Nora Cardona-Castro

Development and Evaluation of a Multiplex Real-Time Polymerase Chain Reaction Procedure to Clinically Type Prevalent Salmonella enterica Serovars

A multiplex real-time polymerase chain reaction procedure was developed to identify the most prevalent clinical isolates of Salmonella enterica subsp. enterica. Genes from the rfb, fliC, fljB, and viaB groups that encode the O, H, and Vi antigens were used to design 15 primer pairs and TaqMan probes specific for the genes rfbJ, wzx, fliC, fljB, wcdB, the sdf-l sequence, and invA, which was used as an internal amplification control. The primers and probes were variously combined into six sets. The first round of reactions used two of these sets to detect Salmonella O:4, O:9, O:7, O:8, and O:3,10 serogroups. Once the serogroups were identified, the results of a second round of reactions that used primers and probes for the flagellar antigen l genes, 1,2; e,h; g,m; d; e,n,x; and z(10), and the Vi gene were used to identify individual serovars. The procedure was standardized using 18 Salmonella reference strains and other enterobacteria. The procedures reliability and sensitivity was evaluated using 267 randomly chosen serotyped Salmonella clinical isolates. The procedure had a sensitivity of 95.5% and was 100% specific. Thus, our technique is a quick, sensitive, reliable, and specific means of identifying S. enterica serovars and can be used in conjunction with traditional serotyping. Other primer and probe combinations could be used to increase the number of identifiable serovars.

Development and evaluation of a multiplex polymerase chain reaction assay to identify Salmonella serogroups and serotypes

To improve limitations of Salmonella serotyping, 2 multiplex polymerase chain reaction (M-PCR) were developed using a strategy that identifies first the genes encoding serogroups (rfbJ, wzx). According to the serogroup determined, a second M-PCR identifies serotype (fliC, fljB, wcdB, and sdf-I sequence). Standardization and evaluation of both M-PCRs were carried out.

IL-10 gene promoter polymorphisms and leprosy in a Colombian population sample.

INTRODUCTIONnPolymorphisms in promoters of genes code for cytokines that affect transcription levels. Several have been associated with leprosy patients that have functional and clinical implications.nnnOBJECTIVEnPolymorphisms in the promoter of the IL10 gene of leprosy patients will be compared frequencies in normal population.nnnMATERIALS AND METHODSnSNPs (single nucleotide polymorphism) -1082 A/G (rs1800896), -819C/T (rs1800871), and -592A/C (rs1800872) were identified in 100 leprosy patients and in a control group of 100 volunteers from a leprosy endemic region of Colombia.nnnRESULTSnThe genotypes C/C and C/T in the SNP -819 were associated together with leprosy (OR=4.34, p<0.001).Similarly, the genotypes C/C and C/A in the -592 SNP showed an association (OR=4.3, p<0.001). The haplotypes -819C-519C and -1082A-819C-592C showed significant association (OR=4.34, p<0.001 and OR=6.25, p<0.001) respectively. These haplotypes in homozygosis conditions were also associated with leprosy: -819C-519C/-819C-519C (OR=4.34, p<0.001), -1082A -819C-592C/-1082A -819C-592C (OR=1.90, p=0.04). The SNP -1082 was not associated with leprosy in this population.nnnCONCLUSIONSnThe haplotypes associated with leprosy, -1082A-819C-592C/-1082A-819C-592C, have been reported as low producers of IL-10. Functionally, the low production of IL-10 may have immune response consequences and clinical implications. Additional haplotypes of IL-10 have been reported as markers for leprosy susceptibility or resistance in other ethnic populations. This suggests that differences in distribution of diverse IL-10 gene polymorphisms among ethnic groups may indicate important gene-disease associations.

Polimorfismos en el gen promotor de IL-10 en una muestra de pacientes colombianos con lepra

