Comparison of three typing methods for clinical and environmental isolates of Aspergillus fumigatus.

To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.     

Ribotyping for differentiating Flavobacterium meningosepticum isolates from clinical and environmental sources.

On the basis of DNA-DNA hybridization data, two main genomic relatedness groups (I and II) have been reported for a geographically varied collection of 52 strains of Flavobacterium meningosepticum. Herein, we have shown that genomic group II can be further divided into four subgroups (II:1 to II:4). To examine the taxonomic relevance of the ribosomal patterns of the 52 F. meningosepticum strains, the patterns were compared with existing DNA-DNA hybridization data with restriction enzymes PstI and HindIII. Ribotyping of the 52 F. meningosepticum strains showed banding patterns that could identify them correctly to one of the five genomic groups or subgroups. To assess the value of ribotyping for the interpretation of epidemiological data, the discriminatory power of the method was investigated for the 52 F. meningosepticum strains. With one to four restriction enzymes (PstI, HindIII, ClaI, EcoRI), a discriminatory index of 0.95 to 0.97 was found. The value of ribotyping in an epidemiological setting was assessed for three clinical isolates of F. meningosepticum from an outbreak of meningitis and bacteremia in the neonatal intensive care unit, Rigshospitalet, Copenhagen, Denmark. The three clinical isolates were shown to belong to the same ribotype, characteristic of genomic subgroup II:1. This ribotyping method will prove to be a useful tool for epidemiological studies concerning F. meningosepticum in the future.     

Detecting change: A comparison of three neuropsychological methods, using normal and clinical samples.

Detecting change in individual patients is an important goal of neuropsychological testing. However, limited information is available about test-retest changes, and well-validated prediction methods are lacking. Using a large nonclinical subject group (N = 384), we recently investigated test-retest reliabilities and practice effects on the Wechsler Adult Intelligence Scale and Halstead-Reitan Battery. Data from this group also were used to develop models for predicting follow-up test scores and establish confidence intervals around them. In this article we review those findings, examine their generalizability to new nonclinical and clinical groups, and explore the sensitivity of the prediction models to real change. Despite similarities across samples in reliability coefficients and practice effects, limits to the generalizability of prediction methods were found. Also, when multiple test measures were considered together, one or more "significant" changes were common in all (including stable) subject groups. By employing normative cut-offs that correct for this, sensitivity of the models to neurological recovery and deterioration was modest to good. More complex regression models were not more accurate than the simpler Reliable Change Index with correction for practice effects when confidence intervals for all methods were adjusted for variations in level of baseline test performance.     

Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods

The genotypes of 52 strains of Aspergillus fumigatus isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.     

Comparative analysis of multilocus sequence typing and pulsed-field gel electrophoresis for characterizing Listeria monocytogenes strains isolated from environmental and clinical sources

One hundred seventy-five Listeria monocytogenes strains were characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) based on loci in actA, betL, hlyA, gyrB, pgm, and recA. One hundred twenty-two sequence types (STs) were identified by MLST based on allelic profiles of the four housekeeping genes (betL, gyrB, pgm, and recA), and 34 and 38 alleles were identified for hlyA and actA, respectively. Several actA and hlyA alleles appeared to be predominantly associated with clinical isolates. MLST differentiated most of the L. monocytogenes strains better than did PFGE, and the discriminating ability of PFGE was better than that of serotyping. Several strains with different serotypes were found, by MLST and PFGE, to have very closely related genetic backgrounds, which suggested possible "antigen switching" among them. MLST can be a useful typing tool for differentiating L. monocytogenes strains (including strains undistinguishable by PFGE typing and serotyping), and it may be of value during investigations of food-borne outbreaks of listeriosis.     

Differences in biofilm production by three genotypes of Candida parapsilosis from clinical sources.

