Characterization of a Tsukamurella Pseudo-Outbreak by Phenotypic Tests, 16S rRNA Sequencing, Pulsed-Field Gel Electrophoresis, and Metabolic Footprinting

Abstract

We report a pseudo-outbreak of Tsukamurella due to improperly wrapped scissors used for processing of tissue specimens. A polyphasic approach, involving biochemical, genetic, and metabolomic techniques, was used in the laboratory investigation. This report highlights that early recognition of pseudo-outbreaks is important in preventing unnecessary and incorrect treatment of patients. Laboratory contamination of clinical specimens can lead to erroneous microbiology reports which can adversely affect patient management (1). Early recognition of such pseudo-outbreaks is important for clinical microbiology laboratories. In 2010, five isolates of Tsukamurella were isolated from the tissue specimens of four patients within 10 days in our clinical microbiology laboratory. Outbreak investigation revealed that the recovery of Tsukamurella was due to laboratory contamination during processing of the tissue samples. In this article, we report the investigation of this pseudo-outbreak and characterization of the outbreak strains using a polyphasic approach.The index patient was a 28-year-old female veterinarian, admitted for fever of unknown origin with right axillary lymphadenopathy (Table 1, patient 1). A direct Gram smear of the right axillary lymph node biopsy specimen in our laboratory revealed Gram-positive bacilli. A lymph node biopsy specimen was culture positive for Tsukamurella. However, the histological section, processed by the pathology laboratory, showed coalescent epithelioid cell granulomas, and no organisms were revealed by Gram staining, Ziehl-Neelsen staining, periodic acid-Schiff diastase staining, or Grocott staining. Bartonella henselae serology showed a Ͼ4-fold rise in IgG level for sera collected 6 days apart. PCR amplification and sequencing for the 16S rRNA gene of B. henselae was also positive on the right axillary lymph node. Since the clinical syndrome was compatible with cat scratch disease, there was a suspicion of contamination of the lymph node biopsy specimen. Contamination at the level of specimen processing in the microbiology laboratory was suspected because it was seen in the direct Gram smear that was processed in the microbiology laboratory but not in the biopsy specimens processed in the pathology laboratory.Within a period of 9 days, 4 additional tissue specimens from 3 patients also grew Tsukamurella. A direct Gram smear of these 4 specimens performed in the clinical microbiology laboratory was negative. Line listing was performed for the 4 cases as described previously (Table 1) (2). A review of our laboratory records revealed that in the 24-month period before the outbreak, only 3

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