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Methods

Spoligotyping is a polymerase chain reaction–based reverse‐hybridization blotting technique that assays the genetic diversity of the direct repeat locus of the Mycobacterium tuberculosis complex. When used in association with mycobacterial interspersed repetitive units (MIRU) typing, spoligotyping is a fast, robust, and cost‐effective genotyping technique, an alternative to traditional IS6110 restriction‐fragment–length polymorphism (RFLP) typing.² Since 2005, MIRU typing is routinely performed on all isolates sent to the reference laboratory in Vienna. Presently, approximately 80% of all M. tuberculosis isolates and 100% of multidrug‐resistant isolates are analyzed with this method. As of January 1, 2007, there is a legal obligation to send in isolates for genotyping in Austria.

Results

In March 2006, the reference laboratory received a multidrug‐resistant isolate of M. tuberculosis that yielded a “rare” spoligotype (1111n11111111111111111111111n1n1nnnn1111111) (Figure). The isolate originated from patient A, a 64‐year‐old man (who tested negative for human immunodeficiency virus [HIV]), who was hospitalized in November 2005 in hospital A with a 2‐week history of thoracic pain and received a diagnosis of culture‐positive tuberculosis. In 2003, patient A had experienced the successful treatment of pulmonary tuberculosis due to a fully susceptible strain of M. tuberculosis in a different hospital (hospital B). Patient A’s first episode of tuberculosis in 2003 was caused by a pansusceptible strain of M. tuberculosis (spoligotype 1111111111111111111111111nnnnnn1nnnn1111111), for which the patient received treatment for nearly 10 months and for which contact tracing had not revealed a source among 32 contacts. The treatment was as follows. For the first 8 weeks, starting on February 26, 2003, he received isoniazid (200 mg), rifampin (600 mg), pyrazinamide (2 g), and ethambutol (1.6 g), all administered orally once per day, plus intravenously administered amikacin (500 mg twice per day) during the first 4 weeks only. For the next 4 months, he received isoniazid (200 mg), rifampin (600 mg), and ethambutol (1.6 g), all administered orally once per day. For the remaining 4 months, until December 23, 2003, he received isoniazid (200 mg) and rifampin (600 mg) orally once per day. Control tests done in June and in October revealed no signs of infection. Computed tomography (CT), performed routinely, was done on December 23, 2004, and showed an “unclear infiltration” in the left upper lobe of the lung, but bronchoscopy performed in January 2005 and a CT from the end of May 2005 had results that did not demand therapeutic intervention. A control CT performed at the end of September 2005 showed “increasing infiltration.”

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Figure.  Spoligotyping patterns of 3 Mycobacterium tuberculosis isolates from 2 patients. Strain 1, Fully susceptible isolate from patient A that was cultured in 2003. Strain 2, Multidrug‐resistant isolate from patient A that was cultured in 2005. Strain 3, Multidrug‐resistant isolate from patient B that was cultured in 2003.

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Contrary to results in 2003, the isolate cultured from samples obtained in November 2005 showed resistance to isoniazid, rifampin, ethambutol, and streptomycin and had the “rare” spoligotyping pattern. Additional isolate‐susceptibility testing showed resistance to p‐amino salicylic acid, prothionamide, pyrazinamide, and rifabutin and sensitivity to ofloxacin, moxifloxacin, capreomycin, amikacin, clofazimine, and linezolid. The “rare” spoligotyping pattern was clearly distinguishable from all but one of the approximately 2,500 M. tuberculosis isolates that had been spoligotyped so far in Austria.