Three distinct genotypes of Candida parapsilosis (group I, II, and III) have been identified among clinical isolates but their clinical significance remains unclear. We investigated the distribution of C. parapsilosis genotypes in isolates from blood, all other sites from patients, and the hands of health care workers (HCWs), and we examined the relationship between genotype and biofilm positivity. The 53 bloodstream isolates and 38 of 39 HCW isolates were categorized as group I, whereas the 67 non-blood isolates taken from patients were distributed in groups I (n=43), II (n=13), and III (n=11). Biofilm positivity was observed in 77% (103 of 134) of group I isolates versus 0% (0 of 25) of non-group I (groups II and III) isolates (P < 0.01). There was no difference in biofilm production among group I isolates from blood (81%), other clinical specimens (72%), and the hands of HCWs (73%). This study has shown that biofilm production differs among three genotypes of C. parapsilosis isolates and that a majority of C. parapsilosis isolates from the bloodstream (100%), the hands of HCWs (97%), and all other sites from patients (64%) belong to group I, which has the ability to produce biofilm.     

Mitochondrial DNA fingerprinting of Acanthamoeba spp. isolated from clinical and environmental sources.

Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA fingerprinting) was evaluated as an epidemiologic tool for identifying potential reservoirs of Acanthamoeba infection. Fingerprints for 15 clinical isolates recovered by our affiliated laboratories were compared with those for 25 environmental isolates from western Washington State and 10 American Type Culture Collection (ATCC) strains. Seven different fingerprint groups emerged from the analysis of clinical isolates with six selected restriction enzymes (BamHI, BglII, EcoRI, HindIII, KpnI, and SalI). Fourteen (56%) environmental and 4 (40%) ATCC isolates displayed fingerprints similar to those of clinical isolates. In all, five of the seven groups contained one or more environmental and/or ATCC isolates. Comparisons with published mtDNA fingerprints for Acanthamoeba isolates showed that two groups have counterparts in Europe and Japan and in Europe and Australia. The inclusion of environmental isolates demonstrated that the most common clinical isolates do have counterparts readily recoverable from the surrounding environment and that some of these counterparts appear to be geographically widespread.     

Evaluation of three test procedures for identification of Staphylococcus aureus from clinical sources.

A total of 520 clinical and environmental isolates of the family Micrococcaceae that fermented glucose anaerobically were tested for their ability to produce coagulase, thermostable nuclease, and deoxyribonuclease. Of these, 450 isolates coagulated rabbit plasma, produced thermostable nuclease, and were identified as Staphylococcus aureus, 447 of which produced a 3+ to 4+ clot. The remaining three isolates produced a 2+ clot, deoxyribonuclease, and thermostable nuclease. It was found that three of the S. aureus isolates failed to produce deoxyribonuclease. A total of 70 isolates which did not coagulate rabbit plasma and which were thermostable nuclease negative were identified as S. epidermidis. Three of them produced deoxyribonuclease. It is suggested that the thermostable nuclease test be performed on all isolates producing a 2+ (or 1+) clot in the coagulase test before identifying them as S. aureus.     

Legionnaires’ disease caused by Legionella longbeachae and Legionella pneumophila: comparison of clinical features,host-related risk factors,and outcomes

Legionnaires’ disease remains an important cause of mortality and morbidity worldwide. Disease caused by Legionella pneumophila has been extensively studied, and its clinical characteristics have been well described. There is, however, little information on disease caused by Legionella longbeachae, despite its importance in some countries. We undertook a retrospective review of culture-positive cases of Legionnaires’ disease in the Canterbury region of New Zealand over 10 years, in order to compare the clinical features and outcomes of Legionnaires’ disease caused by these two species.     

Comparison of three typing methods for Pseudomonas aeruginosa isolates from patients with cystic fibrosis

The aim of this study was to compare two traditional pattern matching techniques, pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD), with the more reproducible technique of multilocus sequence typing (MLST) to genotype a blinded sample of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. A blinded sample of 48 well-characterized CF P. aeruginosa isolates was genotyped by PFGE, RAPD, and MLST, each performed in a different laboratory. The discriminatory power and congruence between the methods were compared using the Simpson’s index, Rand index, and Wallace coefficient. PFGE and MLST had the greatest congruence with the highest Rand index (0.697). The discriminatory power of PFGE, RAPD, and MLST were comparable, with high Simpson’s indices (range 0.973–0.980). MLST identified the most clonal relationships. When clonality was defined as agreement between two or more methods, MLST had the greatest predictive value (100?%) in labeling strains as unique, while PFGE had the greatest predictive value (96?%) in labeling strains as clonal. This study demonstrated the highest level of agreement between PFGE and MLST in genotyping P. aeruginosa isolates from CF patients. MLST had the greatest predictive value in identifying strains as unique and, thus, has the potential to be a cost-efficient, high-throughput, first-pass typing method.     

