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Showing papers on "Typing published in 1995"


Journal ArticleDOI
TL;DR: A modified, standardized PFGE methodology should enable typing laboratories to obtain rapid, reliable results in 3 to 4 days when starting with an isolated colony on agar media.
Abstract: Bacteriophage typing (BT) (World Health Organization method) has been used at the Centers for Disease Control and Prevention for over 30 years to type isolates of Staphylococcus aureus. Since studies have shown that BT patterns have poor reproducibility and because BT fails to type a high percentage (15 to 20%) of isolates, the Centers for Disease Control and Prevention has converted from using BT to using pulsed-field gel electrophoresis (PFGE) for strain typing S. aureus. We compared the results of BT with results of PFGE for typing 300 isolates of S. aureus, including strains from several well-characterized outbreaks. Ninety-six isolates were BT group I, 19 were group II, 82 were group III, 7 were group V, and 96 were nontypeable. PFGE identified subgroups within each phage group and thus was more discriminating than BT, which identified no subgroups. PFGE was able to type all isolates and distinguish related from unrelated strains of S. aureus. Our modified, standardized PFGE methodology should enable typing laboratories to obtain rapid, reliable results in 3 to 4 days when starting with an isolated colony on agar media.

567 citations


Journal ArticleDOI
TL;DR: A typing enzyme immunoassay (TYPE-EIA) was used to determine the antigenic types of 64 astrovirus-positive specimens from nine collections from seven countries, finding most strains of the same type were identical.
Abstract: A typing enzyme immunoassay (TYPE-EIA) was used to determine the antigenic types of 64 astrovirus-positive specimens from nine collections from seven countries. Six of the seven known astrovirus types were detected in the collections, with HAstV-1 predominating in all collections for one from the United Kingdom. Selected specimens were analyzed further by reverse transcriptase PCR and nucleotide sequencing of 348 bp within the capsid protein precursor region of the genome. The phylogenetic groupings (genotypes) determined from the sequences were entirely consistent with the antigenic groupings (serotypes) of isolates obtained by using the TYPE-EIA. The genetic variation within genotypes was small compared with the variation between genotypes, allowing unambiguous categorization of all specimens. Although some strains from widely separated geographic areas had identical sequences, in general, within a region most strains of the same type were identical. The TYPE-EIA may help further our understanding of the epidemiology of astrovirus and the possible role of serotype-specific immunity, while further knowledge of sequences could facilitate the development of simpler molecular methods of typing astrovirus strains.

349 citations



Journal ArticleDOI
TL;DR: Despite the high frequency of single- and zero-band isolates in this population, the discriminatory power of RFLP typing with IS6110 is sufficiently high to be useful for clinical and epidemiological studies.

210 citations


Journal ArticleDOI
TL;DR: A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described and two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented.
Abstract: Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.

210 citations


Journal ArticleDOI
K. E. Wright1, Gareth A. Wilson1, D. Novosad1, C. Dimock1, D. Tan1, J. M. Weber1 
TL;DR: A series of oligonucleotide primers able to detect, type, and subtype type A influenza viruses in a single reverse transcription-PCR was developed and was comparable to standard culture techniques in the detection, typing, andSubtyping of influenza viruses.
Abstract: Type A and B influenza viruses can cause a wide spectrum of illness, and these viruses are responsible for considerable mortality and morbidity. Rapid typing of isolates is desirable when amantadine treatment or prophylaxis of contacts of type A influenza virus carriers is considered, but the available rapid techniques lack sensitivity and standard diagnostic methods require expansion of virus in tissue culture or embryonated hens' eggs. We developed a series of oligonucleotide primers able to detect, type, and subtype type A influenza viruses in a single reverse transcription-PCR. RNA was isolated from clinical specimens, and cDNA was generated with random primers. PCR was carried out with a mixture of primers specific for influenza viruses of types B, A/H1 and A/H3, and subtyping of the neuraminidase was carried out on the same cDNA template under identical conditions. Amplified products were detected by ethidium bromide staining of amplified products after agarose gel electrophoresis. When it was used to test 98 clinical specimens, this method was comparable to standard culture techniques in the detection, typing, and subtyping of influenza viruses.

