance in interpreting DNA band clustering [8]. From 1961 through 1967, 205 strains of M. tuberculosis were collected from samples obtained from small groups of epidemiologically related patients. The strains were divided into 2 groups by means of the bacteriophage BK1, and the results were compared with the epidemiologic linkage information [10]. The strains were Variable Collection period Patients in study, no. Age, mean years (SD) RFLP-typed strains,a no. From male patients, no. (%) From Danish-born patients, no. (%) Historical strains NOTE. RFLP, restriction fragmentlength polymorphism. a DNA analysis was done by the RFLP method [11]. b More than 1 strain from some patients was examined. then stored as freeze-dried samples for 33 39 years, until they were recultured in 2001. The DNA patterns of these strains are now being analyzed, and, at present, DNA patterns from 130 of the historical strains have been compared with those from M. tuberculosis strains collected in the 1990s. In 1992, DNA analysis of M. tuberculosis strains was implemented in Denmark on a nationwide basis, using the internationally standardized restriction fragment length polymorphism (RFLP) method [11]. Since then, the RFLP patterns of 4008 strains collected from 3781 patients with TB have been analyzed, representing 97% of all culture-positive patients with TB in Denmark, as well as the Faroe Islands and Greenland. Table 1 gives basic epidemiologic data for both the historical and the recent strains. Specimen processing. Freeze-dried M. tuberculosis strains were resuspended in distilled water, and each was transferred directly into 1 tube of Dubos culture medium containing Tween 80 (SSI Diagnostika) and 2 tubes of Lowenstein-Jensen medium (SSI Diagnostika). Bacteria were harvested after 3 4 weeks of incubation. The recent strains were cultured in the Bactec culture system (Becton Dickinson), and, when species identification by AccuProbe (Gene-Probe) revealed the presence of M. tuberculosis complex, the isolates were subcultured for 3 4 weeks in Dubos medium containing Tween 80. Harvested bacteria were heat killed (90 C for 30 min), and RFLP testing was done by the internationally standardized method [11]. Results DNA analysis. Two of the M. tuberculosis strains that we examined exhibited identical 13-band DNA patterns (figure 1). The strains were isolated, at an interval of 33 years, from a father (in 1961) and son (in 1994). This particular DNA pattern was not found in any other strain, either among the 130 strains that were analyzed from the group of 205 historical strains or among the 4008 recent strains (table 1). Index case: father. The index case patient was born in 1926. According to both hospital and IRLM records, he was given a diagnosis of tuberculous infection of a knee joint in 1946 and of pulmonary TB in 1961. The patient presented in 1961 with a 1-year history of fever, weakness, and productive cough. Chest radiography showed several small nodular pulmonary opacities and a large (4 5 cm) cavity in the apex of the right lung. The diagnosis was confirmed by microscopy of a s (...truncated)