Increasing incidence of Mycobacterium xenopi at Bellevue hospital: An emerging pathogen or a product of improved laboratory methods?

STUDY OBJECTIVES: To investigate the dramatic rise in number of Mycobacterium xenopi isolates identified in our mycobacteriology laboratory, and to determine if this increase was due to emerging clinical pathology or to changes in culture technique. DESIGN: Retrospective chart and laboratory review. SETTING: University-affiliated tertiary-care city hospital. PATIENTS: Eighty-one patients with a single culture positive for M xenopi from 1975 to 1994 (period 1), and 47 patients with two or more cultures positive from 1994 to 1998 (period 2). INTERVENTIONS: The Bellevue mycobacteriology laboratory changed the culture medium from solid Lowenstein-Jensen medium (used from 1975 to 1990) to the Septi-Check AFB System (Becton-Dickinson; Glencoe, MD; used from 1991 to 1994), to the Mycobacteria Growth Indication Tube (MGIT; Becton-Dickinson; used from 1995 to 1998). MEASUREMENTS AND RESULTS: We recovered 29 M xenopi isolates from 1975 to 1990, 12 isolates from 1991 to 1994, and 381 isolates from 1995 to 1998. We subsequently identified and reviewed the medical records of all 81 patients who were culture positive for M xenopi from 1975 to 1994 (period 1), and 46 patients who had two or more isolates culture positive for M xenopi from 1995 to 1998 (period 2). For period 1, 75% of the subjects were male, 80% were minority, and at least 43% were HIV positive. Only one patient had clinical M xenopi lung disease during this period. For period 2, 79% of the subjects were male, 83% were minority, and at least 58% were HIV positive; two additional patients were identified who had clinical M xenopi lung disease. CONCLUSIONS: The dramatic increase in M xenopi isolates noted in our hospital was due to a more sensitive laboratory isolation technique, rather than a true increase in clinical disease. Other hospitals utilizing MGIT systems for mycobacterial recovery should interpret positive M xenopi cultures with caution.

Correction of glycogen storage disease type II by enzyme replacement with a recombinant human acid maltase produced by over-expression in a CHO-DHFR(neg) cell line.

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in the autosomal recessive glycogen storage disease type II (GSD II). To investigate whether we could generate a functional recombinant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cDNAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFR(neg) Chinese hamster ovary cells and established a stable cotransformant that expressed rhGAA. We cultured the recombinant cells in media with progressively increasing concentrations of methotrexate and found that human GAA enzyme activity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein by rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulted in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-phosphate in the media blocked uptake. GAA -/- mice were treated with the rhGAA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, most notably skeletal muscle, heart and diaphragm after two infusions. More importantly, after multiple infusions, hind, and fore-limb muscle weakness was reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II.

Isolation of the gene for the beta subunit of RNA polymerase from rifampicin-resistant Mycobacterium tuberculosis and identification of new mutations.

Tuberculosis (TB) is one of the most important infections worldwide, with an estimated incidence of 10 million active cases per year. Rifampicin is a key component of the first-line therapy used in the treatment of tuberculosis. In Escherichia coli and Mycobacterium leprae, rifampicin has been shown to inhibit the beta subunit of RNA polymerase. The gene (rpoB) encoding this enzyme has been described in both species. We report the isolation of the homologous functional rifampicin resistance gene from M. tuberculosis. A library was constructed with 15 to 25 kb BamHI-digested DNA fragments from a rifampicin-resistant M. tuberculosis clinical isolate that was ligated into an E. coli-mycobacterial shuttle plasmid. Southern analysis of BamHI-digested DNA from 200 recombinant plasmids was performed and filters were hybridized to a 411 bp fragment of the beta subunit of RNA polymerase from M. tuberculosis. Only DNA from one plasmid (#86) hybridized, which suggested that the gene is found as a single copy per genome. This plasmid was able to transfer rifampicin resistance to sensitive M. smegmatis and thus codes for a functional genetic unit. Sequence analysis in the expected "hotspot" region in eight rifampicin-resistant M. tuberculosis strains (including one multidrug-resistant strain) revealed two novel mutations as well as others previously described.