Transmission between HIV-infected patients of multidrug-resistant tuberculosis caused by Mycobacterium bovis

Transmission between HIV-infected patients of multidrug-resistant tuberculosis caused by Mycobacterium bovis Sofía Samper, Carlos Martín, Alfonso Pinedo*, Antonio Rivero, Jesús Blázquez, Fernando Baquero, Dick van Soolingen and Jan van Embden Objective: To investigate outbreaks of multidrug-resistant tuberculosis (TB) by using DNA fingerprint databases. Design: Investigation of two outbreaks of multidrug-resistant TB in separate hospitals in Spain by restriction fragment length polymorphism (RFLP) and spoligotyping. Outbreak strains were compared with more than 1500 RFLPs of Mycobacterium tuberculosis complex strains isolated in Spain and 6000 RFLPs from 30 different countries. Methods: Standardized IS6110 DNA fingerprinting and spoligotyping was used to type multidrug-resistant isolates belonging to the M. tuberculosis complex amongst the outbreak cases. The DNA types were matched against DNA fingerprint databases in Spain and The Netherlands. Results: The DNA typing analysis indicated that a single multidrug-resistant Mycobacterium bovis strain was responsible for a nosocomial outbreak in a hospital in Spain involving at least 16 HIV-infected patients with non-treatable to multidrugresistant TB. Introduction of the fingerprint type of this strain to the international database revealed a single matching strain. This strain was also isolated from an HIV-infected patient in The Netherlands who had died from multidrug-resistant TB. This patient had previously been hospitalized in Spain, where a multidrug-resistant TB nosocomial outbreak involving 20 HIV-infected patients was ongoing. The strains causing this outbreak were also identified as M. bovis with an identical DNA pattern to those strains isolated in the Spanish hospital and the patient in The Netherlands. Conclusions: The use of centralized DNA databases can help to identify rapidly the origin and transmission routes of multidrug-resistant TB across international boundaries and the potential use of such an early warning surveillance system for investigation of nosocomial multidrug-resistant TB outbreaks between HIV-infected patients. To our knowledge this is the first report of transmission of multidrugresistant M. bovis between hospitals. AIDS 1997, 11:1237 1242 Keywords: Tuberculosis, HIV, Mycobacterium bovis, multidrug-resistant, nosocomial transmission, molecular fingerprinting From the Department of Microbiology and Public Health, Saragossa University, the *Unit of Microbiology and the Unit of Infectious Diseases, Hospital Universitario de Malaga, Malaga, the Unit of Microbiology, Hospital Ramon y Cajal de Madrid, Madrid, Spain and the Mycobacterial Department and the Department of Bacteriology, Rijksinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands. Sponsorship: Supported by the Ministry of Health, Spain (Fondo Investigaciones Sanitarias de la Seguridad Social) grant FIS94/0051 and by the European Commission Research Programme BIOMED: Mycobacterial Resistance CT95 1241 and Concerted Action on the Epidemiology of Tuberculosis CT93 1614. Requests for reprints to: Dr Carlos Martín, Departamento de Microbiología Medicina Preventiva y Salud Pública, Facultad de Medicina, C/Domingo Miral, 50009 Zaragoza, Spain. Date of receipt: 7 November 1996; revised: 10 April 1997; accepted: 16 April 1997. Rapid Science Publishers ISSN 0269-9370 1237

1238 AIDS 1997, Vol 11 No 10 Introduction Since 1990, several outbreaks of multidrug-resistant tuberculosis (TB) have been reported in hospitals and prisons in the United States, mainly among HIVinfected patients [1]. The emergence of multidrugresistant TB has the potential to cause serious public health problems, particularly in areas with high rates of HIV infection and a high incidence of TB. Spain records the highest incidence rate of AIDS in Europe (173.6 cases per million in 1995) [2] and in 1994 the reported cases of respiratory TB was also one of the highest in Europe (24.0/100 000) [3]. Outbreaks of multidrug-resistant TB have been reported throughout Europe with cases in Spain [4] the United Kingdom [5] and Italy [6]. The application of strain-specific DNA markers (e.g., IS6110) to differentiate Mycobacterium