[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. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Both brucellosis and tuberculosis are chronic-debilitating systemic granulomatous diseases with a high incidence in many countries in Africa, Central and South America, the Middle East and the Indian subcontinent. Certain focal complications of brucellosis and extrapulmonary tuberculosis are very difficult to differentiate clinically, biologically and radiologically. As the conventional microbiological methods for the diagnosis of the two diseases have many limitations, as well as being time-consuming, multiplex real time PCR (M RT-PCR) could be a promising and practical approach to hasten the differential diagnosis and improve prognosis.
We designed a SYBR Green single-tube multiplex real-time PCR protocol targeting bcsp31 and the IS711 sequence detecting all pathogenic species and biovars of Brucella genus, the IS6110 sequence detecting Mycobacterium genus, and the intergenic region senX3-regX3 specifically detecting Mycobacterium tuberculosis complex. The diagnostic yield of the M RT-PCR with the three pairs of resultant amplicons was then analyzed in 91 clinical samples corresponding to 30 patients with focal complications of brucellosis, 24 patients with extrapulmonary tuberculosis, and 36 patients (Control Group) with different infectious, autoimmune or neoplastic diseases. Thirty-five patients had vertebral osteomyelitis, 21 subacute or chronic meningitis or meningoencephalitis, 13 liver or splenic abscess, eight orchiepididymitis, seven subacute or chronic arthritis, and the remaining seven samples were from different locations. Of the three pairs of amplicons (senX3-regX3+ bcsp3, senX3-regX3+ IS711 and IS6110+ IS711) only senX3-regX3+ IS711 was 100% specific for both the Brucella genus and M. tuberculosis complex. For all the clinical samples studied, the overall sensitivity, specificity, and positive and negative predictive values of the M RT-PCR assay were 89.1%, 100%, 85.7% and 100%, respectively, with an accuracy of 93.4%, (95% CI, 88.3-96.5%).
In this study, a M RT-PCR strategy with species-specific primers based on senX3-regX3+IS711 sequences proved to be a sensitive and specific test, useful for the highly efficient detection of M. tuberculosis and Brucella spp in very different clinical samples. It thus represents an advance in the differential diagnosis between some forms of extrapulmonary tuberculosis and focal complications of brucellosis.
[Show abstract][Hide abstract] ABSTRACT: Miliary tuberculosis after intravesical Calmette-Guerin bacilli (BCG) therapy is rare. To date, only 23 cases have been reported. We describe the case of a patient on hemodialysis, review the literature, and call attention to the potential hazard of intravesical BCG therapy in patients on renal replacement therapy and the value of polymerase chain reaction-based methods for early diagnosis of this serious complication.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: The aim of this article has been to analyze the clinical and radiological data suggesting tuberculous vertebral osteomielitis (TVO), and then discuss the steps to be followed to achieve an aetiological diagnosis. METHODS: A thorough literature search was carried out to identify the best clinical and microbiological evidence for a fast and efficient diagnosis of TVO. RESULTS: The clinical and radiological diagnosis of spinal tuberculosis suffers from serious limitations, with a high percentage of cases requiring vertebral biopsy to reach a definitive diagnosis. The increasing incidence of multidrug-resistant tuberculosis has highlighted the insufficiency of the histopathological diagnosis and the need for microbiological diagnosis. Unfortunately, the maximum sensitivity of spinal tuberculosis cultures is 80 %, and traditional methods require 6 to 8 weeks for the isolation, identification and sensitivity study. New culture media and identification methods have improved sensitivity and reduced the time required for the identification. Molecular methods have now been integrated into a single test, with identification of the mycobacterium responsible and its sensitivity to rifampicin. Additionally, multiplex-PCR tests have been developed that allow a rapid differential diagnosis between granulomatous spondylodiscitis. CONCLUSIONS: All patients with subacute inflammatory back or neck pain showing suggestive radiological findings should be studied to rule out TVO. If there is no clear evidence of tuberculosis from another location or indication for surgery, a percutaneous vertebral biopsy should be performed. When TVO is suspected, all spinal or paravertebral tissue samples should be sent simultaneously to pathology and microbiology laboratories for appropriate processing.