194 citations


Journal ArticleDOI
TL;DR: It is concluded that macrorestriction analysis of P. aeruginosa with SpeI provides the best means of discrimination between epidemiologically unrelated strains and DNA probe typing with either toxA or rDNA reveals information on the strain population structure and evolutionary relationships.
Abstract: We assessed the capacity of three DNA typing techniques to discriminate between 81 geographically, temporally, and epidemiologically unrelated strains of Pseudomonas aeruginosa. The methods, representing powerful tools for hospital molecular epidemiology, included hybridization of restricted chromosomal DNA with toxA and genes coding for rRNA (rDNA) used as probes and macrorestriction analysis of SpeI-digested DNA by pulsed-field gel electrophoresis. The probe typing techniques were able to classify all strains into a limited number of types, and the discriminatory powers were 97.7 and 95.6% for toxA and rDNA typing, respectively. Strains that were indistinguishable on the basis of both toxA and rDNA types defined 12 probe type homology groups. Of these, one contained five strains, three contained three strains each, and eight groups were represented by two strains each. Strains in 10 of the homology groups had the same O serotype. SpeI macrorestriction patterns discriminated between all strains with at least four band differences, which corresponded to a similarity level of 85%. Fifteen pairs of strains were similar at a level of > 75% and differed by only four to seven bands. Of these pairs, 11 belonged to the same probe type homology group, indicating their clonal relatedness. We conclude that macrorestriction analysis of P. aeruginosa with SpeI provides the best means of discrimination between epidemiologically unrelated strains. However, DNA probe typing with either toxA or rDNA reveals information on the strain population structure and evolutionary relationships.

167 citations


Journal ArticleDOI
TL;DR: A comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions is described.
Abstract: Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.

163 citations


Journal ArticleDOI
TL;DR: An analysis of the constraints of PCR typing of field Plasmodium falciparum isolates by using a few highly polymorphic markers, MSA-1, M SA-2, TRAP, and CS shows that the reactions are specific for the P. falcIParum species.
Abstract: We report on an analysis of the constraints of PCR typing of field Plasmodium falciparum isolates by using a few highly polymorphic markers, MSA-1, MSA-2, TRAP, and CS. We show that the reactions are specific for the P. falciparum species. The detection threshold (minimum number of parasites required to detect a visible band by ethidium bromide) differed from one marker to the other and, within one locus, from one primer combination to the other. Importantly, the various MSA-1 and MSA-2 reference alleles were amplified with the same efficiency. Amplification from reconstituted allele mixtures indicated that at certain allele ratios, the most abundant allele interfered with the amplification of the less abundant one. An analysis of nine isolates collected from patients with acute malaria in Dielmo, Senegal, during a transmission season when the inoculation rate was one infective bite every second night is presented and discussed. All samples contained more than one parasite type. A significant polymorphism was observed for the four markers. Novel TaqI restriction fragment length polymorphisms were found for the TRAP gene, and TRAP gene typing alone allowed a distinction between the various isolates. MSA-1 and MSA-2 gave multiple band patterns specific for each sample.