tuberculosis strains is a powerful tool for carrying out epidemiological studies [7,8]. These molecular typing methods can facilitate the identification of the origin and spread of multidrug-resistant strains. In a study in New York City involving 253 isolates, the strains were shown to be disseminated throughout the city and across state lines to at least four additional cities. The same isolate types were even recorded in Europe [9]. Although Mycobacterium bovis is the primary cause of TB in animals, human TB caused by M. bovis infection is relatively rare. However, immunosupression caused by HIV infection has resulted in less common mycobacterial infections such as M. bovis being encountered in clinical samples. To date, cases of HIV-related human TB caused by M. bovis infection have been reported in England, France and the US [10] and human-tohuman transmission of M. bovis in HIV-positive patients has been confirmed [11]. A nosocomial outbreak of multidrug-resistant M. bovis among HIVinfected patients was described in 1993 in France [12], but when the strains were characterized at the molecular level, they were identified as M. tuberculosis (Veronique Vincent, personal communication, 1996). Because of the low IS6110 copy number in M. bovis, strain differentiation using this marker is generally inconclusive. Alternative techniques based on hybridization with other repetitive DNA elements have been shown to be more useful to differentiate M. bovis strains [11]. The polymerase chain reaction (PCR)- based spoligotyping method [13] is a relatively new technique with the power to differentiate between M. bovis strains that are indistinguishable by other means [14]. Recently, one outbreak of M. bovis-associated multidrug-resistant TB involving HIV-positive patients has been reported in a Spanish hospital [15]. Here we demonstrate, by using a DNA fingerprinting database, that the same multidrug-resistant M. bovis strain is responsible of the multidrug-resistant TB outbreaks among HIV-infected patients in two different Spanish hospitals. The outbreak also involved an HIV-infected patient in The Netherlands. This is the first report of transmission of multidrug-resistant M. bovis between hospitals. Methods Nineteen nosocomial cases of bacteriologically-confirmed multidrug-resistant M. bovis were studied from Ramón y Cajal Hospital, Madrid, Spain (Hospital 1) in Spain between December 1993 and February 1995. Twenty nosocomial cases were studied of bacteriologically-confirmed multidrug-resistant M. tuberculosis complex from our second hospital (Hospital Clínico de Málaga, Spain; Hospital 2) between January 1995 and February 1996. The bacterial isolates were typed at the University of Saragossa (Spain). The 36 strains were confirmed, by spoligotyping, M. bovis with an identical spoligotype. These strains were compared with data held in large DNA fingerprinting databases in Spain and The Netherlands. Restriction fragment length polymorphism (RFLP) analysis was carried out by probing genomic DNA extracted from mycobacteria on Southern blots. The DNA probe was derived from the insertion sequence IS6110 which is routinely used to differentiate between strains of M. tuberculosis. This method has been standardized to allow the comparison of data generated in different laboratories [16]. The use of spoligotyping, a more rapid system than RFLP, was also used for typing. Spoligotyping relies on the amplification of the polymorphic direct repeat (DR) region containing 36-bp direct repeats and interspersed 35 41 bp variable spacer sequences. Spoligotyping may be used to differentiate M. tuberculosis complex strains with low IS6110 copy numbers and also to distinguish M. bovis from M. tuberculosis [13]. An international DNA fingerprinting database has been established at the Rijksinstituut voor Volksgezondheid en Milieuhygiene in Bilthoven (The Netherlands). This database contains over 6000 RFLPs of M. tuberculosis complex strains from The Netherlands and from 30 other countries. In the University of Saragossa (Spain) a national fingerprint database contains over 1500 RFLPs from M. tuberculosis complex strains isolated in Spanish hospitals. Fingerprinting and computer-assisted analysis were carried out as previously described [13,16].

