Arbitrarily Primed Polymerase Chain Reaction Research Articles

PCR identification of Trichophyton mentagrophytes var. interdigitale and T. mentagrophytes var. mentagrophytes dermatophytes with a random primer.

Dermatophytes are a group of keratinophilic fungi falling within the genera of Epidermophyton, Microsporum and Trichophyton. The genus Trichophyton is particularly important and complex; it comprises at least 15 recognised species. In addition, there are several different variants in the species T. mentagrophytes, which occur both in man and animals. The current methods of determining T. mentagrophytes varieties may require several different culture media and time-consuming procedures, as well as specialist skills. This study used a random primer, 5'-GAGCCCGACT-3', in the arbitrarily primed polymerase chain reaction (AP-PCR) and showed that the two common T. mentagrophytes varieties (var. interdigitale and var. mentagrophytes) can be clearly identified on the basis of their characteristic DNA band patterns. The relative reproducibility, ease of use and precision of this method make the AP-PCR a valuable tool in the laboratory diagnosis of human dermatophytosis.

Prevalence of Actinobacillus actinomycetemcomitans serotypes in Japanese patients with periodontitis.

Oral Actinobacillus actinomycetemcomitans strains are serologically classified into 5 distinct groups, a to e. We examined the distribution of A. actinomycetemcomitans serotypes in Japanese patients with periodontitis. A total of 157 A. actinomycetemcomitans clinical isolates from diseased sites of 39 patients with periodontitis were serotyped by using serotype-specific rabbit antisera against A. actinomycetemcomitans serotypes a, b, c, d and e strains. In the immunodiffusion assay, autoclaved extracts of 42, 6, 39, 9 and 41 A. actinomycetemcomitans clinical isolates reacted with serotypes a, b, c, d and e antisera, respectively. Although 37 patients were infected with a serotype strain, 2 patients harbored 2 different serotype strains, b/e and b/untypeable. To establish a correlation between serotype and genotype of A. actinomycetemcomitans clinical isolates from 2 patients who had different serotype strains, we used arbitrarily primed polymerase chain reaction (AP-PCR) to fingerprint clinical isolates of different serotypes. The AP-PCR genotypes among 4 clinical isolates (b/e and b/untypeable) were identical to that of A. actinomycetemcomitans Y4 (serotype b), indicating the presence of multiple A. actinomycetemcomitans serotypes which are genetically homogeneous in the periodontally diseased sites of patients with periodontitis.

Vaginal carriage of enterotoxigenic Bacteroides fragilis in pregnant women.

Bacteroides fragilis is an anaerobic bacterial species that is involved in gynecological infections and pathology. The incidence of vaginal carriage is largely unknown, and in order to study this, 120 pregnant women attending a general hospital for delivery were examined. Cultures were positive for eight of these women (6.6%). Interestingly, potential clonal relatedness could be demonstrated among several of the nonenterotoxigenic B. fragilis strains. Among the strains, only one produced metalloprotease enterotoxin. The presence of the gene for the metalloprotease, giving rise to the pathogenic effect on cultured eukaryotic HT29/C1 cells, was confirmed by a newly designed specific PCR assay. The enterotoxigenic B. fragilis (ETBF) strain was analyzed with the help of arbitrarily primed PCR (AP-PCR) and PCR-mediated ribotyping. The ETBF strain was shown to be genetically different compared to several other strains obtained from diverse sources. Our data indicate a relatively high vaginal B. fragilis carriage rate among pregnant women in Warsaw, Poland. Although neither ETBF nor B. fragilis colonization presented a clinical problem, the possible genetic relatedness among the colonizing B. fragilis strains indicates the need for additional research in the field of ETBF transmission and molecular epidemiology.