Introduccion. Se han reportado polimorfismos en los genes promotores que codifican para citocinas y que afectan los niveles de transcripcion, con implicaciones clinicas y funcionales en pacientes con lepra. Objetivo. Detectar los polimorfismos en el gen promotor de la interleucina 10 (IL-10), de los polimorfismos de un solo nucleotido (Single Nucleotide Polymorphisms, SNP) -1082 A/G (rs1800896), -819C/T (rs1800871) y -592A/C (rs1800872), en 100 pacientes con lepra y un grupo control de 100 voluntarios, de una region endemica de Colombia. Resultados. Los haplotipos -819C-519C y -1082A-819C-592C mostraron asociacion significativa con lepra: OR=4,34, p=1 x 10-3, y OR=6,25, p=5 x 10-4, respectivamente. Estos haplotipos en condiciones de homocigoto, estan tambien asociados con lepra: -819C-519C/-819C-519C (OR=4,34 p=1 x 10-3), -1082A -819C-592C/-1082A -819C-592C (OR=1,90 y p=0,04). El SNP -1082 no se encontro asociado con lepra en esta poblacion. Los genotipos C/C y C/T en el SNP -819, se encontraron asociados a lepra (OR=4,34, p=1 x 10-3); de igual manera, los genotipos C/C y C/A en el SNP -592 mostraron asociacion (OR=4,34, p=1 x 10-3). Conclusiones. El haplotipo que encontramos asociado con lepra, -1082A-819C-592C/-1082A-819C-592C, se ha relacionado con baja produccion de IL-10. Funcionalmente, esta baja produccion de IL-10 puede tener consecuencias en la respuesta inmunitaria, ademas de implicaciones clinicas. Se han reportado diferentes haplotipos de IL-10 como marcadores de vulnerabilidad y resistencia de lepra en otras poblaciones, lo cual sugiere que las diferencias en la distribucion de diversos polimorfismos del gen de IL-10 entre grupos etnicos, es un factor importante al determinar la asociacion entre enfermedad y genes. DOI:xa0 http://dx.doi.org/10.7705/biomedica.v32i1.386

Gallbladder microbiota variability in Colombian gallstones patients

INTRODUCTIONnGallbladder stones are a very frequently occurring condition. Despite bile bactericidal activity, many bacteria have been detected inside the gallbladder, and gallstones facilitate their presence. Between 3% and 5% of the patients with Salmonella spp. infection develop the carrier stage, with the bacteria persisting inside the gallbladder, shedding bacteria in their feces without signs of infection. The aim of this study was to isolate bacteria from Colombian patients with gallstones, using standard culturing methods, and to identify Salmonella spp. carriers by molecular techniques.nnnMETHODOLOGYnA total of 149 patients (120 female and 29 male) diagnosed with gallstones who underwent cholecystectomy and who did not have symptoms of acute inflammation were included. Gallbladder tissue and bile were cultured and used for DNA extraction and Salmonella spp. hilA gene detection.nnnRESULTSnOf the 149 patients 28 (19%) had positive cultures. Twenty-one (75%) patients with positive cultures were from Medellins metropolitan area. In this geographical location, the most frequent isolations were Pseudomonas spp. (38%), Klebsiella spp. (23%), and Proteus spp. (9%) in addition to unique cases of other bacteria. In Apartado, the isolates found were Enterobacter cloacae (50%), Raoultella terrigena (32%), and both Enterobacter cloacae and Raoultella terrigena were isolated in one (18%) male patient. Five (3.3%) of the 149 patients had positive polymerase chain reaction (PCR) results for the hilA gene of Salmonella spp., all of whom were female and residents of the Medellín metropolitan area.nnnCONCLUSIONSnThe gallbladder microbiota variability found could be related to geographical, ethnic, and environmental conditions.

Detection of Salmonella human carriers in Colombian outbreak areas

INTRODUCTIONnSalmonellosis, a zoonotic and foodborne disease, is a public health problem in developing countries. With the aim of identifying human carriers of Salmonella, a survey was performed in five regions of Colombia with reported salmonellosis outbreaks.nnnMETHODOLOGYnThe general population and cholecystectomy surgical patients were included in this study. Stool samples from 667 volunteers and gallbladder bile samples from 199 surgical patients were examined. Detection of Salmonella from cultured stool and bile samples was determined by polymerase chain reaction (PCR). Multiplex PCR and biochemical and serological tests were performed to identify the serovars of the isolates.nnnRESULTSnNine (1.35%) stool samples were positive for Salmonella: two S. Newport, two S. Anatum, one S. Sinstorf, and four Salmonella spp. A total of 11 gallbladder bile samples were positive: S. Enteritidis was isolated from 3 bile cultures (1.5%), and 8 samples (4%) were positive for Salmonella spp.nnnCONCLUSIONSnOur results show the presence of Salmonella carriers in the inhabitants of regions with reported outbreaks and suggest that these carriers are potential sources of infection in endemic and epidemic cases. Carriers also suggest Salmonella zoonotic transmission, since broiler and beef cattle are hosts to the Salmonella serotypes isolated. It is important to establish the source of infection in regions where salmonellosis is endemic in order to control transmission.