Multidrug-resistant TB caused by M. bovis Samper et al. Results Between 1977 and 1992 no cases of primary multidrug-resistant TB were diagnosed in Hospital 1. Between November 1993 and February 1995, a multidrug-resistant TB nosocomial outbreak in Hospital 1 was detected among 19 HIV-infected patients (16 males and three females) with a mean age of 31 years. Pulmonary TB was detected in 15 cases and in eight cases the location was disseminated. The 19 cases were confirmed as multidrug-resistant TB. Sixteen cases were attributed to nosocomial contact (same ward) with a multidrug-resistant TB patient. The three other cases had attended another hospital in Madrid ( Hospital 3 ) in addition to Hospital 1. This hospital had previously reported a multidrug-resistant TB epidemic [4]. Sixteen strains isolated from the 19 cases were shown to have an identical IS6110 RFLP pattern with two IS6110 bands at identical positions and were identified as M. bovis (Fig. 1a). This included the three cases who had attended Hospital 3. All 16 M. bovis strains isolated displayed the following identical multiple drug resis(a) tance phenotype: isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, para-aminosalicylic acid, clarithromycin, ethionamide, ofloxacin, capreomycin and amikacin. This suggested that a single strain was responsible for the outbreak in 16 patients. The RFLP pattern of the multidrug-resistant TB was compared with more than 1500 RFLPs of M. tuberculosis complex strains isolated in Spain and deposited in the data-base of the University of Saragossa. Approximately 800 RFLP patterns corresponded to isolates from the Saragossa Province in Spain, and the remainder to isolates from reference laboratories in Madrid, Andalucia, Galicia and other regions of Spain. Analysis of the RFLP profiles from the 16 cases revealed no matching patterns. When the comparison was carried out against approximately 6000 RFLPs in the Bilthoven IS6110 fingerprint database, the pattern matched a single strain isolated from an HIV-infected patient (Fig. 2a). This patient has died in Amsterdam of multidrug-resistant TB caused by M. bovis [17]. The spoligotype pattern of the Amsterdam patient s isolate was identical to strains from Hospital 1, all of which were characteristic of M. bovis. The unique pattern was (b) Fig. 1. (a) Restriction fragment length polymorphism (RFLP) pattern obtained in Mycobacterium bovis isolates in Hospitals 1 and 2 by DNA typing with IS6110. Lanes 1 and 2, DNAs from M. bovis isolates in Hospital 1; lanes 3 and 4, isolates in Hospital 2. M, reference M. tuberculosis strain Mt14323 [16]. (b) Spoligotype obtained in M. bovis isolates in Hospitals 1 and 2 by DNA typing by hybridization with 43 oligonucleotides derived from spacer sequences in the direct repeat region. Lanes 1 10, M. bovis isolates in Hospital 1; lanes 11 20, M. bovis isolates in Hospital 2. BCG, M. bovis bacille Calmette-Guerin Pasteur; H37, M. tuberculosis H37Rv. 1239