Genotyping of Clostridium difficile isolates from a hospital in Warsaw: A preliminary study

Background: Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhea. It is believed that patient-to-patient transmission and environmental contamination are risk factors for the spread of this disease in hospitals. The development of precise typing schemes has provided some understanding of the epidemiology of this nosocomial infection. Objective: The purpose of this study was to identify strain types of C. difficile present in the environment of a maternity ward and surgical wards of a hospital in Warsaw, and to correlate these types with those isolated from neonates and surgical patients. Methods: Arbitrarily primed polymerase chain reaction (APPCR), with the arbitrary primer PG-05, was used to genotype 14 isolates of C. difficile obtained from different sources in a hospital in Warsaw. These included clinical isolates from neonates and surgical patients, as well as environmental isolates from the maternity and surgical wards. Results: A predominant toxigenic isolate was found in the maternity environment, and this strain type was also isolated from the stools of two of five newborns. Unique and different DNA banding patterns were observed in the remaining three isolates from neonates. In addition, isolates from an infant with diarrhea also showed a unique AP-PCR type. The isolates from three surgical patients analyzed had the same AP-PCR profile. This type was also found in the surgery ward environment. Conclusion: These data support environmental contamination as an important factor in the etiology of C. difficile-associated diarrhea in this hospital.

Polymerase chain reaction for identification and typing of Clostridium difficile isolated from Chinese patients

Objective: Toxigenic Clostridium difficile, the etiologic agent of C. difficile-associated diarrhea (CDAD), is the most common cause of hospital-acquired diarrhea in many developed countries. In spite of the endemic nature of diarrhea in China, few reports of C. difficile infection in this country have been documented in the literature. Methods: In this study, a polymerase chain reaction (PCR) assay was used to determine the presence of toxigenic C. difficile in the stools of 19 patients who developed diarrhea after antibiotic use in a hospital in China. Clostridium difficile was also cultured from the stool of these patients. After DNA extraction, PCR was performed with primers targeting C. difficile toxin A and toxin B gene sequences. Results: Toxin sequences were detected in 5 of 19 (26%) specimens analyzed. To determine strain identity, these isolates were genotyped using arbitrarily primed PCR (AP-PCR). Three APPCR profiles were identified among these isolates. Three of these isolates had the same DNA banding pattern, and they were isolated from patients who were at the same institution. Conclusions: These preliminary results indicate that C. difficile may be involved in some diarrhea cases in China. This study may aid in the understanding of the nature of endemic diarrhea in this country.

Assessment of genomic damage in colorectal cancer by DNA fingerprinting: prognostic applications.

Here we evaluate the prognostic significance of the relative value of genomic damage assessed by DNA fingerprinting in colorectal cancer. Sixty-three tumor and paired normal mucosa samples were included in the study. Genomic damage was assessed by comparative analysis of paired normal and tumor tissue DNA fingerprints by the arbitrarily primed polymerase chain reaction (AP-PCR). Decreases and increases of intensity in bands were computed and referred to the total number of visualized bands per case. An index reflecting the genomic damage fraction (GDF), with separated values for losses and gains, was obtained for each tumor. This index was used to determine molecular and clinicopathologic correlates after exclusion of eight cases displaying microsatellite instability. Fifty-five cases were considered for the statistical analysis. The average fraction of altered bands per tumor was 0.287+/-0.121. When losses and gains were computed separately, the average fraction of changes was 0.126+/-0.113 and 0.161+/-0.120, respectively. Tumors lacking a ras mutation showed an increased GDF, primarily because of a higher fraction of gains. Tumors that were at advanced Dukes' stages and that were poorly differentiated also displayed a higher GDF. Finally, disease-free survival was significantly diminished in tumors with a GDF greater than 0.314 (P < .001). The prognostic significance of the GDF was independent of Dukes' stage (Cox multivariate analysis, P = .005). The degree of genomic damage assessed by unbiased DNA fingerprinting correlates with genotypic, phenotypic, and clinical variables in colorectal carcinoma and may be useful in assessing prognosis in colorectal cancer.

Molecular determination of dermatophyte fungi using the arbitrarily primed polymerase chain reaction.