Multilocus sequence typing for characterization of clinical and environmental salmonella strains

Multilocus sequence typing (MLST) based on the 16S RNA, pduF, glnA, and manB genes was developed for Salmonella, and its discriminatory ability was compared to those of pulsed-field gel electrophoresis (PFGE) and serotyping. PFGE differentiated several strains undifferentiable by serotyping, and 78 distinct PFGE types were identified among 231 Salmonella isolates grouped into 22 serotypes and 12 strains of undetermined serotype. The strains of several PFGE types were further differentiated by MLST, which suggests that the discriminatory ability of MLST for the typing of Salmonella is better than that of serotyping and/or PFGE typing. manB-based sequence typing identified two distinct genetic clusters containing 32 of 54 (59%) clinical isolates whose manB gene sequences were analyzed. The G+C contents and Splitstree analysis of the manB, glnA, and pduF genes of Salmonella indicated that the genes differ in their evolutionary origins and that recombination played a significant role in their evolution.     

Characterization of bovine MHC class II polymorphism using three typing methods: serology, RFLP and IEF.

Various methods, with different strengths and weaknesses, are currently used to define polymorphism of the bovine major histocompatibility complex (MHC) class II genes. A more complete characterization of bovine lymphocyte antigen (BoLA) haplotypes can be achieved by combining several of these methods. In this study BoLA class II polymorphism was characterized using three typing methods: serology, restriction fragment length polymorphism (RFLP), and isoelectric focusing (IEF). Twenty six Holstein-Friesian and 15 Angus cattle that carried an array of serologically defined BoLA haplotypes were selected for the study. The panel included 12 BoLA complex homozygotes. The three class II typing methods recognized polymorphism associated with the same or very tightly linked genes in the DQ-DR class II subregion. In total 25 BoLA-A locus (class I)--DQ-DR subregion (class II) haplotypes were defined. Three of the serological class II specificities, Dx1, Dx3, and Dx4, were associated with more than one RFLP defined DQ-DR haplotype. The other 4 class II specificities behaved as private specificities. One BoLA haplotype was found in both Holstein and Angus cattle. Two other BoLA haplotypes defined here have previously been described in other breeds. This suggests that these haplotypes exist in strong linkage disequilibrium.     

Indirect immunofluorescence test for serodiagnosis of Legionnaires disease: evidence for serogroup diversity of Legionnaires disease bacterial antigens and for multiple specificity of human antibodies.

Evidence obtained by others who used direct immunofluorescence staining to demonstrate serological differences among strains of Legionnaires disease bacterium prompted this study of parameters influencing the ability of the indirect immunofluorescence test to detect human antibodies to Legionnaires disease bacterium. A total of 25 Legionnaires disease bacterium strains, representing four serogroups, were used as immunofluorescence antigens to test selected human sera. The use of diethyl ether in preparing the antigens was discontinued when it was found that titers against ether-killed group 2 (Togus 1-like) antigens were impossible to determine. Instead, heat-killed suspensions of Legionnaires disease bacterium in 0.5% buffered normal chicken yolk sac were used to show the serogroup diversity of the strains and the serogroup specificity of the antibody response of some, but not all, patients with serological evidence of Legionnaires disease. These studies suggest that multiple antigens should be used in serological tests for Legionnaires disease. Furthermore, the fact that some sera contain antibodies that bind equally well to strains of all four serogroups implies that demonstration of a fourfold increase in titer of paired sera when tested with a single antigen should not be interpreted as evidence of infection with a strain of the same serogroup.     

Molecular typing of clinical and environmental isolates of Scedosporium prolificans by inter-simple-sequence-repeat polymerase chain reaction.

Invasive infections by Scedosporium prolificans have increased alarmingly in recent years, mainly in immunosuppressed patients. The epidemiology, pathogenesis and the natural habitat of this pathogen are practically unknown. Isolates of S. prolificans were distinguished from one another by inter-simple-sequence-repeat (ISSR) fingerprinting, a technique based on the high degree of polymorphism of the multisatellite genetic markers used. This technique was found useful for typing 84 isolates of S. prolificans from different countries and sources. The assemblage of S. prolificans isolates tested was extremely diverse, with 35 genotypes present. Several patients were found to have been infected or colonized by more than one strain. Overall, this technique facilitates the epidemiological study of S. prolificans infection.     

