Western immunoblot analysis of protein antigens of Penicillium marneffei.

Protein antigens of Penicillium marneffei prepared during the yeast and mould phases of in vitro growth were analyzed by gel electrophoresis and immunoblot assay. More than 20 yeast phase proteins were detected by Coomassie staining; among these, at least 10 reacted with IgG in the pooled sera of 28 AIDS patients with penicilliosis. Four immunogenic proteins of 200, 88, 54 and 50 kDa were produced in large quantity during the deceleration and early stationary phases of growth. When these proteins were reacted with individual sera derived from 33 AIDS patients with penicilliosis, reactivities to the 200, 88, 54 and 50 kDa protein were detected in 72.7, 93.9, 60.6 and 57.6%, respectively. The bands of 88, 54 and 50 kDa gave strong reactions with about a half of serum samples. In one serum derived from an AIDS patient, reactivities to the 54 and 50 kDa proteins could be strongly detected two months before the definite diagnosis by fungal culture. Protein components from the mould form were of lower yield and gave weaker signal in immunoblot analysis. These results indicate that at least two yeast-phase immunoreactive proteins (54 and 50 kDa) are relatively specific to the P. marneffei infection, thereby suggesting its potential for clinical application to the diagnosis of this emerging disease.

Efficiency of the flotation method in the isolation of Penicillium marneffei from seeded soil.

The efficiency of a modified flotation method for the isolation of Penicillium marneffei from soils has been evaluated. About 80% was recovered from sterilized soil freshly seeded with P. marneffei (100 conidia/1.5 g soil). When seeded non-sterile soil was used (at least 100 seeded conidia/1.5 g soil), P. marneffei could be effectively recovered by employing a combination of the flotation method and the mouse inoculation method.

Serological reactions of leptospirosis-positive (MAR and CFT) bovine sera in ELISA.

The collection of test sera for measuring ELISA results was composed of bovine sera with MAT titres of greater than or equal to 1:200 in the leptospirosis MAT and of greater than or equal to 1:5 in the CFT together with sera from a serologically negative and clinically non-suspicious cattle herd. To establish cut-off ODs, the geometric mean net-extinction of the negative serum collection plus 1, 2, and 3 standard deviations were calculated. By comparison of 3 different conjugates from rabbits, it was demonstrated that results from anti-total bovine Ig were superior to anti-IgG and anti-IgM conjugates. Considerations regarding sensitivity and specificity led to the recommendation to use a test serum dilution of 1:160, to apply anti-total bovine Ig conjugates, and to establish the cut-off OD at the geometric mean net-extinction of negative sera plus 3 standard deviations. Under such conditions, agreement between leptospirosis MAT/CFT positivity on the one side and ELISA positivity on the other was reached in 74%. This recommendation is made for cross-sectional studies but not for examinations of clinically suspicious cattle herds.