Dermatophytes are keratinophilic fungi capable of causing dermatophytosis (commonly known as tinea or ringworm) in humans and animals. Previously, we reported the differentiation of the common dermatophytes Trichophyton rubrum, T. mentagrophytes and T. tonsurans using a random primer 5'-ACCCGACCTG-3' (OPAA11) in the arbitrarily primed polymerase chain reaction (AP-PCR). In the present study, by examining additional dermatophytes including eight Microsporum spp., 16 Trichophyton species/subspecies and Epidermophyton floccosum using both OPAA11 and a second random decamer 5'-GAGAGCCAAC-3' (OPD18) in AP-PCR, we show that except for T. rubrum and T. gourvilli, and three T. mentagrophytes varieties, most of the dermatophyte fungi investigated formed distinct DNA band patterns on gel electrophoresis. The amplification of specific DNA bands in AP-PCR appeared to be independent of culture variations shown by dermatophyte isolates. These results provide the basis for the rapid identification of dermatophytes at the genetic level, supplementing existing laboratory methods and improving the diagnosis of human dermatophytosis.

Use of different PCR-based DNA fingerprinting techniques and pulsed-field gel electrophoresis to investigate the epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii complex

Acinetobacter calcoaceticus-Acinetobacter baumannii complex is an important nosocomial pathogen for which optimal typing methods in epidemiologic investigations have not been defined. We compared DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) with different PCR-based DNA fingerprinting techniques, including enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR), repetitive extragenic palindromic (REP) PCR, arbitrary-primed PCR with primer M13, and multiplex PCR with primers REP-1, REP-2 and M13, for characterization of 98 clinical isolates (including 10 apparent outbreak-related isolates and 68 presumed epidemiologically unrelated isolates) in a tertiary-care hospital over a 4-year period. The PFGE patterns after SmaI restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the unweighted pair group method with arithmetic averages clustering and the Dice coefficient. A cluster of 48 isolates (cluster A), including 9 outbreak isolates, linked at a level of 83.4% similarity was observed. This epidemic strain and its variants were also found among the 68 presumed epidemiologically unrelated isolates, and this may represent ongoing endemic infection in this institution. The discrimination index for the PCR-based DNA fingerprinting techniques was 0.75 for enterobacterial repetitive intergenic consensus 1, 0.71 for M13, 0.77 for REP-1, 0.77 for REP-2, and 0.87 for multiplex PCR. The discriminatory power of PFGE was found to be higher than those of PCR-based techniques. It was concluded that both PFGE and PCR-based fingerprinting are useful for typing of A. calcoaceticus-A. baumannii complex. However, PFGE can detect minor mutations among outbreak strains, and this is important for epidemiological study of this species in a complex endemic setting.

Simultaneous persistence of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus in a neonatal ward of a Warsaw Hospital

Fifty-seven methicillin-resistant Staphylococcus aureus (MRSA) isolates from babies ( N = 31), carriers amongst health care workers ( N = 16; 10% of all staff members) and the environment ( N = 10); 39 MSSA isolates, from babies ( N = 18), health care workers ( N = 5) and environment ( N = 16) were analysed. The strains were from the neonatal ward of a teaching hospital in Warsaw and were collected over a period of 16 months (1993/1994). The isolates were characterized by phage-typing, arbitrary-primed polymerase chain reaction (AP PCR), DNA repeat polymorphism within the protein A gene and the resistance pattern to antimicrobial agents. The presence of the mecA gene was determined by PCR. MRSA were classified as heterogeneously resistant to methicillin, susceptible to other antimicrobial agents and, except for three isolates, appeared to be genotypically almost identical. The first example of mupirocin resistant MRSA in Poland was documented. Amongst MSSA isolates, increased variability was seen, however, the persistence of one predominate clone of MSSA was shown. In this particular hospital environment, several different strains of both MRSA and MSSA were capable of maintaining persistent colonization.

The role of arbitrarily primed PCR in identifying the source of an outbreak of Legionnaires' disease.

