Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.The use of electric field pulsing techniques in conjunction with agarose gel electrophoresis for discrimination of large DNA molecules was introduced by Schwarz and Cantor in 1984 (9). During the past decade the methodology has been adapted and improved by various research groups to the point that pulsed-field gel electrophoresis (PFGE) for bacterial strain typing is now utilized with relative ease in a variety of laboratories (1). The combination of contour-clamped homogeneous field electrophoresis and PFGE for the molecular analysis of Staphylococcus aureus has been reported since the late 1980s (7, 19). At present, PFGE is considered to have both the reproducibility and resolving power of a standard technique for the epidemiological typing of bacterial isolates (10, 15).Molecular typing systems can identify different strains within a species, generating data useful for taxonomic or epidemiologic purposes (10, 14). A frequently observed shortcoming of typing systems in general is their lack of reproducibility: most typing systems do not provide a definitive strain identification, which is usually due to the variability of the technique and the lack of large databases containing fragment patterns from a wide variety of organisms to which unknowns can be compared. These problems were recently described in detail for two molecular typing systems. A multicenter study on random amplification of polymorphic DNA for discrimination of S. aureus strains revealed a lack of interlaboratory reproducibility among the banding patterns generated by the participating centers, although the epidemiological interpretation of the data was similar for all the centers involved (16). For PFGE, a similar lack of interlaboratory reproducibility of patterns was observed, although the interpretation of the experimental data also differed per participating center (2). The latter study analyzed 12 different methicillin-resistant S. aureus (MRSA) strains with different techniques optimized in each center and different sources and types of equipment. Since interlaboratory discrepancies with respect to classification of the strains were observed, the study concluded that there is a clear need for standardization of the technique, including the construction of a panel of reference strains to assist the individual researcher in the optimization of the PFGE protocol.The aim of the present study was to compare the fragment patterns of a well-defined collection of MRSA isolates in 12 laboratories using in-house and a standard set of PFGE parameters to determine whether standardization of experimental parameters (DNA preparation and switching protocols) would improve intercenter reproducibility of PFGE analysis.     

Integrative transformation of the mycotoxin-producing fungus,Penicillium paxilli

A high frequency transformation system has been developed for Penicillium paxilli using pAN7-1. Up to 44% of the primary transformants were heterokaryons. Loss of hygromycin resistance was observed in primary transformants that were sub-cultured on non-selective media, but single spores of these primary transformants were mitotically stable on both selective and non-selective media. A molecular analysis of the transformants generated showed that 78% had single-site integrations, with half of these containing a single copy of pAN7-1. CHEF-gel electrophoresis showed that P. paxilli has at least six chromosomes with a total genome size of about 23.4 Mb.     

Construction of cosmid contigs and high-resolution restriction mapping of the Huntington disease region of human chromosome 4

The gene responsible for Huntington disease (HD) has been localizedto a 2.2 million base pair (Mbp) region between the loci D4S10and D4S98 on the short arm of human chromosome 4. As part ofa strategy originally designed to clone the gene based on itschromosomal location, we and others previously identified overlappingyeast artificial chromosome (YAC) clones covering most of thisregion. While these YAC clones were useful for initially obtaininglong-range clone continuity, a number of features of the YACsindicated that smaller clones are generally more useful in thesubsequent steps of the positional cloning strategy. In thispaper, we use these YAC clones to generate sets of overlappingcosmid clones covering most of the HD region. We Isolated alarge number of cosmids by screening a chromosome 4-specificcosmld library with labeled DNA from a minimal overlapping setof YAC clones. These cosmid clones were further analyzed byrestriction mapping and hybridization experiments, leading tothe assembly of 185 cosmids Into eleven contigs covering morethan 1.65 Mbp and to a fine-structure restriction map of theregion. Nine of these contigs cover 90 percent of the 1.7 Mbpsubregion between loci D4S125 and D4S98 where the HD gene isnow known to lie. The detailed restriction map and the cosmidclones should facilitate the identification and localizationof cDNAs and polymorphic markers, and they provide reagentsfor large scale DNA sequencing of this region of the human genome.Our results suggest that this strategy should be generally usefulfor converting YAC clones into cosmid contigs and generatinghigh-resolution restriction maps of genomioc regions of interest.     

Evidence of genetic heterogeneity in the autosomal recessive adult forms of limb-girdle muscular dystrophy following linkage analysis with 15q probes in Brazilian families.

The autosomal recessive limb-girdle muscular dystrophies (LGMD) represent a heterogeneous group of diseases which may be characterised by one or more autosomal loci. A gene at 15q has recently been found to be responsible for a mild form of LGMD in a group of families from the isolated island of Réunion, now classified as LGMD2. Based on results of eight out of 11 large Brazilian LGMD families of different racial background (which were informative for the closest available probe to the LGMD2 gene), we confirmed linkage to the LGMD2 gene at 15q in two of these families and exclusion in six others. These data provide the first evidence of genetic heterogeneity for the autosomal recessive limb-girdle muscular dystrophies.     

