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J C Palomares

Publications and source records attributed to J C Palomares.

At least 19 recordsLinked to original sources
PubMed2002

Rifampin and isoniazid resistance associated mutations in Mycobacterium tuberculosis clinical isolates in Seville, Spain.

The susceptibility phenotypes of 964 clinical isolates of Mycobacterium tuberculosis were studied over a 7-year period in Seville, Spain. Thirty-eight (3.9%) strains were rifampin (RMP) resistant, 79 (8.2%) were isoniazid (INH) resistant and 22 (2.3%) were resistant to at least both antimicrobials (multidrug-resistant, MDR). We studied the mechanisms of resistance to these drugs in 94 resistant clinical isolates of M. tuberculosis using three molecular methods: 1) PCR-single strand conformation polymorphism (SSCP) analysis, 2) RFLP analysis using B1/B2 primers, and 3) sequence analysis. Five different mutations were detected in the rpoB gene: Ser531-->Leu (72.3%), His526-->Asp (12.8%), Asn518-->Ser (2.1%), Gln513-->Leu (2.1%) and a nine-nucleotide deletion (2.1%). In the case of resistance to INH, four different mutations in the katG gene were detected, Ser315-->Thr (58.0%), Ser315-->Leu (2.9%), partial deletion (5.8%) and Ile304-->Val (1.4%), while in the inhA regulatory region the only mutation was the nucleotide substitution C209T (4.3%). No mutation was found in the ahpC promoter.

DNA, Bacterial↗
PubMed2000

Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis.

Very fast amplification of DNA in small volumes can be continuously monitored with a rapid cycler that incorporates fluorimetric detection. Primers were designed to amplify a 157-bp fragment of the rpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium tuberculosis. Most mutations associated with resistance to rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. Two pairs of hybridization probes were synthesized; one in each pair was 3' labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5' labeled with LightCycler-Red 640. Each pair of probes recognized adjacent sequences in the amplicon. After DNA amplification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH, RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial agents) were amplified, and the presence of mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the LightCycler method correctly genotyped all the strains without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.

Antitubercular Agents↗
PubMed1999

Molecular analysis of rifampin and isoniazid resistance of Mycobacterium tuberculosis clinical isolates in Seville, Spain.

In this study we examined the mechanisms of resistance to rifampin (RMP) and isoniazid (INH) in 352 clinical isolates of Mycobacterium tuberculosis from Sevilla, Spain, using three different molecular methods: 1) PCR-single strand polymorphism analysis; 2) the commercial system Inno-LiPA RTB for RMP resistance; and 3) sequence analysis. Resistance to RMP was found in 21 strains, where the following mutations in the rpoB gene were detected: Ser531-->Leu (n = 14 strains); His526-->Asp (n = 3), Asn518-->Ser (n = 1), Gln513-->Leu (n = 1) and a nine nucleotide deletion (n = 1). Resistance to INH occurred in 29 strains, with mutations observed in: a) katG gene: Ser315-->Thr (n = 12), Ile304-->Val (n = 1), and a partial deletion (n = 4); b) regulatory region of the inhA gene: nucleotide substitution C209T (n = 3). No mutation was found in the ahpC promoter.

Antibiotics, Antitubercular↗
PubMed1998

[Characterization of the rpoB gene mutations in clinical isolates of rifampicin-resistant Mycobacterium tuberculosis].

OBJECTIVES: Characterization and frequency of the rpoB gene mutations associated with rifampin resistance in Mycobacterium tuberculosis clinical isolates in Sevilla. METHODS: Characterization of rpoB mutations in 21 rifampicin-resistant strains of M. tuberculosis isolated during a three-year period (1994-1996) by three different molecular methods: a nonradioactive Single-strand conformation polymorphism (SSCP) analysis, DNA sequence analysis and a commercial method the line probe assay InnoLiPA. RESULTS: Five distinct rpoB mutations were identified. Ser531-->Leu mutation was detected in 14 strains (66.7%), H526-->Asp in 3 strains (14.3%), Ans512-->Ser in 1 strain (4.8%), Glu513-->Leu in 1 strain (4.8%). A nine nucleotide deletion (codon 510-513) was found in one strain (4.8%) while in the remaining resistant strain (4.8%) no mutation was detected. CONCLUSIONS: The frequency of the different mutations found in the rpoB gene, associated with rifampicin resistance in Mycobacterium tuberculosis clinical isolates in Seville, are similar to those previously reported. However, two new mutations has been detected: a nine nucleotide deletion (codon 510-513), and the Asn512-->Ser point mutation. The characterization of the mutations in the rpoB gene could serve as epidemiological marker for the rifampicin resistant clinical isolates of M. tuberculosis.

Amino Acid Substitution↗
PubMed1997

Molecular epidemiology of Mycobacterium tuberculosis strains isolated during a 3-year period (1993 to 1995) in Seville, Spain.