152 citations


Journal ArticleDOI
TL;DR: The first evidence of dual HIV-1 infection in humans is provided and the need for polyvalent vaccines is reinforced, suggesting that antiviral immunity evoked by one subtype can be incompletely protective against a second.
Abstract: Multiple genetic subtypes of human immunodeficiency virus type 1 (HIV-1) have been identified among internationally collected isolates The HIV-1 epidemic in Thailand is largely due to B and E subtypes of virus Dual infection with distinct HIV-1 subtypes would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second Polymerase chain reaction typing and serologic typing were used to screen a panel of specimens from HIV-1-infected subjects in Thailand Two persons simultaneously harbored HIV-1 of env subtypes B and E, and this was confirmed by colony hybridization with subtype-specific probes and nucleotide sequence analysis of a 630-bp fragment of gp120 from multiple molecular clones In addition, both subtypes were identified in cocultured peripheral blood mononuclear cells from 1 individual These data provide the first evidence of dual HIV-1 infection in humans and reinforce the need for polyvalent vaccines

148 citations


Journal ArticleDOI
TL;DR: The NA2 gene is more frequent in the German population than the NA1 gene, as determined by genotyping using PCR‐SSP, in contrast to GIFT, which showed an error rate for NA typing of 15 percent.

Journal ArticleDOI
TL;DR: The data demonstrate that DNA exposed to a variety of environmental insults yields reliable PM typing results and can be used in forensic analyses and paternity tests to estimate the frequency of a multiple locus DNA profile in various general United States populations.
Abstract: Studies were performed to evaluate the forensic applicability of multiplex amplification of the loci low density lipoprotein receptor, glycophorin A, hemoglobin G gammaglobin, D7S8, and group-specific component (PM loci) and simultaneous typing of these loci using a reverse dot blot approach where allele specific oligonucleotide probes are immobilized on a nylon membrane strip. These results were obtained by using the AmpliType® PM PCR Amplification and Typing Kit. The experiments included: mixed body fluid studies; chemical contaminant effects on the DNA in body fluid samples; the effect of typing DNA from body fluid samples deposited on various substrates; the effect of microorganism contamination on typing DNA derived from blood and semen; the effect of sunlight and storage conditions on DNA typing; determination of the sensitivity of detection of the PM test kit; determination of cross-reactivity of DNA from species other than human; typing DNA derived from various tissues from an individual; and an evaluation of the hybridization temperature of the assay. The data demonstrate that DNA exposed to a variety of environmental insults yields reliable PM typing results. Allele and genotype frequencies for six loci (PM loci and HLA-DQα) were determined in African Americans, Caucasians, southeastern Hispanics, and southwestern Hispanics. All loci meet Hardy-Weinberg expectations and there is little evidence for association of alleles between the loci. The frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a multiple locus DNA profile in various general United States populations.

Journal ArticleDOI
TL;DR: A novel method for molecular typing of organisms, amplified fragment length polymorphism analysis, was tested for its suitability in epidemiological studies in medical microbiology and is fast, efficient, and reproducible for typing strains of Legionella pneumophila isolated from both humans and the environment.
Abstract: A novel method for molecular typing of organisms, amplified fragment length polymorphism analysis, was tested for its suitability in epidemiological studies in medical microbiology. Amplified fragment length polymorphism analysis, originally developed for typing crop plants, consists of a simple restriction-ligation reaction and a subsequent PCR amplification. In a single-step reaction, the genomic DNA is digested and the restriction fragments are ligated to specially constructed adapters. PCR amplification of such tagged restriction fragments with primers complementary to the adapters allows the detection of restriction fragment length polymorphisms upon resolution on agarose gels. The method is fast, efficient, and reproducible for typing strains of Legionella pneumophila isolated from both humans and the environment. The accuracy of the method was tested by comparison with standard restriction fragment length polymorphism typing performed with both a ribosomal and a genomic probe.

Journal ArticleDOI
TL;DR: PFGE is suggested to be the most discriminatory of the three subtvping methods examined in an epidemiological investigation of Campylobacter jejuni in the UK.
Abstract: In this study we have evaluated the ability of three typing methods, pulsed field gel electrophoresis (PFGE), phage-typing and ribotyping, to discriminate not only between strains of differing serotypes but also between strains within a single serotype, heat stable serotype 2 (HS2). Forty-five isolates derived from cases of campylobacter enteritis occurring in the Cardiff area were examined. These included 18, mostly HS2, strains associated with an outbreak. The typing results for these and a further 39 epidemiologically unrelated strains of serotype HS2 were compared. This is the first report documenting the use of PFGE in an epidemiological investigation of Campylobacter jejuni in the UK. The results presented suggest that this technique is the most discriminatory of the three subtyping methods examined.