1240 AIDS 1997, Vol 11 No 10 not found among any spoligotypes of 500 other M. bovis strains isolated from animals and humans (200 of these isolates originated in The Netherlands, the others from several countries including New Zealand, Spain and Ireland) (Fig. 2b). In the clinical report of the Amsterdam HIV-infected patient it was recorded that he had died in Amsterdam in June 1995 from multidrug-resistant TB. Six months previously (January 1995) he had been admitted to Hospital 2 [17]. The distance between Hospital 1 in Madrid and Hospital 2 in Malaga is 540 km. Since he had not previously attended Hospital 1, no direct epidemiological relationship between patients in these hospitals could be ascertained. Between January 1995 and February 1996, 20 HIVpositive patients were diagnosed with multidrug-resistant TB in the Infectious Diseases Unit of Hospital 2. The 20 cases presented fever and in 19 cases the chest X-ray was suggestive of TB. All 20 cases had fewer than 200 106/l CD4 cells. All patients died fewer than 3 months after diagnosis. The mean age of the patients was 34.3 years. Nineteen were male, 12 were intravenous drug users, four were homosexuals, three were heterosexuals engaging in high-risk sexual behaviour and one patient did not apparently belong to a (a) high risk group associated with HIV infection. From the 20 cases studied, 18 had nosocomial contact (same ward) with a multidrug-resistant TB patient in the Unit of Infectious Diseases in Hospital 2 and one was a voluntary health worker. The remaining patient lived with multidrug-resistant TB patients. In March 1996 strains from these 20 patients from Hospital 2 were sent for typing to the University of Saragossa. The 20 strains shared the IS6110 type (Fig. 1a) and also the spoligotype found among the M. bovis strains isolated in Madrid, and the Amsterdam isolate (Fig. 2b). A total of 20 multidrug-resistant TB isolates from different HIV-infected patients shared identical spoligotypes, indicating an multidrug-resistant TB nosocomial outbreak given that all of the patients had attended the same hospital around the same time (10 are showed in Fig. 1b). An indirect epidemiological link between the multidrug-resistant TB patients in Hospitals 1 and 2 was established since three patients from Hospital 1 and one from Hospital 2 had previously been admitted to a third hospital in Madrid (Hospital 3). In 1992, the first outbreak of multidrug-resistant TB in Spain was reported from this hospital [4,18]. All 48 reported cases of multidrug-resistant TB at Hospital 3 were among (b) Fig. 2. (a) Comparison of the multidrug-resistant (MDR)-Mycobacterium bovis restriction fragment length polymorphism (RFLP) with RFLPs of nine Mycobacterium tuberculosis complex strains in the central IS6110 fingerprint database Rijksinstitunt voor Volksgezondheid en Milieuhygiene (RIVM) Bilthoven (The Netherlands). Lane 1, M. bovis isolate from Burundi; lane 2, M. bovis isolate from The Netherlands; lane 3, M. bovis isolate from Mexico; lane 4, MDR M. bovis isolate Hospital 1 in Spain; lane 5, MDR M. bovis isolate from the patient in The Netherlands; lanes 6 and 8, M. tuberculosis isolated from The Netherlands; lane 7, M. tuberculosis isolate from South Korea; lane 9, M. tuberculosis isolate from Honduras. (b) Comparison of the M. bovis spoligotype with the same nine M. tuberculosis complex strains spoligotypes in the central database RVIM Bilthoven. Lane 1, M. tuberculosis isolate from Honduras; lane 2, M. tuberculosis isolated from The Netherlands; lane 3, M. tuberculosis isolate from South Korea; lane 4, M. tuberculosis isolated from The Netherlands; lane 5, MDR M. bovis isolate from the patient in The Netherlands; lane 6, MDR M. bovis isolate Hospital 1 in Spain; lane 7, M. bovis isolate from Mexico; lane 8, M. bovis isolate from The Netherlands; lane 9, M. Bovis isolate from Burundi.