An outbreak of community-acquired Legionnaires' disease (LD) occurred in Providence, R.I., in fall 1993. To find the outbreak source, exposures of 17 case patients were compared to those of 33 matched controls. Case patients were more likely than controls to have visited a section of downtown (area A) during the 2 weeks before illness (11 [65%] versus 9 [27%]; matched odds ratio, 6.5; P = 0.01). Water samples were cultured from 27 aerosol-producing devices within area A. Legionella pneumophila serogroup 1 isolates underwent monoclonal antibody (MAb) subtyping and arbitrarily primed PCR (AP-PCR). All four L. pneumophila serogroup 1 isolates available from case patients who visited area A had identical MAb and AP-PCR patterns. Among 14 environmental isolates, 5 had MAb patterns that matched the case patient isolates, but only 1 had a matching AP-PCR pattern. This investigation implicates a cooling tower in area A as the outbreak source and illustrates the usefulness of AP-PCR for identifying sources of LD outbreaks.

Differential display reveals transcriptional up-regulation of the motor molecules for both anterograde and retrograde axonal transport during nerve regeneration.

RNA fingerprinting using an arbitrary primed polymerase chain reaction was carried out to compare differences in expression of mRNAs between axotomized and normal hypoglossal motoneurons in the mouse. In this survey, the kinesin light chain (KLC) was identified as a nerve injury-associated molecule. This was also confirmed by in situ hybridization using hemihypoglossal nerve-transected brain sections. In order to identify the exact species of molecules belonging to the KLC family, in situ hybridization was carried out with oligonucleotide probes specific to rat KLC A, KLC B and KLC C, using the rat hypoglossal nerve injury model. In addition, expression of both ubiquitous and neuron-specific kinesin heavy chain and cytoplasmic dynein which is a retrograde motor, was also examined. Expression of all the members of the KLC (A-C) family and dynein was up-regulated during nerve regeneration, whereas the abundant expression of the neuron-specific KHC mRNA was not changed. The present results indicate that the molecules associated with both anterograde and retrograde axonal transport are up-regulated in their expression during efferent motor nerve regeneration, suggesting that the retrograde transport of growth factors and anterograde transport of vesicles, providing membrane material, could be increased during motor nerve regeneration.

Outbreak of scarlet fever at a hospital day care centre: analysis of strain relatedness with phenotypic and genotypic characteristics

An outbreak of scarlet fever involving 12 children occurred at a hospital day care centre from February to March 1996. Twenty-five throat isolates of Streptococcus pyogenes (GAS, group A streptococcus) available from 24 children, including 10 children with scarlet fever and 14 asymptomatic carriers, and one asymptomatic staff member were studied for the presence of genes encoding streptococcal pyrogenic exotoxin types A ( speA), B ( speB), and C ( speC) and for protease activity. Antimicrobial susceptibilities using the E-test, cluster analysis by cellular fatty acid composition and random amplified polymorphic DNA (RAPD) patterns by means of arbitrarily-primed polymerase chain reaction (APPCR) of the isolates were performed to investigate the outbreak. Only one isolate from an asymptomatic child possessed the speA gene. All isolates possessed the speB gene and 24 (96%) isolates were positive for the speC gene. There was no difference in protease activity between isolates from children with scarlet fever and from asymptomatic carriers. Thirteen isolates (10 recovered from children with scarlet fever, two from asymptomatic children, and one from the staff member) were considered to be the same strain according to the identical antimicrobial susceptibility profile and RAPD patterns and were also considered to be similar by cluster analysis of fatty acid composition. These findings suggest that the outbreak was caused by a unique clone of GAS. We conclude that RAPD typing and cluster analysis by cellular fatty acids composition both provide a powerful tool for epidemiological investigation of GAS infections.

Comparative study of Mycobacterium paratuberculosis strains isolated from Crohn's disease and Johne's disease using restriction fragment length polymorphism and arbitrarily primed polymerase chain reaction

To obtain insights into the pathogenic mechanisms involving Mycobacterium paratuberculosis in Crohn's disease (CD) we questioned if the strains of M. paratuberculosis isolated from CD are distinguishable from those involved in Johne's disease (JD), a chronic granulomatous enteritis in cattle. Accordingly we compared human and animal strains at the DNA level, both by the analysis of restriction fragment length polymorphism (RFLP) in and around the insertion sequence IS 900 and by the arbitrarily primed chain reaction (AP-PCR). Results are in favour of a common clonal origin for the 4 strains isolated from CD and for 8 of the 11 strains isolated from cattle and sheep JD.

