Molecular and Cellular Probes (2000) 14, 355–363 doi:10.1006/mcpr.2000.0325, available online at http://www.idealibrary.com on Description of a novel Mycobacterium simiae allelic variant isolated from Caribbean AIDS patients by PCR-restriction enzyme analysis and sequencing of hsp65 gene E. Legrand, K. S. Goh, C. Sola and N. Rastogi* Unite ´ de la Tuberculose et des Mycobacte ´ ries, Institut Pasteur, BP 484, F-97165 Pointe a ` Pitre-Cedex, Guadeloupe (Received 15 May 2000, Accepted 12 September 2000) A sudden upsurge in the isolation of Mycobacterium simiae from terminally ill AIDS patients was recently reported on the Caribbean island of Guadeloupe. Identification of these M. simiae isolates was achieved using biochemical tests and further confirmed by PCR-restriction analysis (PRA) of a 439-bp fragment of hsp65. A novel PRA profile III (three Bst EII fragments of 240/125/80 bp and four HaeIII fragments of 145/125/40/25 bp) was observed in four blood isolates from two patients. The 16 S rRNA gene sequencing of the hypervariable A region confirmed that all the pattern III isolates were indeed M. simiae species, and the hsp65 sequencing confirmed the existence of a new hsp65 allele in these caribbean isolates. A hsp65 sequence-based phylogenetic tree was also created for 39 species including M. simiae and related mycobacterial species as well as other rapid and slow growing mycobacteria, and may serve as an useful tool for identification of mycobacteria to species level.  2000 Academic Press KEYWORDS: Mycobacterium simiae, hsp65, PCR-restriction analysis, 16 S rRNA gene sequencing. INTRODUCTION M. simiae infection have only been rarely described; one involved a patient with multiple sclerosis, 8 In humans, Mycobacterium simiae is an opportunistic whereas others concerned patients with AIDS. 9–13 In this context, contrary to a single case of M. simiae pathogen infecting the lung and/or causing dis- seminated infection, that can be isolated from tap infection identified at the Pasteur Institute of Gua- deloupe for the period 1986–1991, a sudden upsurge water, the environment, and other suitable animal hosts. 1–8 With a variable response to niacin pro- in M. simiae infections was observed for the com- parable period of 1992–1997 (22 isolates from nine duction, it is one of the rare nontuberculous myco- bacteria that may be positive for niacin, however, patients). 14 Since this study, three more isolates from two patients were added to the previously described based on phenotypic characters and in case of a negative niacin test, it may sometimes be erroneously pool of M. simiae isolates. We hypothesized that this increase was essentially linked to the AIDS epidemic identified a member of the Mycobacterium avium complex. 1–8 Despite limited reports concerning Myco- as 9 of 11 patients were simultaneously coinfected with the HIV with CD4 counts below 50/mm 3 , an bacterium simiae infections associated with human disease, 1–7 cases of disseminated disease due to observation supported by a similar increase in * Author to whom all correspondence should be addressed at: Unite ´ Tuberculose et Mycobacte ´ries, Institut Pasteur, Morne Jolivie `re, BP 484, F-97165 Pointe-a `-Pitre Cedex, Guadeloupe. Tel: +590 893 881; Fax: +590 893 880; E-mail: rastogi@pasteur.gp 0890–8508/00/060355+09 $35.00/0  2000 Academic Press