Fourty-five sputum specimens collected at the National Sanatorium Hiroshima Hospital were subjected to cultivation using either BACTEC 460 TB System (BACTEC method; Becton Dickinson Co., Towson, Md., U.S.A.) or 3% Ogawa egg medium. Test suptum was treated with four volumes of 4% NaOH for approximately two minutes, after which 0.1ml of the treated sputum was immediately inoculated onto 3% Ogawa egg medium. After neutralizing the remaining pretreated sputum with 1N HCl, and diluting with 1/15 M phosphate buffer PB; pH 6.8), it was then centrifuged at 3, 000rpm for 20min and the sediment was suspended in 1.5 ml of PB. Volumes of 0.5 ml each were inoculated into BACTEC 12B medium (4ml), containing PANTA for prevention of contamination and POES for promoting the growth of mycobacteria.
In the BBCTEC method, bacterial growth was measured in terms of increases in the Growth Index (GI) values which were determined by the amount of 14CO2 released from the 14C -labelled palmitate during cultivation at 37°C (positive growth;GI≤50). Moreover, ρInitro-α-acetylamino-β-hydroxy-propiophenone (NAP)-sensitivity testing was done by transferring a part of the BACTEC 12B culture showing positive growth to a NAP vial, and thereafter subjected to further cultivation.
Among the 45 sputum specimens, the number of positive specimens for mycobacterial growth in the afore mentioned cultivation methods and time required for growth were as follows: 3% Ogawa egg medium, 12 specimens (27%), seven M. tuberculosis complex strains at 12-35 days (average 21 days), five M. avium complex strains at 14-21 days (average 18 days): BACTEC method, 18 specimens (40%), 11 M. tuberculosis complex strains at 3-28 days (average 14 days), six M. avium complex strains at 3-10 days (average 6 days) and one M. scrofulaceum strain at 28 days. There were no specimens that tested positive for mycobacterial growth on 3% Ogawa egg medium but negative in BACTEC 12B medium. The BACTEC method was most efficacious in cultivating acid-fast bacilli from smear-negative
When NAP-sensitivity testing was done using the BACTEC method, mycobacteria in 11 test sputa were deteremined as NAP-sensitive, thereby belonging to M. tuberculosis complex. The fact that all of the organisms determined as NAP-sensitive using the BACTEC method were rough and nonphotochromogenic, and identified as M. tuberculosis complex by AccuProbeTM testing, confirmed the reliability of NAP-testing. sputa.
The mycobacteria in seven sputum specimens detected using the BACTEC method were determined as NAP-resistant. Six of them were smooth and nonphotochromogenic, and identified as M. avium complex by AccuProbe testing. The one remaining strain was a scotochromogen with a smooth colony morphology, and had no reaction to either the M. tuberculosis complex- or M. avium complex-AccuProbe tests. This strain was identified as M. scrofulaceum using an α-antigen analysis.
These results indicate the usefulness of the BACTEC 460 TB system in the rapid diagnosis of mycobacteria including M. tuberculosis complex and M. avium complex.
