Mycobacterium reverse hybridization line-probe assay used to diagnose disseminated Mycobacterium haemophilum infection in a child with acute lymphoblastic leukemia.

Abstract

Special growth requirements make it difficult to isolate Mycobacterium haemophilum in a routine laboratory setting. In the case presented here a Mycobacterium sp. was detected directly on skin biopsy tissue using a broadspectrum polymerase chain reaction assay. Mycobacterium genotyping via a reverse hybridization line-probe assay, combined with conventional microbiological and molecular techniques, led to the identification of Mycobacterium haemophilum. A 6-year-old Dutch girl was diagnosed with B-cell precursor acute lymphoblastic leukemia in November 1999 and treated according to a protocol of the Dutch Childhood Leukemia Study Group. Following complete remission in January 2000, maintenance therapy was started with 6-mercaptopurine, methotrexate and pulses with vincristine and dexamethasone. In August 2001 she was admitted to hospital with weight loss and episodes of diarrhea, vomiting and coughing. Physical examination revealed a temperature of 37.4°C, distension of the abdomen and erythema with slight desquamation on both wrists. A small infiltrate in the left lung was seen on chest radiograph. Bone marrow aspiration showed normal hematopoiesis and no excess lymphoblasts. A Cryptosporidium sp. was found in the feces and in a duodenal biopsy, and treatment with azithromycin was started. During the course of admission, painful suppurative skin lesions developed on the patient’s knees, elbows and face, accompanied by fever of up to 39.5°C. Histopathological examination of skin biopsy tissue showed granulomatous inflammation, and Ziehl-Neelsen staining for acid-fast bacilli was positive. Based on these findings, empirical antimycobacterial treatment was started with the agents ethambutol, rifampicin and clarithromycin. Initially, repeated bacterial, fungal and mycobacterial cultures of ulcera specimen and blood were negative with the BACTEC 9000 MB System (Becton Dickinson Benelux, Erembodegem, Belgium) and solid-phase Coletsos base medium (Bio-Rad, Veenendaal, The Netherlands) with and without osseine, incubated at both 37°C and 32°C. The line-probe assay for determination of Mycobacterium spp. (LiPA MYCOBACTERIA; Innogenetics, Ghent, Belgium) is a reverse hybridization line-probe assay for the identification of non-tuberculous mycobacteria, based on amplification of the mycobacterial 16-23S ribosomal RNA spacer region using a broad-spectrum PCR assay combined with hybridization of biotinylated mycobacterial DNA products with 14 specific oligonucleotide probes on a nitrocellulose strip [1]. Although the test has only been validated for use on cultured mycobacteria rather than on clinical material [1, 2], we performed the LiPA assay directly on biopsy tissue obtained from a skin lesion of our patient. Upon completion of the assay, clear bands were present at the marker line, the Mycobacteria genus positive control, and band number 9, the MAIS-group. Since the LiPA assay cannot differentiate within this group between Mycobacterium malmoense,Mycobacterium haemophilum and Mycobacterium intermedium, we used the biopsy tissue for 16S rRNA gene sequencing, which led to the identification ofMycobacterium haemophilum. Because of the special growth requirements of Mycobacterium haemophilum, iron-containing compounds (X-factor tablets; Rosco, Cardinal Health, Zutphen, The Netherlands) F. Bosma (*) . T. Schulin . W. J. G. Melchers Department of Medical Microbiology, University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands e-mail: F.Bosma@mmb.umcn.nl Tel.: +31-24-3614356 Fax: +31-24-3540216