Detecting patients with Alzheimer''s disease suitable for drug treatment: comparison of three methods of assessment.

BACKGROUND. Therapy to enhance cholinergic function in the brain is under evaluation for the treatment of Alzheimer's disease. Tetrahydroaminoacridine (tacrine) has recently received a product licence in the United States of America for the treatment of Alzheimer's disease, and the licence application in the United Kingdom will shortly be reviewed. It is therefore possible that this drug will become available for use in the UK in due course. There will then be a need for screening procedures for a large number of elderly patients to decide whether or not they have dementia and, if so, whether it is the result of Alzheimer's disease and is suitable for treatment with the new drug. METHOD. A total of 246 patients aged 75 years or over in two general practices in Bristol were assessed to investigate the potential workload such screening would engender. Three different assessment schedules for the diagnosis of dementia were compared--the mini-mental state examination, the Kew test, and the abbreviated mental test score. RESULTS. None of the assessment schedules was found to be particularly onerous, with median times for administration of five, three and two minutes, respectively. A score of 23 or less on the mini-mental state examination was taken as the main cut-off point for further evaluation. Sixty six patients obtained this score--in 25 the low score reflected factors other than dementia, and 11 others declined further assessment. Of the remaining 30 patients only four had probable Alzheimer's disease at an appropriate level of severity for treatment, and lived with a carer who could ensure compliance and monitor side effects. Two of these patients were receiving conflicting medical treatment and a third declined therapy, leaving only one person for whom treatment could be prescribed. CONCLUSION. It seems likely that of those medically suitable for treatment, it may not be possible to prescribe tacrine for an appreciable proportion. Nevertheless, all potential patients should be screened as the procedures involved are not onerous and at least some of those found suitable for treatment are likely to benefit from this new approach.     

Correlation of typing methods for Acinetobacter isolates from hospital outbreaks.

Four methods, namely, biotyping, cell envelope protein electrophoresis, ribotyping, and comparison of antibiograms, were used for strain identification of Acinetobacter isolates from five outbreaks in hospitals. There was good agreement among the methods for the identification of an index strain, but biotyping and the comparison of antibiograms were the least discriminatory.     

Latex agglutination-negative methicillin-resistant Staphylococcus aureus recovered from neonates: epidemiologic features and comparison of typing methods.

An unusual strain of methicillin-resistant Staphylococcus aureus (MRSA) was repeatedly isolated from infants in a newborn special care unit (NBSC) and a newborn intensive care unit. Between January 1989 and March 1990, approximately 100 isolates from infected or colonized infants were recovered. Surveillance cultures taken during this time revealed a 20% colonization rate, which was defined as recovery of MRSA from the nares, umbilicus, or groin. Isolates were identified as S. aureus by tube coagulase reactivity and heat-stable nuclease production but were unreactive in a latex agglutination assay. Representative isolates that were collected during the outbreak and that were found to share the latex agglutination assay-negative phenotype were compared by antibiogram (12 isolates), bacteriophage typing (20 isolates), capsular polysaccharide typing (30 isolates), and plasmid as well as chromosomal DNA analyses (20 isolates). All isolates known to be associated with the outbreak had nearly identical antibiograms and were notably susceptible to clindamycin. Staphylococcal bacteriophage typing was not useful in determining the relatedness of the isolates, since the majority were nontypeable. Plasmid pattern analysis revealed one large plasmid (approximately 100 kb) of equivalent size among the isolates. Capsular polysaccharide typing revealed that 14 of 30 isolates tested were type 5. Isolates identified in children at two other hospitals in the city which were also unreactive by the latex agglutination assay and clindamycin susceptible had plasmid and antibiogram patterns identical to those of isolates from the NBSC. Pulsed-field gel electrophoresis of restriction enzyme-digested genomic DNAs from the outbreak isolates demonstrated identical patterns which could be clearly differentiated from those of other unrelated MRSA. The strain from the NBSC is, therefore, unique and underscores the need for caution in interpreting the latex agglutination reactivities of MRSA isolates.