Journal ArticleDOI
TL;DR: The results indicate that the ERIC-PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis ofplasmids, and ribotyping.
Abstract: Shigella sonnei is a major cause of diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. In this study, we have compared different molecular typing methods, i.e., plasmid profile analysis, restriction endonuclease analysis of plasmids, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis (PFGE), and enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR (ERIC-PCR) for typing 20 clinical isolates of S. sonnei collected from six incidents of infection. PFGE and ERIC-PCR fingerprintings had the highest discriminatory power for discrimination of epidemiologically related isolates from epidemiologically unrelated strains of S. sonnei, and both gave seven distinct strain types among these isolates and the type strain of the species. Plasmid study and ribotyping produced only six and typing techniques demonstrated two distinct patterns, respectively, among these strains. All of these molecular an identical fingerprint for eight temporally related sporadic isolates. It is possible that these temporally related isolates belonged to a single bacterial clone and circulated obscurely through the community. Our results indicate that the ERIC-PCR technique represents a rapid and simple means for typing S. sonnei with a level of discrimination equivalent to that of PFGE but greater than those of plasmid profile analysis, restriction endonuclease analysis of plasmids, and ribotyping.

Proceedings ArticleDOI
16 May 1995
TL;DR: This paper describes a simple, software based keyboard monitoring system for the IBM PC for the continuous analysis of the typing characteristics of the user for the purpose of continuous authentication.
Abstract: This paper describes a simple, software based keyboard monitoring system for the IBM PC for the continuous analysis of the typing characteristics of the user for the purpose of continuous authentication. By exploiting the electrical characteristics of the PC keyboard interface together with modifications to the internal system timer, very accurate measurements can be made of keystroke interval and duration, including measurements of rollover. Rollover patterns, particularly when typing common diphthongs, can be highly characteristic of individual users and provide quite an accurate indication of the users identity.

Journal ArticleDOI
TL;DR: Restriction fragment length polymorphism was used to study 75 clinical isolates identified as Mycobacterium avium and two repetitive insertion sequences, IS1311 and IS900, exhibited marked polymorphism with both probes.
Abstract: Restriction fragment length polymorphism (RFLP) was used to study 75 clinical isolates identified as Mycobacterium avium. Two repetitive insertion sequences, IS1311 and IS900, were used as DNA probes. Although less than 25% of isolates showed RFLP patterns with IS900, all strains gave banding patterns with IS1311. M. avium strains isolated from patients with AIDS exhibited marked polymorphism with both probes.

Journal ArticleDOI
Inger Kühn1, L. G. Burman1, S. Haeggman1, K. Tullus1, Barbara E Murray1 
TL;DR: The PhP system is useful for epidemiological studies of enterococcal isolates and is suitable for analyzing large numbers of isolates, yielding results similar to those obtained with DNA typing by pulsed-field gel electrophoresis.
Abstract: The Phene Plate (PhP) biochemical fingerprinting system for bacteria is based on measurements of the kinetics of bacterial biochemical reactions. This system was modified for typing of enterococci and was compared with DNA typing by pulsed-field gel electrophoresis and with ribotyping by using 45 Enterococcus faecalis isolates from international collections. It was also used to study 170 fecal enterococcal isolates from healthy individuals and 28 isolates of E. faecalis from the blood of neonates. The PhP system showed a high degree of discriminatory power for unrelated enterococcal isolates. Among the 170 unrelated fecal isolates, 107 isolates from international collections, PhP typing discriminated 19 types, and ribotyping discriminated 5 types. In most cases, when isolates were of the same DNA type, they were also of the same PhP type, and the level of agreement between these two methods was high (96%). A combination of PhP typing and DNA typing identified 34 different types, but ribotyping did not yield any further discrimination. PhP typing of E. faecalis isolates from healthy individuals (n = 89) and from the blood of neonates with septicemia (n = 28) yielded a diversity of 0.93 for both populations and similar major PhP types in both populations. Thus, the isolates from blood seemed to consist of a normal E. faecalis population, without a dominance of certain strains associated with virulence. We conclude that the PhP system is useful for epidemiological studies of enterococcal isolates, yielding results similar to those obtained with DNA typing by pulsed-field gel electrophoresis. Since PhP typing is a method that is simple and rapid and that is based on automatic evaluation of the data, it is suitable for analyzing large numbers of isolates and can be used alone or in combination with DNA typing or epidemiological and ecological studies of enterococci.

