Showing papers on "Typing published in 2000"
TL;DR: PCR-ribotyping offers the best combination of advantages as an initial typing tool for C. difficile infections, and PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE.
Abstract: Clostridium difficile is now recognized as the major agent responsible for nosocomial diarrhea in adults. Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of C. difficile infections. However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated. We compared the results of three methods—AP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with SmaI endonuclease)—to differentiate 99 strains of C. difficile that had been previously serogrouped. Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G. Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5. Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C. PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE. We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for C. difficile.
231 citations
TL;DR: It is concluded that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus from bovine mammary secretions and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovines S.Aureus.
Abstract: Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus.
224 citations
TL;DR: It is concluded that Invader chemistry using PCR products as template represents a useful technology for typing large numbers of SNPs rapidly and efficiently.
Abstract: Large-scale pharmacogenetics and complex disease association studies will require typing of thousands of single-nucleotide polymorphisms (SNPs) in thousands of individuals. Such projects would benefit from a genotyping system with accuracy >99% and a failure rate <5% on a simple, reliable, and flexible platform. However, such a system is not yet available for routine laboratory use. We have evaluated a modification of the previously reported Invader SNP-typing chemistry for use in a genotyping laboratory and tested its automation. The Invader technology uses a Flap Endonuclease for allele discrimination and a universal fluorescence resonance energy transfer (FRET) reporter system. Three hundred and eighty-four individuals were genotyped across a panel of 36 SNPs and one insertion/deletion polymorphism with Invader assays using PCR product as template, a total of 14,208 genotypes. An average failure rate of 2.3% was recorded, mostly associated with PCR failure, and the typing was 99.2% accurate when compared with genotypes generated with established techniques. An average signal-to-noise ratio (9:1) was obtained. The high degree of discrimination for single base changes, coupled with homogeneous format, has allowed us to deploy liquid handling robots in a 384-well microtitre plate format and an automated end-point capture of fluorescent signal. Simple semiautomated data interpretation allows the generation of approximately 25,000 genotypes per person per week, which is 10-fold greater than gel-based SNP typing and microsatellite typing in our laboratory. Savings on labor costs are considerable. We conclude that Invader chemistry using PCR products as template represents a useful technology for typing large numbers of SNPs rapidly and efficiently.
219 citations
TL;DR: A single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products.
Abstract: We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.
151 citations
TL;DR: A novel method, which allows simultaneous detection and genotyping of NLVs, is useful in the diagnosis and typing ofNLVs obtained from outbreaks and in large-scale epidemiological studies.
Abstract: “Norwalk-like viruses” (NLVs) are the most common cause of outbreaks of nonbacterial gastroenteritis worldwide. To date, the method most widely used for typing of NLV strains is sequencing and subsequent phylogenetic analysis of reverse transcription (RT)-PCR products, which has revealed the existence of stable distinct lineages (genotypes). This typing method is rather costly, not routinely used in clinical laboratories, and not very suitable for the analysis of large numbers of samples. Therefore, we have developed a rapid and simple method for genotyping of NLVs. The method, designated reverse line blot hybridization, is based on the nucleotide divergence of a region of the gene for RNA polymerase which can be used to classify NLVs into genotypes. NLV RNA was amplified by RT-PCR and then hybridized to 18 different membrane-bound oligonucleotides that were able to discriminate among 13 NLV genotypes. Application of the method to a panel of 132 positive stool samples from 34 outbreaks and 20 sporadic cases of gastroenteritis collected in a 6-year period (1994 to 1999) resulted in successful genotyping of 124 samples (94%), as confirmed by phylogenetic analysis. The nucleotide sequences of the remaning eight strains (6%) from three outbreaks did not cluster with the known NLV genotypes. Phylogenetic analysis of the complete and partial open reading frame 2 (capsid gene) sequences of these strains revealed the existence of one novel genotype (Alphatron) and one potentially novel genotype (Amsterdam). This novel method, which allows simultaneous detection and genotyping of NLVs, is useful in the diagnosis and typing of NLVs obtained from outbreaks and in large-scale epidemiological studies.
149 citations
TL;DR: A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolate of this apicomplexan.
145 citations
TL;DR: The REP-PCR technique is a useful and expeditious method for the epidemiologic characterization of A. baumannii nosocomial outbreaks, the results being comparable to those obtained with the PFGE technique.
131 citations
TL;DR: It is concluded that AFLP is a highly discriminatory typing method and well suited for computer-assisted data analysis; however, for optimal typing of campylobacters, a combination of multiple typing methods is needed.