European Spine Journal 05/2012; · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rapid diagnosis of individuals involved in brucellosis outbreaks can sometimes be difficult with conventional microbiological techniques. We analyzed, for the first time, the diagnostic yield of a real-time polymerase chain reaction (PCR) assay in a family outbreak of brucellosis due to consumption of unpasteurized goat cheese. PCR correctly identified all symptomatic cases.
[Show abstract][Hide abstract] ABSTRACT: Case-control study for assessing a diagnostic test.
The aim of this study was to analyze the diagnostic yield of a multiplex real-time polymerase chain reaction (PCR) assay in the differential diagnosis of tuberculous vertebral osteomyelitis (TVO) and brucellar vertebral osteomyelitis (BVO).
Vertebral osteomyelitis (VO) is one of commonest osteoarticular complications of tuberculosis and brucellosis. However, the very similar clinical, radiologic, and histologic characteristics of these entities mean that diagnosis requires etiological confirmation, but conventional microbiologic methods have important limitations.
Fifteen vertebral samples from patients with TVO or BVO and 9 from pyogenic and nontuberculous mycobacteria VO were studied by multiplex PCR and conventional microbiologic techniques. To identify Brucella DNA, we used a fragment of 207 bp from the conserved region of the gene coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and for Mycobacterium tuberculosis complex, a fragment of 164 bp from the intergenic region SenX3-RegX3.
The histopathologic findings were inconclusive in 4 of 14 cases (28.6%) with TVO or BVO and cultures were positive in 11 of 15 cases (73.3%). Multiplex PCR correctly identified 14 of the 15 samples from patients with TVO and BVO and was negative in all the control samples. Thus, the overall sensitivity and specificity of the multiplex PCR were 93.3% and 90%, respectively, with an accuracy of 92% (95% CI, 81.4%-100%).
These results suggest that multiplex real-time PCR is far more sensitive than conventional cultures, and this, together with its speed, makes this technique a very practical approach for the differential diagnosis between TVO and BVO.
[Show abstract][Hide abstract] ABSTRACT: Arduous to differ clinically, extrapulmonary tuberculosis and focal complications of brucellosis remain important causes of morbidity and mortality in many countries. We developed and applied a multiplex real-time PCR assay (M RT-PCR) for the simultaneous detection of Mycobacterium tuberculosis complex and Brucella spp.
Conventional microbiological techniques and M RT-PCR for M. tuberculosis complex and Brucella spp were performed on 45 clinical specimens from patients with focal complications of brucellosis or extrapulmonary tuberculosis and 26 control samples. Fragments of 207 bp and 164 bp from the conserved region of the genes coding for an immunogenic membrane protein of 31 kDa of B. abortus (BCSP31) and the intergenic region SenX3-RegX3 were used for the identification of Brucella and M. tuberculosis complex, respectively.
The detection limit of the M RT-PCR was 2 genomes per reaction for both pathogens and the intra- and inter-assay coefficients of variation were 0.44% and 0.93% for Brucella and 0.58% and 1.12% for Mycobacterium. M RT-PCR correctly identified 42 of the 45 samples from patients with tuberculosis or brucellosis and was negative in all the controls. Thus, the overall sensitivity, specificity, PPV and NPV values of the M RT PCR assay were 93.3%, 100%, 100% and 89.7%, respectively, with an accuracy of 95.8% (95% CI, 91.1%-100%). Since M RT-PCR is highly reproducible and more rapid and sensitive than conventional microbiological tests, this technique could be a promising and practical approach for the differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.
PLoS ONE 02/2009; 4(2):e4526. · 3.53 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: Diagnosis of brucellosis can be difficult in certain scenarios where conventional microbiological techniques have important limitations. The aim of this study was to develop a LightCycler Quantitative PCR assay in serum samples to discriminate between active and past brucellosis. In total, 110 serum samples from 46 brucellosis patients and 64 controls, including persons who had recently been treated for brucellosis, asymptomatic persons exposed to brucellosis, and patients with febrile syndromes involving a differential diagnosis with brucellosis, were studied. Brucella spp.-specific sequences of the PCR primers and probe were selected from the gene encoding an immunogenic membrane protein of 31 kDa (BCSP31). The analytical sensitivity was 1 x 10(1) fg of Brucella DNA. The mean threshold cycles for brucellosis patients and controls were 31.8 +/- 1.7 and 35.4 +/- 1.1, respectively (p <0.001). The best cut-off for bacterial DNA load was 5 x 10(3) copies/mL. At this cut-off, the area under the receiver operating characteristic curves was 0.963 (95% CI 0.920-1.005), with a sensitivity of 93.5% and a specificity of 98.4%. Under the assay conditions, the LightCycler Quantitative PCR in serum samples seems to be highly reproducible, rapid, sensitive and specific. It is therefore a useful method for both the initial diagnosis and the differentiation between past and active brucellosis.