Journal ArticleDOI
TL;DR: The results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization.
Abstract: Arbitrarily primed PCR fingerprinting was carried out on 43 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients. Seventeen major groups of strains that coincided with groups also distinguished by macrorestriction (pulsed-field gel electrophoresis) typing were identified. Our results illustrated that a CF patient can carry more than one strain and can carry a given strain for long periods of time and that strains can evolve by changes in drug resistance or other phenotypic traits during long-term colonization. The arbitrarily primed PCR method is recommended for first-pass screening of P. aeruginosa isolates from CF patients, especially when many strains are to be typed, because of its sensitivity and efficiency.

Journal ArticleDOI
TL;DR: 12 isolates of Streptococcus pneumoniae with various levels of susceptibility of penicillin and extended-spectrum cephalosporins are characterized by antimicrobial susceptibility patterns, serotypes, ribotypes, chromosomal DNA restriction patterns, multilocus enzyme electrophoresis patterns,Penicillin-binding protein (PBP) profiles, and DNA restriction endonuclease cleavage profiles of pbp1a, pbp2x, and pBP2b.
Abstract: We characterized 12 isolates of Streptococcus pneumoniae with various levels of susceptibility of penicillin and extended-spectrum cephalosporins by antimicrobial susceptibility patterns, serotypes, ribotypes, chromosomal DNA restriction patterns by pulsed-field gel electrophoresis, multilocus enzyme electrophoresis patterns, penicillin-binding protein (PBP) profiles, and DNA restriction endonuclease cleavage profiles of pbp1a, pbp2x, and pbp2b. Seven cefotaxime-resistant (MIC, > or = 2 micrograms/ml) serotype 23F isolates were related on the basis of ribotyping, pulsed-field gel electrophoresis, and multilocus enzyme electrophoresis, but they had two slightly different PBP patterns: one unique to strains for which the MIC of penicillin is high (4.0 micrograms/ml) and one unique to strains for which the MIC of penicillin is low (0.12 to 1.0 micrograms/ml). The pbp1a and pbp2x fingerprints were identical for the seven isolates; however, the pbp2b fingerprints were different. An eighth serotype 23F isolate with high-level resistance to cephalosporins was not related to the other seven isolates by typing data but was a variant of the widespread, multiresistant serotype 23F Spanish clone. The PBP profiles and fingerprints of pbp1a, pbp2x, and pbp2b were identical to those of the Spanish clone isolate. An additional serotype 6B isolate with high-level resistance to cephalosporins had unique typing profiles and was unrelated to the serotype 23F cephalosporin-resistant isolates but was related on the basis of genetic typing methods to a second serotype 6B isolate that was cephalosporin susceptible. The serotype 6B isolates had different PBP profiles and fingerprints for pbp1a, but the fingerprints for pbp2x and pbp2b were the same.