Abstract: For epidemiological tracing of the thermotolerant Campylobacter species C. jejuni and C. coli, reliable and highly discriminatory typing techniques are necessary. In this study the genotyping techniques of flagellin typing (flaA typing), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and amplified fragment length polymorphism (AFLP) fingerprinting were compared. The following aspects were compared: computer-assisted analysis, discriminatory power, and use for epidemiological typing of campylobacters. A set of 50 campylobacter poultry isolates from The Netherlands and neighboring countries was analyzed. Computer-assisted analysis made cluster analysis possible and eased the designation of different genotypes. AFLP fingerprinting was the most discriminatory technique, identifying 41 distinct genotypes, while PFGE identified 38 different types, flaA typing discriminated 31 different types, and ribotyping discriminated 26 different types. Furthermore, AFLP analysis was the most suitable method for computer-assisted data analysis. In some cases combining the results of AFLP fingerprinting, PFGE, and flaA typing increased our ability to differentiate strains that appeared genetically related. We conclude that AFLP is a highly discriminatory typing method and well suited for computer-assisted data analysis; however, for optimal typing of campylobacters, a combination of multiple typing methods is needed.
123 citations
TL;DR: The RFLP method was the most reliable, due to the easily recognisable pattern produced by the two virus species present in Europe, and dot-blot hybridisation is less expensive for identifying TYLCVs in large numbers of samples, particularly when a mixture of two probes is used.
Abstract: Tomato yellow leaf curl disease is spreading in southern Europe, where it has quickly become a serious problem. In recent years, several virus isolates have been characterised. Although with some genetic variability, all isolates found in Europe belong to one of two species Tomato yellow leaf curl-Sardinia (TYLCV-Sar) or Tomato yellow leaf curl-Israel (TYLCV-Is). Several methods were tested to identify and type TYLCV isolates from field samples: (1) RFLP of a DNA fragment amplified from the coat protein gene; (2) PAGE of a fragment amplified from the C2 gene; (3) dot-blot hybridisation. All methods enabled the detection of the TYLCVs and provided good indications for attributing them to one species or the other. However, for typing purposes, the RFLP method was the most reliable, due to the easily recognisable pattern produced by the two virus species present in Europe. Dot-blot hybridisation is less expensive for identifying TYLCVs in large numbers of samples, particularly when a mixture of two probes is used. PAGE of the C2 fragment is the fastest of the methods tested.
118 citations
TL;DR: Community-acquired gentamicin-sensitive MRSA strains were found to be related by PFGE and coagulase typing and had similar phage typing patterns, which suggest they are related to GS-MRSA previously reported in New Zealand.
Abstract: Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) susceptible to gentamicin has been reported in a number of countries in the 1990s. To study the acquisition of gentamicin-sensitive MRSA (GS-MRSA) in southeast Queensland and the relatedness of GS-MRSA to other strains of MRSA, 35 cases of infection due to GS-MRSA from October 1997 through September 1998 were examined retrospectively to determine the mode of acquisition and risk factors for MRSA acquisition. Thirty-one isolates from the cases were examined using a variety of methods (antibiotyping, phage typing, pulsed-field gel electrophoresis [PFGE] fingerprinting, and coagulase typing by restriction analysis of PCR products) and were compared with strains of local hospital-acquired gentamicin-resistant MRSA (GR-MRSA) and of Western Australian MRSA (WA-MRSA). Only 6 of 23 cases of community-acquired GS-MRSA had risk factors for MRSA acquisition. Twenty of 21 isolates from cases of community-acquired infection were found to be related by PFGE and coagulase typing and had similar phage typing patterns. Hospital- and nursing home-acquired GS-MRSA strains were genetically and phenotypically diverse. Community-acquired GS-MRSA strains were not related to nosocomial GR-MRSA or WA-MRSA, but phage typing results suggest that they are related to GS-MRSA previously reported in New Zealand.
115 citations
TL;DR: In this article, the authors used coagulase gene (coa) short sequence repeat region sequencing to measure relatedness among a collection of temporally and geographically diverse methicillin-resistant Staphylococcus aureus isolates.
Abstract: Coagulase gene (coa) short sequence repeat region sequencing was used to measure relatedness among a collection of temporally and geographically diverse methicillin-resistant Staphylococcus aureus isolates. The results show that coa polymorphism is free of strong selective pressure and has a low index of variation that may be useful for long-term epidemiological investigations. coa typing is a useful addition to spa typing for analysis of S. aureus, including methicillin-resistant strains.
TL;DR: The majority of phenotypic and genotypic techniques developed for MRSA are not sufficiently discriminatory, reproducible, stable or useful in an outbreak to be acceptable.
