[Polymerase chain reaction. Should it be used in clinical microbiologic laboratories?].
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Clinical Laboratory Techniques↗
Biomedical subjects
J C Palomares
Publications and source records attributed to J C Palomares.
Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae.
This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded gene cppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 of Neisseria gonorrhoeae. A total of 119 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All Neisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains of Neisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 x 10(4) cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100% while the specificity was 97.5%. Positive and negative predictive values were 91.2% and 100%, respectively. The fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.
Base Sequence↗
Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.
This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.
Alkaline Phosphatase↗
[Chlamydia trachomatis detection by DNA-RNA hybridization].
BACKGROUND: A one-chain DNA probe, that complements ribosomal RNA of Chlamydia trachomatis was used as a detection method for this microorganism on clinical samples. We compare the method with the cell culture one. METHODS: A total of 175 samples (cervix swabs) from women seen at the STD center of the Facultad de Medicina de Sevilla were examined by both diagnostic techniques. When the results were different, a third method (ELISA) was also used. RESULTS: Using serial dilutions of a C. trachomatis cell culture as reference pattern, we determine the minimum number of inclusion forming units needed in order to be detected by the probe was 1000. Of all 175 samples, in 24 (14%) cell culture was positive for C. trachomatis, and 26 were positive using the DNA probe test. Sensitivity and specificity for this test were 93% and 95%, respectively. CONCLUSIONS: We believe that the DNA probe test was similar to the cell culture test as screening test in Chlamydia trachomatis infections diagnosis, specially among high risk populations.
Chlamydia Infections↗
DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.
A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.
Alkaline Phosphatase↗
Evaluation of a DNA probe of plasmid origin for the detection of Neisseria gonorrhoeae in cultures and clinical specimens.
This study evaluates a cryptic plasmid-derived DNA probe in a dot-blot hybridization assay of 4-h duration, using both known bacterial isolates and clinical specimens. The probe, consisting of a 237 bp segment of the plasmid-encoded gene cppB, sequences of which are also found in the chromosome, was labelled with digoxigenin-11-dUTP. The sensitivity of the probe was approximately 25 pg of DNA or 500 cfu of Neisseria gonorrhoeae. A total of 170 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All N. gonorrhoeae strains, including three plasmid-free strains, hybridized with the probe. Among the heterologous bacterial cultures, only one strain of N. cinerea reacted with the probe when the cell concentration was 5 x 10(6) cfu. The probe was also evaluated in a clinical study. A total of 201 patients visiting the STD clinic at the University Hospital, University of Seville, participated in the study. The sensitivity of the assay was 95% while the specificity was 98%. Positive and negative predictive values were 97% and 98%, respectively. It appears that the plasmid-derived probe used in this study could serve as a useful tool in the rapid and specific detection of Neisseria gonorrhoeae in clinical specimens.
Cloning, Molecular↗
Antibiotic resistance, plasmid profile, auxotypes and serovars of Neisseria gonorrhoeae strains isolated in Sevilla (Spain).
The antibiotics resistance pattern, the plasmid profile, the auxotypes and serotypes of 116 Neisseria gonorrhoeae clinical isolates obtained in one year were examined. The incidence of penicillinase producing (PPNG) strains was 12% (14 strains). The most frequent plasmid pattern was the combination of 4.5, 2.6 and 24.5 MDa plasmids. The conjugative plasmid of 24.5 MDa showed a high prevalence (32% of the total strains), and almost all the PPNG strains harboured this plasmid. The strains with the 4.5 MDa plasmid belonged to the auxotypes Pro-, Zero and Pro-Hyx-Ura-, whereas that with the 3.2 MDa plasmid was of auxotype Pro-Hyx-His-. The serotypes Aedih/Arst (WI serogroup) and Bak/Bropt, Back/Bropyt and Bak/Bropyt (WII/III serogroup) were predominant.
Drug Resistance, Microbial↗
[Nucleic acid hybridization techniques].
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DNA↗
[In situ hybridization with DNA probe for the diagnosis of Neisseria gonorrhoeae infections].
A comparative study of a DNA probe for the detection of Neisseria gonorrhoeae with Gram stain and modified Thayer-Martin medium was performed. The probe was the 2.6 megadaltons (Mda) cryptic plasmid of N. gonorrhoeae, labeled with a nonradioactive system combining sulphonation of DNA with the antibody detection of sulphonated groups (Organics Chemiprobe). Overall 101 samples were evaluated: 39 urethral exudates, 3 rectal exudates, and 59 cervical exudates, from 42 males and 54 females in whom gonococcal infection was suspected. Simultaneously, Gram stain and culture of the several exudates were carried out. The in vitro sensitivity of the method was evaluated with different dilutions of N. gonorrhoeae, from 1 to 1000 cfu/ml, and a minimum of 50 cfu/ml were detected. In the detection of N. gonorrhoeae in clinical samples, there was a 100% sensitivity and a specificity of 83% (males) and 79% (females), with positive predictive value of 97% in males and 53% in females. The negative predictive value was 100% both for males and females.
DNA Probes↗
In vitro activity of 14 antimicrobial agents against Neisseria gonorrhoeae from Spain.