Exchange of Pseudomonas aeruginosa strains among cystic fibrosis siblings

The molecular epidemiology of Pseudomonas aeruginosa infection in cystic fibrosis (CF) siblings was analysed by DNA fingerprinting using arbitrary primed polymerase chain reaction. A total of 306 strains collected from six pairs of siblings over a period of 20-126 months (median 64) was studied. Fifty-four different P. aeruginosa genotypes were recognized. Two out of six pairs of siblings were ultimately colonized by identical strains, and it was shown that a single P. aeruginosa clone can persist in an individual patient for over ten years. No overlap in P. aeruginosa genotypes was encountered between families, whereas in all families at least transient cross-colonization with the same genotype was observed. This finding demonstrates that P. aeruginosa cross-infection or acquisition of the same strain from an identical environmental source exists within the family situation, but does not always result in a long-term colonization by identical genotypes in all family members suffering from CF.

Molecular epidemiology of Staphylococcus aureus strains colonizing the lungs of related and unrelated cystic fibrosis patients.

BACKGROUND: Cystic fibrosis (CF) patients can become persistently colonized with Staphylococcus aureus. This is initiated at an early age and may continue until or sometimes even during adolescence. Little is known about the epidemiology and cross-infectivity of S. aureus in CF patients, whether via the environment or person to person. METHODS: S. aureus isolates (n=189) from six unrelated CF patients and six pairs of CF siblings were genetically typed by arbitrary primed polymerase chain reaction (AP-PCR) assays. RESULTS: This longitudinal study revealed 35 different genotypes among the 189 isolates; the median number of types in a patient was three (range 1--6). One common S. aureus genotype was found in six patients and involved 20% of all isolates analyzed. Ultimately, in most of the patients long-term colonization with a single genotype was observed. In several, but certainly not all, pairs the siblings became persistently colonized with isolates that could not be discriminated by the typing method used; different S. aureus genotypes were isolated on an incidental but relatively frequent basis. Only one pair of siblings never shared identical isolates at any time during the screening period. CONCLUSIONS: In five of six cases, identical isolates were shared by CF siblings at a certain time. This suggests intra-family transmission or the presence of a common environmental source. The fact that in most of the CF sibling pairs different genotypes of S. aureus caused the ultimate long-term colonization indicates that, despite regular cross-colonization, patient characteristics select the S. aureus strain best adapted to the affected lung. Some genotypes may be particularly prevalent in the CF patient population, but additional studies are needed to confirm this.

Arbitrary primed PCR rules out Clostridium difficile cross-infection among patients in a haematology unit

Eight out of 20 (40%) patients with haematological malignancies hospitalized in the same unit of our hospital from 24 January to 24 April 1995, suffered from diarrhoea due to Clostridium difficile. The C. difficile isolates were characterized by serotyping and by arbitrary primed polymerase chain reaction (AP-PCR) using three different 10-mer oligonucleotides. It was found by serotyping that five patients had non-typeable isolates and three had serogroup H isolates. The AP-PCR typed all the isolates and yielded various patterns suggesting that there had been no cross-transmission between the patients. Control faecal sample cultures showed that two patients were still carrying the same isolates after specific treatment with vancomycin or metronidazole, and that one patient had acquired an isolate with a new AP-PCR type. AP-PCR was found to be a rapid, effective discriminative method for the immediate epidemiological tracking of hospital-acquired infections due to C. difficile.

Differentiation of Leptospira species and serovars by PCR-restriction endonuclease analysis, arbitrarily primed PCR and low-stringency PCR.