TL;DR: Two DNA typing methods were used to study the genetic relationships among 90 Moraxella catarrhalis strains, suggesting the existence of subpopulations of M. catarr Halis that differ in virulence, and they may have implications for vaccine development.
Abstract: Two DNA typing methods, probe-generated restriction fragment length polymorphism analysis and single-adapter amplified fragment length polymorphism analysis, were used to study the genetic relationships among 90 Moraxella catarrhalis strains. Both methods were found to be highly concordant, generating a dendrogram with 2 main branches. The division of the M. catarrhalis population into 2 subspecies was supported by analysis of the 16S rRNA sequences. Both beta-lactamase-positive and beta-lactamase-negative strains were found in all main branches, suggesting horizontal transfer of the beta-lactamase gene. In contrast, 2 virulence traits, complement resistance and adherence to epithelial cells, were strongly associated with 1 of the 2 subspecies. The branch depth suggested that complement-resistant adherent strains diverged from a common ancestor more recently than did complement-sensitive nonadherent strains. These findings suggest the existence of subpopulations of M. catarrhalis that differ in virulence, and they may have implications for vaccine development.
TL;DR: Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.
Abstract: The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.
TL;DR: The performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility.
Abstract: Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.
TL;DR: The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in thisserovar was low.
Abstract: Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.
TL;DR: Although the method was not sufficiently discriminatory for epidemiological typing, AFLP analysis reconfirmed the observation that L.monocytogenes comprises two major genetic groups: group 1 includes strains of serovars 1/2a and1/2c, while group 2 serovar 1/ 2b, 3b and 4b.
TL;DR: Multilocus sequence typing was used to characterize isolates of the major Spanish clones of penicillin-resistant and multiple-antibiotic-resistant Streptococcus pneumoniae to allow resistant pneumococci to be assigned to the Spanish clones if they have the typical allelic profile of the clone or if their profiles differ from that profile at a single locus.
Abstract: Multilocus sequence typing was used to characterize isolates of the major Spanish clones of penicillin-resistant and multiple-antibiotic-resistant Streptococcus pneumoniae. Isolates of the multidrug-resistant Spanish serotype 23F clone and serotype variants of this clone either had identical allelic profiles or their allelic profiles differed from this typical allelic profile at only one of the seven housekeeping loci. Similarly, isolates of the Spanish serotype 6B and 14 clones and the penicillin-resistant serotype 9V clone (and serotype variants of this clone) each had the same allelic profiles or profiles that differed at a single locus. Multilocus sequence typing therefore allows resistant pneumococci to be assigned to the Spanish clones if they have the typical allelic profile of the clone or if their profiles differ from that profile at a single locus. A few resistant isolates that had allelic profiles typical of that of a Spanish clone or whose profiles differed from that of the typical profile at only a single locus possessed penicillin-binding protein pbp1a, pbp2b, or pbp2x genes that differed from those that are characteristic of the clone. In most cases these isolates could be assigned as variant members of the clone. Since almost all serotype 9V isolates have very similar genotypes, independently emerging penicillin-resistant clones of this serotype will inevitably appear to be similar by molecular typing procedures. Analysis of the pbp genes, in addition to multilocus sequence typing (or any other molecular typing procedure), is therefore required to assign isolates unambiguously to the penicillin-resistant Spanish serotype 9V clone.
TL;DR: For smaller laboratories that are investigating outbreaks of fowl cholera, it would appear that REA and REP-PCR are the typing methods of choice and MLEE supported by two of the other methods would appear the most suitable techniques.
Abstract: Blackall and Miflin critically examine recent developments in new-generation tests for the identification and typing of Pasteurella multocida. Two polymerase chain reaction (PCR) tests have been reported for P. multocida as both show promise as diagnostic tests that could be considered for routine use.
TL;DR: A method for distinguishing among Pseudomonas aeruginosa strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power.
Abstract: A method for distinguishing among Pseudomonas aeruginosa strains using random amplified polymorphic DNA (RAPD) typing was evaluated for reproducibility and discriminatory power. A total of 200 isolates, blinded in triplicate, were evaluated by RAPD. All 600 samples were typeable; 197 of 200 isolates gave identical results on all three occasions, and 131 distinct RAPD types were identified.
TL;DR: Opa-typing is a more powerful tool for epidemiological investigation of gonorrhoea transmission than earlier methods and can link infections that would otherwise remain unlinked, and may aid interventions to control endemic disease.
TL;DR: Analysis of sequential isolates from patients with cystic fibrosis or with invasive aspergillosis showed the clonality of the colonization or infection by A. terreus, and this straightforward typing method demonstrated the clonal origin of a massive contamination of the environment in a haematology unit.
TL;DR: In this article, a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified using exon 2 group-specific primers.