The genetic polymorphism of Mycobacterium tuberculosis strains isolated in Seville, Spain, was studied by using computer-assisted analysis of the IS6110 fingerprint in order to determine the current situation and to evaluate the human-to-human transmission of this pathogen. One hundred seventy-six isolates from 175 patients among the 205 patients diagnosed with tuberculosis (TB) during a 3-year period (1993 to 1995) were cultured and analyzed. One hundred nine patients (62%) were infected with genetically different isolates, and 67 isolates (38%) were grouped into 19 clusters. These results demonstrate that the level of clustering of strains in Seville is intermediate between those in developed and developing countries. Epidemiological relatedness was shown for isolates from only 10 of these clusters. Active and high transmission rates exist in children and in human immunodeficiency virus (HIV)-infected adults, while in non-HIV-infected adults this transmission rate is moderate. Although transmission from children to adults is uncommon, the probability of transmission from HIV-infected patients to young adults not infected with HIV may be higher. On the basis of these observations, we predict a constant rise in the rate of TB transmission among HIV-infected patients and probably in young adult patients not infected with HIV if measures for the effective prevention of TB among the HIV-infected population are not implemented.

Adolescent↗
PubMed1997

[Subspecific classification of 11 clinical strains of Klebsiella pneumoniae based on their biochemical, antibiotic and plasmid profiles].

The paper summarizes at a subspecific level 11 clinical strains of K. pneumoniae. The objective of the work was to determine in a simple and effective way differences between different strains of the mentioned taxon. Biochemical characteristics, antibiogram and part of the plasmid spectrum were used for assessment of inter-species differences between different strains and at the same time their use as simple markers of epidemiological analyses is presented.

Bacterial Typing Techniques↗
PubMed1997

[False tuberculosis outbreak caused by specimen contamination in a micro-bacteriology laboratory: confirmation by molecular techniques].

BACKGROUND: The authors describe a false outbreak of tuberculosis by contamination in sample processing. METHODS: The longitudinal polymorphisms of restriction fragments (RFLPs) of 6 strains of Mycobacterium tuberculosis isolated in different patients over a three week period and which were apparently implicated in an outbreak of tuberculosis were analyzed. RESULTS: Four of the strains studied presented identical restriction pattern and the remaining two presented totally different patterns. Following study of the clinical histories and the epidemiologic relationships three cases of tuberculosis were confirmed. The other three strains isolated corresponded to contamination during the sampling process. CONCLUSIONS: In a possible outbreak of six cases of tuberculosis, molecular techniques have allowed identification of three true cases of tuberculosis and have demonstrated contamination during the sampling process in three other cases. The latter could not have been shown with the clinical and phenotypical data of the strains.

Adult↗
PubMed1995

Nosocomial transmission of tuberculosis infection in pediatrics wards.

Analysis of restriction fragment length polymorphisms is a well-established method of "DNA fingerprinting" that has been used to trace the transmission of particular strains of Mycobacterium tuberculosis during investigations of outbreaks. This report describe the use of restriction fragment length polymorphisms and arbitrarily primed polymerase chain reaction analysis to investigate two outbreaks of tuberculosis that affected six children who attended two pediatric wards in our hospital. In both outbreaks a history of household exposure to an adult with M. tuberculosis was obtained and suspected tuberculous contacts were identified. We have demonstrated unequivocally the strain relationship among the isolates in all the cases by restriction fragment length polymorphisms and arbitrarily primed polymerase chain reaction analysis. These techniques are very useful for performing epidemiologic studies of tuberculosis in children where natural history of tuberculosis infection is different from that in adults in that it is almost always primary infection rather than reactivation.

Child↗
PubMed1993

Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods.

This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100-extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS200. The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55 degrees C, the microwells were washed and an alkaline phosphatase-labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 microliters of 1 mmol 1(-1) AttoPhos (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1-10 cfu. A total of 172 food samples were tested, both by culture and FD-PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD-PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD-PCR assay described here can be useful to screen a large number of food samples for contamination by salmonellas.

Alkaline Phosphatase↗
PubMed1993

Dual genitotropic human papillomavirus infections in genital warts.

BACKGROUND AND METHODS: We have carried out a prospective study of dual genitotropic human papillomavirus (HPV) infections by means of two different DNA detection methods in biopsy specimens obtained from patients who were examined for genital warts at the STD clinic of the School of Medicine in Seville, between January 1990 and December 1991. RESULTS: 100 patients with a clinical diagnosis of condilomata acuminata were seen during the study period. DNA of the genitotropic HPV 6/11, 16/18 and 31/33/35 was detected by an in situ hybridisation method in 75 (77%) of the 98 evaluable samples; one of the genotypes tested in 59 (61%) samples, and two or more genotypes tested in the remaining 16 (15%) samples. In 21 (98%) of the 23 negative samples by in situ hybridisation, we were able to detect DNA of genital HPV using a polymerase chain reaction amplification method (PCR). Among the 34 samples where PCR was applied we confirmed the presence of two different HPV genotypes in eight samples. CONCLUSIONS: The frequency of dual infections with human genitotropic papillomavirus in genital warts was 8%, although we believe that this rate should be higher as we have not used the PCR method in all of the samples.

Adolescent↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

[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↗
PubMed1992

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↗