Reference strains from 30 serovars representing seven species of Leptospira and 48 recent isolates from human patients, dogs and rats, were characterised by polymerase chain reaction-restriction endonuclease analysis (PCR-REA), arbitrarily primed PCR (AP-PCR) and low stringency PCR (LS-PCR). PCR-REA analysis yielded seven groups among 29 serovars of pathogenic Leptospira; the non-pathogenic L. biflexa serovar patoc was not amplified with the primer pairs studied. AP-PCR and LS-PCR fingerprinting resulted in 25 and 21 distinct profiles, respectively, among the 30 reference strains. The results of the three PCR-based techniques were highly concordant and were in general agreement with those from previous DNA studies, confirming the high level of polymorphism among Leptospira species and serovars, and supported the concept of the serovar as the basic taxonomic unit of leptospiral classification. Results of the PCR-based typing methods for 11 randomised leptospiral strains, 36 clinical isolates from human patients and dogs and 12 survey isolates from trapped rats agreed with those from serological identification. With one exception, isolates of the same serovar gave identical profiles irrespective of the source. AP-PCR and LS-PCR are simple to perform and interpret, and appear to be useful for characterising isolates of Leptospira spp. for diagnostic and epidemiological purposes.

DD/AP-PCR: Combination of Differential Display and Arbitrarily Primed PCR of Oligo(dT) cDNA

In this report we describe the first direct comparison of differential display (DD) and arbitrarily primed PCR (AP-PCR) amplification of oligo(dT)-primed cDNA. Our results indicate that both of these widely used RNA fingerprinting techniques have their respective advantages and limitations. DD produces profiles specific to the anchored oligo(dT) primer used for cDNA synthesis. AP-PCR displays significant redundancy of profiles generated from different oligo(dT) cDNA pools, but is not as biased to the isolation of A/T-rich or 3′ sequences. It was found that both techniques can utilize cDNA synthesized using a generic anchored oligo(dT) primer (dT12VN; equimolar amounts of dT12VA, dT12VC, dT12VG, and dT12VT, where V is dA, dC, or dG); this efficiently selects for poly(A)+sequences from total RNA, and significantly reduces the number of cDNA preparations required per experiment. Using dT12VN cDNA pools generated from rat liver, spleen, and brain, the two approaches (AP-PCR and DD) were used in combination. Several known mRNAs were identified; some were unique to either technique and some were common to both. Since it is the RNA which is usually the limiting resource, maximum utilization may be achieved by generating a single pool of dT12VN-primed cDNA and performing both AP-PCR and DD (DD/AP-PCR).

メチシリン耐性黄色ブドウ球菌のパルスフィールドゲル電気泳動およびAP-PCRによるゲノムタイピング

Genotyping of 67 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) separated from patients in a hospital in Mizusawa City in 1994 and 1995 was studied by pulsed field gel electrophoresis (PFGE) and arbitrarily primed-polymerase chain reaction (AP-PCR). Two main genotypes were observed by PFGE, and more than 70% of the 67 MRSA isolates produced coagulase type II. One group diverged well and gained higher tolerance in 1994, but was not isolated in 1995. The other group was continually isolated during the two-year period and showed moderate tolerance in 1994, and higher tolerance in 1995. AP-PCR was able to classify the genotypes of MRSA into 6 subgenotypes under the present conditions, which supported the results obtained by PFGE. These results suggest that AP-PCR could become a convenient and useful typing method by improving both sequence and length of a primer.

Arbitrary primed PCR fingerprinting and serotyping of clinicalPseudomonas aeruginosastrains

Arbitrary primed PCR (AP-PCR) analysis was compared with serotyping as a means of high-resolution typing of Pseudomonas aeruginosa. Seventy-four isolates from 3 different hospitals and 18 reference strains were studied. Serotyping provided good index of discrimination, although eleven isolates could not be serotyped. Genomic DNA was amplified with a single 10 nucleotide primer (sequence 5'-AGG GGT CTT G-3'). The strains were genetically diverse and 61 different AP-PCR profiles of 2-7 bands between 0.3 and 2.4 kb were obtained. AP-PCR profiles were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype. Numerical analysis of AP-PCR patterns defined 7 groups at the 55% similarity level, and identified predominant strains in each hospital. The results show that AP-PCR analysis provides a simple and practical approach to typing P. aeruginosa that is more discriminatory than traditional serotyping scheme. We suggest that maximum discrimination can be achieved by a combination of both methods.