Utilization of Single Nucleotide Polymorphisms To Detect and Speciate Atypical Mycobacteria in Formalin Fixed, Paraffin Embedded Specimens
JM Havens, LJ Gilbrech, SA Schichman, LW Lamps. Univ. Arkansas for Medical Sciences, Little Rock, AR; Central Arkansas Veterans Healthcare System, Little Rock, AR
Background: Non-tubucular mycobacterial (MOTT) infections are increasingly recognized as important causes of pneumonia and extrapulmonary infections, particularly in immunocompromised patients. Distinction of MOTT from M. tuberculosis, as well as identification of the MOTT species, is important because drug therapy differs. Diagnosis often requires time-consuming cultures and/or PCR and sequencing. Furthermore, many assays cannot be used in formalin fixed, paraffin-embedded (FFPE) tissues. Our goal was to develop a molecular assay that detects and distinguishes between 12 species of MOTT (including M. avium-intracellularae [MAI]; M. gordonae [MG]; M. simiae [MSi]; M. kansasii; M. malmiennse [MMa]; M. gastri [MGa]; M. marinum; M. scrofulaceum [MSc]; M. asiaticum; M. szulgai; and M. leprae), for use in FFPE tissues.
Design: Novel primers were designed exploiting single nucleotide polymorphisms (SNPs), which allows for subtle distinction between genetically similar species that cannot be detected by conventional PCR. The primers were used in conjunction with the ABI SNaPshot kit, which is designed for use with multiplex assays. 27 known MOTT positive FFPE specimens (including skin, lung, and intestine) were analyzed. Following deparaffinization and DNA extraction, a 130-bp fragment of the IST1 interspace region was amplified by PCR. The amplicons were then subjected to a second multiplex reaction using the novel primers. Capillary electrophoresis using an ABI 3100 genetic analyzer was used for detection. Known culture strains obtained from ATCC and known positive cases served as positive controls. Reagent blanks served as negative controls.
Results: 23 of 27 cases contained MOTT DNA by PCR; 4 cases did not contain amplifiable DNA. Species was determined in all 23 amplified cases as well (7 MAI, 3 MG, 4 MGa, 3 MSi, 2 MMa, 4 MSc). All positive and negative controls worked appropriately.
Conclusions: Using our novel assay, the MOTT species was determined in all 23 cases containing amplifiable DNA. Since the diagnosis of MOTT was considered retrospectively in these cases, culture was not performed, and thus comparison of this assay to other methods (including culture, AFB stains, and conventional PCR/sequencing) requires further study. However, our preliminary data indicate that the use of single nucleotide polymorphisms is an excellent method for detection and speciation of MOTT in FFPE tissues.
Wednesday, March 24, 2010 1:00 PM
Poster Session VI # 217, Wednesday Afternoon