Journal ArticleDOI
TL;DR: A new procedure for typing Streptococcus pyogenes, by amplifying the entire 5- to 7-kb variable vir regulon by long PCR, and the stoichiometric yield of restriction fragments in Vir typing allows unambiguous interpretation of results.
Abstract: We have developed a new procedure (Vir typing) for typing Streptococcus pyogenes, by amplifying the entire 5- to 7-kb variable vir regulon by long PCR. The amplified DNA is then cleaved with HaeIII and visualized by ethidium bromide fluorescence after agarose gel electrophoresis. A simple procedure for preparing DNA of sufficiently high quality from 96 samples was employed simultaneously. This DNA was also used to develop a random amplified polymorphic DNA (RAPD) procedure. The discriminatory power of the two DNA-based procedures was compared with previous methods, M typing, and multilocus enzyme electrophoresis. Both procedures were highly discriminatory, but the stoichiometric yield of restriction fragments in Vir typing allows unambiguous interpretation of results.

Journal ArticleDOI
TL;DR: The value of five different typing methods, including phage, bio- or ribotyping or all three methods in combination, in discriminating 105 Staphylococcus aureus strains from bovine milk samples obtained from 105 different Danish dairy herds was investigated, showed that some subdivision of types had taken place.

Journal ArticleDOI
TL;DR: Although an ideal epidemiological typing technique applicable to a wide range of fungal pathogens is not yet available, several molecular-typing methods may permit rapid, simple, and sensitive discrimination of specific strains among the most clinically important species of fungi.
Abstract: As the incidence of invasive fungal disease-particularly nosocomial candidal infection-has increased significantly over the past two decades, molecular typing has become increasingly important for the development of rational infection-control measures and therapeutic strategies. Numerous molecular methods have been used to subtype Candida species, including restriction endonuclease analysis of genomic DNA, Southern hybridization analysis, pulsed-field gel electrophoresis, and polymerase chain reaction-based approaches. Increasingly, typing techniques are being applied to other fungal organisms as well. Although an ideal epidemiological typing technique applicable to a wide range of fungal pathogens is not yet available, several molecular-typing methods may permit rapid, simple, and sensitive discrimination of specific strains among the most clinically important species of fungi.

Journal ArticleDOI
Rainer Blasczyk1, U. Hahn1, J. Wehling1, Dieter Huhn1, A. Salama1 
TL;DR: A PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the H LA-A locus is presented.
Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.

Journal ArticleDOI
TL;DR: The use of random amplified polymorphic DNA to type isolates of C. albicans in the Hôpital Cochin burn unit underline the importance of fungal surveillance in patients and the need to inform nursing staff of measures to prevent the spread of Candida spp.
Abstract: Burn patients are particularly exposed to deep-seated nosocomial infections caused by Candida species. Superficial carriage of C. albicans is a potential source of infection and dissemination, and typing methods could be useful to trace the different isolates. We report the use of random amplified polymorphic DNA to type isolates of C. albicans in the Hopital Cochin burn unit. This molecular typing method, which is based on PCR with arbitrary short primers, was evaluated on a panel of 32 C. albicans strains isolated from various anatomical sites of unrelated patients, and the strains showed 22 different patterns. Random amplified polymorphic DNA was then used in the epidemiological surveillance of the patients in the burn unit over a 9-month period. Seven patterns were identified among 84 isolates from 18 patients. One pattern (pattern A) corresponding to isolates from 7 of the 18 patients (68% of isolates) predominated throughout the 9-month study, while some strains with other profiles were isolated only once. Some profiles appeared to show a particular geographic pattern within the unit, suggesting transmission from room to room. These results underline the importance of fungal surveillance in such patients and the need to inform nursing staff of measures to prevent the spread of Candida spp. from patient to patient.