Clinical Microbiology and Infection 12/2008; 14(12):1128-34. · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We compared the relative recovery of extraction of bacterial DNA from serum using seven commercial kits (UltraClean DNA BloodSpin Kit, Puregene DNA Purification System, Wizard Genomic DNA Purification Kit, High Pure PCR Template Preparation Kit, GFX Genomic Blood DNA Purification Kit, NucleoSpin Tissue Kit, and QIAamp DNA Blood Mini Kit). Human serum samples were spiked with known concentrations of Brucella melitensis Rev 1; the DNA was extracted and tested in genus-specific LightCycler polymerase chain reaction (PCR). The UltraClean DNA BloodSpin Kit proved to be as sensitive as the QIAamp DNA Blood Mini Kit isolation method and could detect down to 100 fg of DNA, though only the former had no contamination. All the other procedures yielded DNA isolation results that were less sensitive and were always contaminated. Our results show that the UltraClean DNA Blood Spin Kit was the commercially available assay tested that yielded the best sensitivity, purity, and lack of contamination for Brucella DNA isolation from serum.
European Journal of Clinical Microbiology 03/2008; 27(2):109-14. · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.
[Show abstract][Hide abstract] ABSTRACT: We studied the diagnostic yield of a real-time polymerase chain reaction assay in urine samples for the rapid diagnosis of brucella epididymo-orchitis compared to that of conventional microbiological techniques.
We used an SYBR Green I LightCycler based real-time polymerase chain reaction to retrospectively study 10 urine samples from patients with Brucella epididymo-orchitis. The assay amplifies a 223 bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for Brucella genus (BCSP31). After amplifying this 223 bp sequence we performed melting curve analysis to verify the specificity of polymerase chain reaction products.
Brucella melitensis was isolated from blood cultures in 9 cases (90%). Wright's seroagglutination was negative or inconclusive in 30% of cases. Brucella was isolated from urine in only 1 case, whereas real-time polymerase chain reaction assay in urine was positive in 9 (90%). Also, results were available in 4 hours, whereas mean time to availability of the final blood culture results was 5.8 days (range 4.5 to 7).
SYBR Green I LightCycler based real-time polymerase chain reaction assay in urine samples is highly sensitive and specific, and easy to perform. It could provide the clinician with results in less than 5 hours. The technique could be a practical and useful tool for the rapid diagnosis of genitourinary complications of human brucellosis.
The Journal of Urology 12/2006; 176(5):2290-3. · 3.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to develop a LightCycler-based real-time PCR (LC-PCR) assay and to evaluate its diagnostic use for the detection of Brucella DNA in serum samples. Following amplification of a 223-bp gene sequence encoding an immunogenetic membrane protein (BCSP31) specific for the Brucella genus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. The intra- and inter-assay variation coefficients were 1.3% and 6.4%, respectively, and the detection limit was 5 fg of Brucella DNA (one genome equivalent). After optimisation of the PCR assay conditions, a standard curve was obtained with a linear range (correlation coefficient=0.99) over seven orders of magnitude from 10(7) to 10 fg of Brucella DNA. The LC-PCR assay was found to be 91.9% sensitive and 95.4% specific when tested with 65 negative control samples and 62 serum samples from 60 consecutive patients with active brucellosis. The assay is reproducible, easily standardised, minimises the risk of infection in laboratory workers, and has a total processing time of <2 h. It could therefore form a promising and practical approach for the rapid diagnosis of human brucellosis.
Clinical Microbiology and Infection 10/2005; 11(9):713-8. · 5.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We compared the diagnostic yield of a real time polymerase chain reaction (PCR) assay in cerebrospinal fluid (CSF) samples with conventional microbiological techniques for the diagnosis of neurobrucellosis. Following amplification of a 223 bp sequence specific for Brucella genus, melting curve analysis was performed to verify the specificity of the PCR products.
