Multicenter Comparison of Three Different Analytical Systems for Evaluation of DNA Banding Patterns from Cryptococcus neoformans

Abstract

The enormous improvement of molecular typing techniques for epidemiological and clinical studies has not always been matched by an equivalent effort in applying optimal criteria for the analysis of both phenotypic and molecular data. In spite of the availability of a large collection of statistical and phylogenetic methods, the vast majority of commercial packages are limited by using only the unweighted pair group method with arithmetic mean algorithm to construct trees and by considering electrophoretic pattern only as migration distances. The latter method has serious drawbacks when different runs (separate gels) of the same molecular analysis are to be compared. This work presents a multicenter comparison of three different systems of banding pattern analysis on random amplified polymorphic DNA, (GACA) 4 , and contour-clamped homogeneous electric field patterns from strains of Cryptococcus neoformans var. neoformans isolated in different clinical and geographical situations and a standard Saccharomyces cerevisiae strain employed as an outgroup. The systems considered were evaluated for their actual ability to(i) recognize identities, (ii) define complete differences (i.e., the ability to place S. cerevisiae out of the C. neoformans cluster), and (iii) estimate the extent of similarity among different strains. The ability to cluster strains according to the patient from which they were isolated was also evaluated. The results indicate that different algorithms do indeed produce divergent trees, both in overall topology and in clustering of individual strains, thus suggesting that care must be taken by individual investigators to use the most appropriate procedure and by the scientific community in defining a consensus system.

Optimization of Computer Software Settings Improves Accuracy of Pulsed-Field Gel Electrophoresis Macrorestriction Fragment Pattern Analysis

“…However, in agreement with the findings of Cardinali et al (3), there is a clear need for further development of software packages and algorithms used for gel analysis. Improvements, combined with optimization of the software algorithms prior to use, will further facilitate fragment pattern comparisons on multiple gels.…”

“…Much of the interlaboratory variation reported for PFGE results can be attributed to individual user technique, different types of PFGE instrumentation, and different protocols used for testing (4,21). However, variation can also be attributed to fragment position normalization and matching tolerance algorithms used for pattern interpretation (3,21). Random lane and fragment distortions occur within PFGE libraries (2,4).…”

Use of RAPD and killer toxin sensitivity in Saccharomyces cerevisiae strain typing

“…This procedure of merging different matrices in a single comprehensive matrix resulted quite effective in a previous work in which matrices from different typing experiments were merged together (Cardinali et al 2002).…”

“…DNA was extracted as previously described (Bolano et al 2001;Cardinali et al 2002) and amplified on a Hybaid Statistical analyses of electrophoretic data first requires that identical bands be located in different lanes and on separate gels. This was done using existing methodology (Cardinali et al 2002), which can be briefly outlined as follows.…”

“…This was done using existing methodology (Cardinali et al 2002), which can be briefly outlined as follows. Migration distances are measured and transformed into molecular weight data, which are then classified in a binary matrix according to a nonsubjective classification scheme.…”