[Show abstract][Hide abstract] ABSTRACT: Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a practical approach for the rapid differential diagnosis between extrapulmonary tuberculosis and complicated brucellosis.
PLoS ONE 03/2013; 8(3):e58353. DOI:10.1371/journal.pone.0058353 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The PTPN22 gene encodes for an intracellular lymphoid-specific phosphatase (Lyp) that has a negative regulatory effect on T-cell activation. The minor allele of the single nucleotide polymorphism (SNP) in the PTPN22 gene encoding the Lyp-tyrosine phosphatase has been associated with multiple autoimmune disorders and with susceptibility to M. tuberculosis. It is possible, therefore, that variants of this gene may also be involved in susceptibility to another intracellular pathogen, B. melitensis, which gives rise to human brucellosis. Accordingly, we studied 111 patients with brucellosis and 150 healthy controls who had had no prior contact with the pathogen. Genotyping of the PTPN22 1858CT was performed by an allele discrimination assay with TaqMan 5'. We found no statistically significant differences between the patients and the controls in genotype or allele frequencies of PTPN22 1858CT. These data suggest that this variant is not associated with human brucellosis.
[Show abstract][Hide abstract] ABSTRACT: Polymorphisms in the cytokine genes have allowed for the understanding of the genetic determinants in several diseases. We investigated the polymorphism of the transforming growth factor (TGF)-beta1 and IL-6 genes in relation to susceptibility to human brucellosis. We typed 82 Spanish brucellosis patients and 102 healthy controls for TGF-beta1 polymorphisms in codons 10 and 25, and IL-6 promoter polymorphism at position -174 by PCR-SSP methods. The T/T G/G genotype of the TGF-beta1 gene was significantly increased in patients compared to controls (49% vs. 32%) P=0.02; OR=1.99 (1.05-3.80) and the T/C G/G genotype was significantly less common in the patients compared to the controls (32% vs. 49%) P=0.01; OR=0.48 (0.25-0.92). The CC genotype of codon 10 was significantly increased in the patients who had focal forms of the disease as compared with those who did not develop focal forms (19% vs. 4%), P=0.03; OR=0.19 (0.02-1.10). No differences were found in the IL-6 variants between the patients and the controls. These results suggest that polymorphism of the TGF-beta1 gene may be involved in susceptibility to brucellosis and to developing focal forms of the disease in a group of patients from southern Spain.
[Show abstract][Hide abstract] ABSTRACT: Genetic susceptibility to human brucellosis has so far been localized to variants of genes, which participate in the specific response and the innate immune response. The Nramp1 gene, which participates in the innate response, is related to susceptibility and protection in bovine brucellosis. We examined the polymorphism of the human NRAMP1 gene in 65 patients with brucellosis and 89 healthy controls and found no significant differences in the alleles studied. Thus, variants of the NRAMP1 gene do not appear to affect susceptibility or protection in human brucellosis.
Microbes and Infection 02/2006; 8(1):154-6. DOI:10.1016/j.micinf.2005.06.010 · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several HLA alleles are associated with susceptibility or protection in breast cancer. The particular allele varies depending on the geographical region. A study in a small group of Spanish patients using serological methods found an association with HLA-B7. We undertook a larger study in southern Spain using molecular biology techniques.
Genotype variants of HLA class I and II were typed by PCR-SSP in 132 breast cancer patients and 382 healthy controls.
The frequency of the HLA-B7 allele was increased in the patients compared to the controls (P=0.0019; 95% confidence interval, 1.337-3.409, relative risk=2.135). Bonferroni correction of the P showed it was still significant (P(c)=0.0285).
These results support previous suggestions that HLA-B7 is associated with the development of breast cancer in our area.
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO), produced by the inducible nitric oxide synthase (NOS2) protein, is a defence mechanism against various pathogens, including bacteria of the genus Brucella. The aim of this study was to investigate the possible association between the NOS2 gene promoter polymorphism, TAAA(n) and CCTTT(n) microsatellites, and the predisposition to human brucellosis. We performed a case-control study in 85 patients with brucellosis and 100 healthy individuals, matched for age and sex, living in the same geographic area, in whom the NOS2 promoter was genotyped by using a polymerase chain reaction (PCR)-based method combined with fluorescent technology. No statistically significant differences were observed in the distribution of TAAA(n) alleles between the groups under study. When the overall NOS2 CCTTT(n) allele distribution of the brucellosis patients was compared with that of the control subjects, a significant skewing was observed (P = 0.04, by chi(2) test from 2 x 9 contingency table). Interestingly, we observed a trend towards Brucella infection protection with the 9 repeat (181 bp) allele (1.8% patients vs. 7.5% controls; P = 0.01, odds ratios = 0.22, 95% confidence interval = 0.05-0.83), which turned out to be non-significant after applying multiple testing. We concluded that the NOS2 microsatellite polymorphism might not have a major effect on brucellosis; nevertheless, the fact that a non-significant trend towards protection was detected in the CCTTT(n) alleles may be an indication for a follow-up study.
Microbes and Infection 12/2003; 5(13):1165-9. DOI:10.1016/j.micinf.2003.08.010 · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the contribution of HLA gene polymorphism toward susceptibility to osteoarticular focal forms of human brucellosis.
A total of 57 patients with brucellosis, of whom 23 had osteoarticular complications, and 73 healthy volunteers were genotyped for HLA class I and class II antigens by a polymerase chain reaction-sequence specific primer technique.
The HLA-B*39 allele was present in significantly more patients with osteoarticular complications than in the other patients (35% vs 3%; p = 0.0006, OR 15.684, 95% CI 3.453-71.231), or in the controls.
The increased presence of the HLA-B39 genotype in patients with brucellosis with clinical osteoarticular manifestations suggests that this genotype confers susceptibility to developing severe osteoarticular focal forms of the disease.
The Journal of Rheumatology 06/2003; 30(5):1051-3. · 3.17 Impact Factor