Abstract: Due to the expanding number of known HLA class II DQB1 alleles, high-resolution oligotyping is becoming ineffective, therefore a sequence-based typing (SBT) strategy was developed to provide rapid and definitive typing of HLA-DQB1. HLA-DQB1*02, *03, *04, *05, and *06 alleles were individually amplified by polymerase chain reaction (PCR) using exon 2 group-specific primers. Forward and reverse PCR primers were tailed with M13 universal and M13 reverse sequences, respectively. Subsequent bi-directional cycle-sequencing was carried out using Cy5.5-labeled M13 universal primer and Cy5.0-labeled M13 reverse primer. Automated sequencing was performed in 30 min using a Visible Genetics, Inc. (VGI) MicroGene Clipper Sequencer. Full concordance was observed between this SBT method and oligotyping among 151 individuals.
TL;DR: The data obtained by three independent typing methods confirm the existence of two distinct M. pneumoniae genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.
Abstract: Three methods for genotyping of Mycoplasma pneumoniae clinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the M. pneumoniae cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of M. pneumoniae genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the M. pneumoniae isolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct M. pneumoniae genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.
TL;DR: The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.
TL;DR: Six systems, which operate independently of each other, are intended for use as secondary typing systems following HLA-B identification with a medium resolution PCR-SSOP technique.
TL;DR: Typing with PFGE is well suited for detailed investigations of colonization with Pseudomonas aeruginosa in cystic fibrosis patients, and can provide additional information for epidemiologic purposes.
TL;DR: It is concluded that the high rate of tetracycline resistance among Iranian S. pyogenes isolates is due to multiclonal dissemination of resistance within the streptococcal population rather than epidemic spread of single clones.
Abstract: Streptococcus pyogenes, a major human pathogen, is still considered susceptible to beta-lactams, but for other relevant antibiotics, highly variable resistance rates have been reported. Since no data were available from Iran, we tested 1,335 throat isolates from two different regions of the country for their antibiotic susceptibilities and, for comparison, a collection of 80 strains isolated from 1989 to 1991. Erythromycin resistance was uncommon (0.6%), whereas an overall high rate of tetracycline resistance was found, increasing between 1989-1991 and 1995-1997 from 23 to 42%. The tetracycline-resistant strains belonged to more than 10 different T types, the majority being types 4, 11, and B3264. By conventional M typing of 406 tetracycline-resistant isolates, more than 20 different M types were found. Approximately 50% of the strains were nontypeable by T agglutination as well as serological M typing; however, by genotyping by a combined PCR-capture-enzyme-linked immunosorbent assay, many of these strains were successfully emm typed. We conclude that the high rate of tetracycline resistance among Iranian S. pyogenes isolates is due to multiclonal dissemination of resistance within the streptococcal population rather than epidemic spread of single clones.
TL;DR: It is concluded that LCR typing may permit defining the SHV families with simplicity and reliability and can be applied to the detailed characterization and molecular epidemiology of SHV-type β-lactamases.
Abstract: Ligase chain reaction (LCR) is a recently developed technique that employs a thermostable ligase and allows for the discrimination of DNA sequences differing in only a single base pair. The method has been adapted and applied to differentiation of bla(SHV) genes. We have developed an LCR typing method to characterize point mutations in genes for SHV-derived extended-spectrum beta-lactamases with four different sets of biotinylated LCR primers. To evaluate the applicability of the current technique, we tested seven Escherichia coli strains producing SHV-1, SHV-2, SHV-2a, SHV-3, SHV-4, SHV-5, and SHV-12. With the LCR typing, seven SHV genes can be distinguished according to their incorporating point mutations. In an attempt to characterize SHV beta-lactamases by LCR typing in clinical isolates, 46 strains carrying bla(SHV) genes (32 Klebsiella pneumoniae, 10 Enterobacter cloacae, and 4 E. coli) were subjected to antibiotic susceptibility testing, isoelectric focusing, and LCR typing. LCR typing allowed the characterization of beta-lactamases, and genotypes obtained by LCR typing were in accordance with phenotypes such as antibiotic resistance profile and pI value of beta-lactamase. Therefore, we concluded that LCR typing may permit defining the SHV families with simplicity and reliability and can be applied to the detailed characterization and molecular epidemiology of SHV-type beta-lactamases.
TL;DR: A method for typing polymorphisms at the T-cell epitopes within the Th2R and Th3R regions of the Plasmodium falciparum circumsporozoite protein (CSP) that combines the use of PCR and sequence specific oligonucleotide probes (PCR-SSOP), and allows the identification of single nucleotide polymorphisms in these epitope regions.