Presence of autoantibodies to apolipoprotein A-1 in patients with acute coronary syndrome further links autoimmunity to cardiovascular disease

Anti-apolipoprotein A-1 (Apo A-1) autoantibodies were described in autoimmune disorders such as systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and might be involved in the genesis of arterial and venous thrombotic events. To investigate the presence of these autoantibodies in patients with acute coronary syndrome (ACS) without other features of autoimmunity, we set up an enzyme-linked immunosorbent assay (ELISA) for anti-Apo A-1 antibodies. We used it to investigate their prevalence in ACS as compared to SLE and APS and correlated them to plasma Apo A-1 and serum amyloid A protein (SAA) concentrations. The prevalence of anti-Apo A-1 autoantibodies in the healthy control group was 1% (1/92), but was significantly higher in other groups: 21% (11/53) in ACS group (P=0.001), 13% (12/92) in SLE and/or APS group (P=0.005). Multiple linear regression revealed a significant correlation between plasma Apo A-1 (r=-0.72, P=0.013), plasma SAA concentration (r=0.76, P=0.0066) and anti-Apo A-1 IgG titre in ACS patients. The presence of anti-Apo A-1 autoantibodies in patients with ACS highlights an additional link between autoimmunity, inflammation and atherosclerosis.     

Can population differences explain the contrasting results of the Mwanza, Rakai, and Masaka HIV/sexually transmitted disease intervention trials?: A modeling study

OBJECTIVE: To determine whether population differences can explain the contrasting impacts on HIV observed in the Mwanza trial of sexually transmitted disease (STD) syndromic treatment (ST), the Rakai trial of STD mass treatment (MT), and the Masaka trial of information, education, and communication (IEC) with and without ST as well as to predict the effectiveness of each intervention strategy in each population. METHODS: Stochastic modeling of the transmission of HIV and 6 STDs was used with parameters fitted to demographic, sexual behavior, and epidemiological data from the trials and general review of STD/HIV biology. RESULTS: The baseline trial populations could be simulated by assuming higher risk behavior in Uganda compared with Mwanza in the 1980s, followed by reductions in risk behavior in Uganda preceding the trials. In line with trial observations, the projected HIV impacts were larger for the ST intervention in Mwanza than for the MT intervention in Rakai or the IEC and IEC + ST interventions in Masaka. All 4 simulated intervention strategies were more effective in reducing incidence of HIV infection in Mwanza than in either Rakai or Masaka. CONCLUSIONS: Population differences in sexual behavior, curable STD rates, and HIV epidemic stage can explain most of the contrast in HIV impact observed between the 3 trials. This study supports the hypothesis that STD management is an effective HIV prevention strategy in populations with a high prevalence of curable STDs, particularly in an early HIV epidemic.     

Experimental glomerulopathy alters renal cortical cholesterol,SR-B1, ABCA1, and HMG CoA reductase expression

Previous studies indicate that acute tubular injury causes free cholesterol (FC) and cholesteryl ester (CE) accumulation within renal cortex/proximal tubules. This study assessed whether similar changes occur with glomerulopathy/nephrotic syndrome, in which high-circulating/filtered lipoprotein levels increase renal cholesterol supply. Potential adaptive changes in cholesterol synthetic/transport proteins were also assessed. Nephrotoxic serum (NTS) or passive Heymann nephritis (PHN) was induced in Sprague-Dawley rats. Renal injury (blood urea nitrogen, proteinuria) was assessed 2 and 7 days (NTS), or 10 and 30 days (PHN) later. FC and CE levels in renal cortex, isolated glomeruli, and proximal tubule segments were determined. SR-B1 (a CE influx protein), ABCA1 (a FC exporter), and HMG CoA reductase protein/mRNA levels were also assessed. FC was minimally elevated in renal cortex (0 to 15%), the majority apparently localizing to proximal tubules. More dramatic CE elevations were found ( approximately 5 to 15x), correlating with the severity of proteinuria at any single time point (r >/= 0.85). Cholesterol increments were associated with decreased SR-B1, increased ABCA1, and increased HMG CoA reductase (HMGCR) protein and its mRNA. Tubule (HK-2) cell culture data indicated that SR-B1 and ABCA1 levels are responsive to cholesterol supply. Experimental nephropathy can increase renal FC, and particularly CE, levels, most notably in proximal tubules. These changes are associated with adaptations in SR-B1 and ABCA1 expression, which are physiologically appropriate changes for a cholesterol overload state. However, HMGCR protein/mRNA increments can also result. These seem to reflect a maladaptive response, potentially contributing to a cell cholesterol overload state.     

Interobserver variability in determining MIB-1 labeling indices in oligodendrogliomas

Several studies have shown that MIB-1 labeling indices correlate well with tumor grade and prognosis in a variety of tumor types. Several factors are responsible for some degree of variability in the determination of labeling indices. Interobserver variability is one of the factors often cited as responsible for this variability. A slide from each of 30 oligodendrogliomas, stained with MIB-1 antibody, was distributed to six pathologists. The same set of slides was reviewed by each individual. Each pathologist was instructed to determine a MIB-1 labeling index by evaluating 1,000 tumor cell nuclei from the area of the slide with the most staining. The labeling index record reflected a percentage of positive-staining tumor cells. Interobserver agreement was compared. MIB-1 labeling indices ranged from 0 to 45.7. Overall agreement was good (> or =0.75) with a concordance coefficient of 0.832 (confidence interval, 0.700 to 0.909). Variability was greater among tumors with higher labeling indices as compared with tumors with labeling indices closer to 0. The overall agreement of MIB-1 labeling indices, while not perfect, was good. The generally minor variability among observers may be related to differences in the area of the slide evaluated and in differing lower thresholds for interpreting positivity. Further improvement of concordance may theoretically be attainable by further training and discussion among observers.