The other isolate with this “rare” spoligotype originated from an HIV‐negative Austrian citizen, a native Romanian aged 38 years (patient B), who received treatment in hospital B for smear‐ and culture‐positive tuberculosis from February 14, 2003, to June 14, 2003. Until April 30, 2003, when the susceptibility testing revealed multidrug‐resistant tuberculosis (a susceptibility pattern identical to that given above for the multidrug‐resistant tuberculosis isolate of patient A), patient B was sharing a room with 2 other patients with tuberculosis, one of them patient A. Patient B was employed as a temporary worker in a trucking company, with frequently changing temporary coworkers, most of them from abroad. Contact tracing for patient B did not reveal a source of his multidrug‐resistant tuberculosis infection among 55 contacts; 2 epidemiologically unrelated cases of culture‐positive pulmonary tuberculosis were discovered (involving contacts from a carpool and a club house). From May 1, 2003, to March 5, 2004, patient B received treatment with clarithromycin (250 mg orally 3 times per day), moxifloxacin (400 mg orally once per day), and rifabutin (150 mg orally once per day); thereafter, for another 20 months, patient B received linezolid (600 mg orally once per day), clarithromycin (250 mg orally twice per day), and ciprofloxacin (500 mg orally once per day) (the end of therapy was October 12, 2005). Results of sputum cultures remained positive until February 24, 2004.

Besides the sharing of a room in hospital B for 8 weeks, no other social contacts were found between patients B and A. We consider this to be the first documented case of healthcare‐associated, multidrug‐resistant tuberculosis in Austria.

Discussion

We describe a patient, who, while receiving treatment at a hospital, was infected with a multidrug‐resistant tuberculosis strain and, 33 months later, developed tuberculosis disease due to this multidrug‐resistant tuberculosis strain. The rare spoligotype of this isolate strongly supports this chain of transmission. Mining of the fourth international spoligotyping database (SpolDB4) revealed that none of 39,295 isolates (1,939 shared types) from 141 countries shows this pattern (C. Sola, oral communication, June 2007).²

Nosocomial transmission of tuberculosis to healthcare workers and other patients has been well documented in industrialized countries, including Austria.^3,4 Molecular subtyping, meanwhile, has become an indispensable tool for elucidating chains of transmission.⁵ Spoligotying has the advantage of speed over RFLP or MIRU typing. The rapidity of this method makes it very useful in tuberculosis control. MIRU typing detects polymorphisms at multiple loci and is therefore superior for typing strains containing only a few copies of IS6110, the element targeted in RFLP typing. Gori et al.⁶ evaluated the clinical usefulness of spoligotyping for simulatenous detection and typing of M. tuberculosis from mycobacterial cultures and found a sensitivity and specificity of 97% and 95%, respectively. In comparison with IS6110‐based RFLP, spoligotyping overestimated the number of isolates with identical DNA fingerprints by 50%, but had a 100% negative predictive value.

An airborne‐infection isolation room—formerly called a “negative‐pressure isolation room”—is a single‐occupancy patient‐care room used to isolate persons with suspected or confirmed infectious tuberculosis disease.⁷ Although airborne‐infection isolation rooms are recommended for hospitalized patients with tuberculosis in industrialized countries, these facilities are unlikely to be available in most hospitals in Austria. Even single‐occupancy patient‐care rooms are unavailable for most affected patients; patients with tuberculosis who require admission to a hospital are usually kept isolated from patients without tuberculous until they are no longer infectious, but cohorting of patients with culture‐positive tuberculosis is still widely practiced in Austria, a country where approximately 1% of M. tuberculosis isolates are multidrug resistant. Although cohorting may provide some reduction in tuberculosis transmission risk, this intervention has not been proven effective at this time and must be reconsidered in view of the increasing rate of multidrug resistance.^7‐9 Cohorting patients who receive a diagnosis of culture‐positive tuberculosis must not be performed before susceptibility results exclude the presence of multidrug‐resistant tuberculosis. Cohorting of patients with tuberculosis prior to determination of drug susceptibility is unacceptable because M. tuberculosis superinfection can occur.¹⁰ Cohorting is acceptable only for patients infected with fully drug‐susceptible organisms.

This nosocomial transmission of multidrug‐resistant tuberculosis once more underscores the need for all healthcare facilities to implement published recommendations for prevention of tuberculosis transmission.⁹ Antituberculosis drug‐susceptibility testing must be performed on initial M. tuberculosis isolates from all patients with tuberculosis. Drug‐susceptibility testing should be completed rapidly, and results should be reported promptly to the healthcare provider and the regional department of public health, even if confirmatory tests are planned. In Austria, the world’s 8th richest country, the use of single‐occupancy patient‐care rooms to isolate persons with suspected or confirmed infectious tuberculosis disease should no longer be seen as a mere amenity, provided only for patients with a supplementary hospitalization insurance.