Journal ArticleDOI
TL;DR: Analysis of isolates obtained from symptomatic patients throughout the Clinical Center revealed the existence of 41 distinct and reproducible PCR ribotypes, suggesting that PCR ribotyping provides a discriminatory, reproducible, and simple alternative to conventional molecular approaches for typing strains of C. difficile.
Abstract: From January to March 1993, a suspected outbreak of antibiotic-associated diarrhea occurred on a pediatric oncology ward of the Clinical Center Hospital at the National Institutes of Health. Isolates of Clostridium difficile obtained from six patients implicated in this outbreak were typed by both PCR amplification of rRNA intergenic spacer regions (PCR ribotyping) and restriction endonuclease analysis of genomic DNA. Comparable results were obtained with both methods; five of the six patients were infected with the same strain of C. difficile. Subsequent analysis of 102 C. difficile isolates obtained from symptomatic patients throughout the Clinical Center revealed the existence of 41 distinct and reproducible PCR ribotypes. These data suggest that PCR ribotyping provides a discriminatory, reproducible, and simple alternative to conventional molecular approaches for typing strains of C. difficile.

Journal ArticleDOI
TL;DR: The RAPD patterns of strains appear to be stable during epidemics even over periods of several years, and the discriminatory power of RAPD typing was the best among all the methods tested.

Journal ArticleDOI
TL;DR: The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis, and the repetitive elements pTBN12 and DR proved to be suitable for this purpose.
Abstract: Forty Mycobacterium bovis isolates from cattle and goats were analyzed by using different repetitive genetic markers. The 23 M. bovis strains from goats were found to carry six to eight copies of the insertion sequence IS6110. In contrast, most of the bovine isolates contained only a single copy of this element. The standardized IS6110 fingerprinting by restriction fragment length polymorphism (RFLP), described for Mycobacterium tuberculosis strains, allowed the differentiation of caprine strains. Although this method was not useful for typing bovine isolates, the repetitive elements pTBN12 and DR proved to be suitable for this purpose. A procedure using PCR which amplifies IS6110 in the outward direction was found to be as sensitive as RFLP for typing M. bovis strains from goats. The use of PCR and RFLP methods based on the IS6110 polymorphism would be useful for epidemiological studies of caprine tuberculosis. The results are consistent with different strains of M. bovis being implicated in bovine and caprine tuberculosis.

Journal ArticleDOI
TL;DR: It appears that high resolution B-locus typing may be an important addition for detection of potentially relevant HLA incompatibilities in transplantation and should also be valuable for population studies and for the investigation of HLA associations with diseases.
Abstract: HLA B-locus typing by group-specific PCR and hybridization with SSOP was performed in 81 10th IHWS B cell lines and 334 selected subjects of our local panel, from four ethnic groups. Most of the B-locus serological specificities were well defined. However, some antigens like B41, B58, B56, the splits of B14, and some subtypes of B5, were not accurately assigned by serology. In the panel studied, we found 17 hybridization patterns that corresponded to probable new alleles. New patterns occurred in the four ethnic groups examined. Multiple subtypes of B35, B5, B15, B41, B44, B57, B58, B70, B14, B40, B22 were found in subjects of the same ethnic group. In view of the poor serological definition of some alleles, and the occurrence of multiple subtypes in the same ethnic population, it appears that high resolution B-locus typing may be an important addition for detection of potentially relevant HLA incompatibilities in transplantation. It should also be valuable for population studies and for the investigation of HLA associations with diseases.

Journal ArticleDOI
TL;DR: Modifications have been introduced to a previously reported HLA-B PCR-SSOP typing system, which has enabled further definition of alleles, determination of the probe pattern of some alleles not previously examined and identification of patterns of possible new alleles.
Abstract: Modifications have been introduced to a previously reported HLA-B PCR-SSOP typing system. This has enabled further definition of alleles, determination of the probe pattern of some alleles not previously examined and identification of patterns of possible new alleles. However there are still some alleles that cannot be differentiated and there are several alleles which when present as a homozygote have the same pattern as in combination with another allele. When the method was applied to the typing of 66 consecutive cadaveric donors there were three donors whose type differed from the serological type.