All six patients with neurobrucellosis (three meningitis and three meningoencephalitis) had a positive real time PCR assay, whereas CSF cultures and Wright seroagglutination tests were positive in only two and four cases, respectively. Brucella specific amplicons were easily demonstrated by their characteristic melting temperature in all the real time PCR assays.
LightCycler based real time PCR assay in CSF samples is more rapid and sensitive than conventional microbiological tests. This technique could be useful for the rapid diagnosis of neurobrucellosis.
[Show abstract][Hide abstract] ABSTRACT: To overcome some of the limitations of conventional microbiological techniques, polymerase chain reaction (PCR)-based assays have been proposed as a useful tool for the diagnosis of human brucellosis.
A single-blinded comparative study was undertaken that compared 2 different PCR assays: a SYBR Green I LightCycler-based Real-Time PCR assay (LC-PCR; Roche Diagnostic) with serum samples and a PCR-enzyme-linked immunosorbent assay (ELISA) with whole blood samples. Both assays amplify a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for Brucella genus (BCSP31). We analyzed the diagnostic yield of these assays with 60 samples obtained from patients with active brucellosis and 37 samples obtained from a control group composed of patients with febrile syndromes of other defined etiologies, asymptomatic subjects with past brucellosis or exposure to Brucella infection who had persistently high titers of anti-Brucella antibodies, and healthy subjects.
The sensitivities of LC-PCR with serum samples, PCR-ELISA with whole blood samples, and blood cultures were 93.3%, 90%, and 65%, respectively. Three control samples (8.1%) had a positive PCR-ELISA result, and 2 of these samples (5.4%) also had positive LC-PCR results. The specificity and positive likelihood ratios were 94.6% and 17.3, respectively, for LC-PCR and 91.9% and 11.1, respectively, for PCR-ELISA.
The diagnostic yield of LC-PCR with serum samples was higher than that of PCR-ELISA with whole blood samples. The speed and technical simplicity of LC-PCR in serum samples make it a useful alternative to blood cultures for patients with suspected brucellosis and negative or doubtful serological test results.
[Show abstract][Hide abstract] ABSTRACT: In an attempt to avoid some of the inconveniences associated with conventional PCR, such as electrophoresis in ethidium bromide, we developed and analyzed the yield of a digoxigenin-based enzyme-linked immunosorbent PCR assay (PCR-DIG ELISA) for the detection of specific Brucella target DNA. During the DNA amplification process in healthy subjects and controls (Brucella abortus B-19) non-specific amplification of fragments was formed between genomic DNA and specific biotin-labeled primers. The labeled non-specific fragments bound to streptavidin-coated wells, saturating the solid phase streptavidin by biotin-streptavidin interaction. The formation of these non-specific PCR products was demonstrated by reduction in absorbance with hemin, a Taq polymerase inhibitor, and identified by use of a silver stained method which improves the sensitivity of nucleic acid visualization.
Molecular and Cellular Probes 09/2004; 18(4):243-50. · 1.86 Impact Factor
[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. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to overcome some of the limitations of conventional microbiological techniques in the diagnosis of human brucellosis, a simple PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed. After amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for the Brucella genus (BCSP31), the digoxigenin-labeled amplified product was hybridized with a biotinylated capture probe which was complementary to the inner part of the amplicon. The hybrid was captured on streptavidin-coated microtiter plates and detected by using an antidigoxigenin Fab-peroxidase conjugate. The detection limit of the PCR-ELISA in a background of 3.5 micro g of human genomic DNA was 10 fg (two bacterial cells). The PCR-ELISA showed an analytical sensitivity higher than that of ethidium bromide staining and equal to that obtained by conventional PCR followed by dot blot hybridization. In 59 peripheral blood samples from 57 consecutive patients with active brucellosis and 113 control samples, the PCR-ELISA was found to be 94.9% sensitive and 96.5% specific, whereas the sensitivity of the blood culture was only 70.1%. Since the assay can be performed in 1 day, is very reproducible, is easily standardized, and avoids the risk of infection in laboratory workers, this PCR-ELISA seems to be a practical and reliable tool for the diagnosis of human brucellosis.
Journal of Clinical Microbiology 02/2003; 41(1):144-8. · 4.23 Impact Factor