Multidrug-resistant TB caused by M. bovis Samper et al. 1241 HIV-infected patients hospitalized in the HIV ward between September 1991 to May 1995. Beginning in 1993, strains were found to be consistently resistant to isoniazid, streptomycin, ethambutol and rifampicin. Strains from Hospital 3 were unavailable for study and therefore it was not possible to confirm the identity of the strains by molecular typing. However, the resistance patterns available from antibiograms were consistent with that of the M. bovis strain described in the present study. Discussion The results presented in this study strongly suggest that a single M. bovis strain was responsible for multidrugresistant TB nosocomial outbreaks among HIVinfected patients in hospitals in Madrid and Malaga (Hospitals 1 and 2). It also appears that one of the patients carried the strain to The Netherlands following discharge from the Hospital 2 [17]. Although we were unable to trace a direct epidemiological link between multidrug-resistant TB outbreaks in Hospitals 1 and 2, it is probable that such a link exists. Some patients from both hospitals had previously been admitted to another Madrid Hospital (Hospital 3). It was in Hospital 3 that the first large multidrug-resistant outbreak in Spain was described [4,18]. From the beginning of this epidemic in 1992 until 1995, more than 50% of the M. tuberculosis complex strains isolated in Hospital 3 in 1995 were multidrug-resistant. Since multidrug-resistant TB strains from Hospital 3 were not available for DNA analysis, it was not possible to confirm the link between Hospital 3 and the other two Spanish hospitals. Spoligotyping is a powerful technique for differentiating M. tuberculosis from M. bovis [13]. This is important when identifying strains resistant to isoniazid and pyrazinamide since the resistance/susceptibility tests, to pyrazinamide and thiophene-2-carboxylic acid hidrazide, two important test used to differentiate M. tuberculosis from M. bovis, are unreliable [19]. Spoligotyping is a PCR-based technique and can be used to type strains from pathological samples or from culture collections. When compared with RFLP, spoligotyping is less discriminative if the IS6110 copy number is high in M. tuberculosis complex strains [14]. The spoligotype observed in the multidrug-resistant outbreaks described here displayed a unique pattern that was not found previously among a large number of other strains. Outbreaks of multidrug-resistant TB pose a serious public health threat given the difficulty of administering effective treatment, the resulting high rate of fatality and the potential for multidrug-resistant TB strains to spread within and between hospitals. It has recently been reported that transmission of multidrug-resistant TB between individuals occurred on a commercial airline flight, indicating that short contact periods are sufficient for spread of infective material [20]. These problems are exacerbated in HIV-infected individuals, who are highly susceptible to TB infection [21,22]. The evolution of these multidrug-resistant TB strains probably results from the development of resistance in non-compliant patients and then the transmission of initial drug-resistant strains causing outbreaks [22,23]. Given the potential hazard associated with transmission and dissemination of multidrug-resistant TB across national boundaries, we suggest that it would be desirable to systematically catalogue the DNA types of all multidrug-resistant TB isolates. Our study shows that the generation of large databases can facilitate rapid tracing of the transmission routes of multidrug-resistant TB. This is particularly relevant for isolates, such as M. bovis, which are difficult to type using conventional protocols. The use of centralized databases in this fashion could function as an early warning system in the surveillance of the spread of multidrug-resistant TB as part of TB control programmes. Several European laboratories are currently performing DNA typing of M. tuberculosis isolates as part of epidemiological investigations. The general availability and usage of international databases of DNA types of multidrug-resistant TB isolates could form the basis of a co-operative effort to develop strategies to limit the spread of multidrugresistant TB. Acknowledgements We thank the staff of the Units of Microbiology and Infectious Diseases from the Hospital Clínico (Málaga) and Hospital Rámon y Cajal (Madrid) for their contributions to this investigation; in particular to M. A. Sanchez, E. Gómez Mampaso, M. Márquez, J. Santos, A. Guerrero and J. Cobos. The authors greatly appreciate the expert opinions of the following: A. Ortega from Hospital Carlos III (Madrid), Dr Gómez-Lus who is responsible for the Department of Microbiology of the Universidad de Saragossa. To V. Vincent from the Pasteur Institut (Mycobacterial Reference Laboratory) for communicating unpublished results to E. Gormley and J-A. Gavigan for discussion and correction of the manuscript. References 1. Kent JH: The epidemiology of multidrug-resistant tuberculosis in the United States. Med Clin North Am 1993, 77:1391 1409. 2. World Health Organization European Community Collaborating Centres on AIDS: HIV/AIDS Surveillance in Europe. European Centre for the Epidemiological Monitoring of AIDS. Fourth Quarterly Report. Geneva: WHO; 1995:48.

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