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Anti-Bacterial Agents↗
Effect of subinhibitory concentrations of ampicillin on the R plasmid transfer in Escherichia coli.
The effects of subinhibitory concentrations (sub-MICs) of ampicillin on R plasmid transfer in Escherichia coli were studied. Each donor strain culture was separated into two parts; one was mixed with a recipient strain culture for mating, the other was treated with the sub-MIC of ampicillin and then mixed with the recipient strain culture. In both cases the R plasmid transfer frequency was determined at 30, 60, 90 and 120 min of mating. Results showed that there exists a general decrease in the transfer frequencies under sub-MIC treatment (two plasmids did not transfer at all). The proportion of aggregates and the number of cells that compose them were not affected by the sub-MIC of ampicillin. Our study supports the idea that the changes induced in E. coli by sub-MICs of ampicillin did not affect the function of the surface structures responsible for aggregation but did affect the proteins implicated in DNA transfer, situated on the cell surface.
Ampicillin↗
Activities of new quinoline derivatives against genital pathogens.
The in vitro activities of four quinoline carboxylic acids against 48 strains of Neisseria gonorrhoeae, 10 of Chlamydia trachomatis, and 32 of Ureaplasma urealyticum were compared. Ciprofloxacin was the most active against N. gonorrhoeae and C. trachomatis but had poor bactericidal activity against U. urealyticum, whereas ofloxacin showed the most bactericidal activity against U. urealyticum but was less active than ciprofloxacin against the two former pathogens. Norfloxacin and enoxacin were less active against all the studied pathogens.
Anti-Bacterial Agents↗
In vitro evaluation of new penicillins and cephalosporins upon P. aeruginosa and their interaction with mecillinam.
The in vitro activity of the new semisynthetic ureidopenicillins azlocillin (AZ) and mezlocillin (MZ), and of the new cephalosporin cefsulodin (CEF) were determined against 50 carbenicillin-sensitive (CARs) and 50 carbenicillin-resistant (CARr) P. aeruginosa clinical isolates. In the CARs group the most active antibiotics are AZ and CEF with MICs between 0.5 and 8 microgram/ml. Ticarcillin (TIC) and MZ showed more activity than CAR with MICs from 2 to 32 mu/ml. In this group there is a predominance of pyocine-type groups 1 and 3. In the CARr group, AZ is the most active antibiotic at low concentrations. At 64 microgram/ml of CEF, 72% of strains are inhibited as compared to 70% with AZ, 62% with MZ and 50% with TIC. In this group there is predominance of non-typeable strains. Interaction with mecillinam (MEC) and these antibiotics was studied on three different culture media (MH, NIH and DST). There were few cases of synergism with MEC and TIC combinations, mostly on DST medium. No appreciable synergism was found with other combinations of antibiotics.
Amdinocillin↗
[Use of PCR in the epidemiological identification of Campylobacter spp].
BACKGROUND: With arbitrary primer PCR technique it is possible to obtain amplification lane patterns easily and with good reproducibility from the genomic DNA of bacteria studied. There is also no need for prior information regarding the DNA sequence. METHOD: This method implies two cycles of amplification with low stringency, followed by a PCR of high stringency. RESULTS: Using the above mentioned technique, we were able to show that Campylobacter spp from clinical samples could be separated as well as different strains from the same species. CONCLUSIONS: According to our results as well as the ones from different authors applied to other microorganisms, we can assume that the method could be used in any bacterial species for epidemiologic studies purposes.
Base Sequence↗
[Evaluation of a new method (Filtrachek-UTI) for the detection of bacteriuria].
The diagnostic effectiveness of a new test for the detection of bacteriuria (Filtrachek-UTI) was compared with Gram stain and semiquantitative culture in 955 urine samples. 276 of these were from inpatients (108 males and 168 females), and 677 from outpatients (206 males and 471 females). Overall 229 positive cultures were obtained. Taking the sample culture as the standard, the statistical parameters obtained were very low, particularly the sensitivity and the negative predictive value (75-89% sensitivity and 80-91% NPV in inpatients, and 67-92% sensitivity and 79-98% NPV in outpatients). Other problems of the method are the subjective interpretation, choluria or filter obliteration by the urine, high cost and an approximate false negative rate of 16%.
Adult↗
Comparison between plasmids of Salmonella and other enterobacteria isolated from the same patients.
The ecology of R plasmids was studied in the intestinal flora of 19 patients with salmonellosis without antibiotic treatment. The plasmids found in the Salmonella strains and the accompanying non-pathogenic Enterobacteriaceae were characterized in each patient. We determined the transferability by conjugation, the fi character and the incompatibility group and did enzyme restriction analysis of these plasmids. The results obtained showed that S. typhimurium is the species of this genus with the highest incidence of R plasmids, and Escherichia coli among the non-pathogenic Enterobacteriaceae. The plasmids found in Salmonella are different from the plasmids found in the other Enterobacteriaceae in fi character (50% fi+ in Salmonella and 5% in the other Enterobacteriaceae) and incompatibility group (33% belong to the FII group in Salmonella plasmids and none on the other Enterobacteriaceae), thereby expressing a different origin.
Anti-Bacterial Agents↗