Laboratory Diagnosis of Human Brucellosis.

The clinical presentation of brucellosis in humans is variable and unspecific, and thus, laboratory corroboration of the diagnosis is essential for the patient's proper treatment. The diagnosis of brucellar infections can be made by culture, serological tests, and nucleic acid amplification assays. Modern automated blood culture systems enable detection of acute cases of brucellosis within the routine 5- to 7-day incubation protocol employed in clinical microbiology laboratories, although a longer incubation and performance of blind subcultures may be needed for protracted cases. Serological tests, though they lack specificity and provide results that may be difficult to interpret in individuals repeatedly exposed to Brucella organisms, nevertheless remain a diagnostic cornerstone in resource-poor countries. Nucleic acid amplification assays combine exquisite sensitivity, specificity, and safety and enable rapid diagnosis of the disease. However, long-term persistence of positive molecular test results in patients that have apparently fully recovered is common and has unclear clinical significance and therapeutic implications. Therefore, as long as there are no sufficiently validated commercial tests or studies that demonstrate an adequate interlaboratory reproducibility of the different homemade PCR assays, cultures and serological methods will remain the primary tools for the diagnosis and posttherapeutic follow-up of human brucellosis.

What Should a Spanish Graduate in Medicine in the European Higher Education System Know About Biochemistry and Molecular Biology? A Personal Reflection.

For over 40 years of working in the area of biochemistry and molecular biology, 30 of which have been spent teaching at the University of Malaga, I have been involved in the theoretical and practical teaching of the subject initially called biochemistry and now called biochemistry and molecular biology (BMB). The expansion of this scientific discipline and the renewed interest in research have led to such a large accumulation of knowledge (knowledge base) that the elaboration of a BMB program requires applying notable doses of synthesis; otherwise, it would be impossible to cover all this subject within the strict confines of the bi-semestral term. Advances in medicine and BMB are inseparable, and much of modern medicine would not be practiced as it is if it were not for our understanding of how hereditary, pathogenic, and environmental factors affect the human body at the molecular level. The importance, therefore, of teaching medical students biochemistry is evident. BMB is a subject that corresponds to the area of biomedicine and is taught during the first year of medicine. The main aim is to study the basics of chemical structures from the molecular viewpoint, with special emphasis on regulating and integrating aspects, necessary to understand such disciplines as physiology, pharmacology, or pathology. Thus, based on many years of experience teaching BMB to medical undergraduates, my aim is to define what it is that a graduate in medicine should know about the subject.

Lessons learned with molecular methods targeting the BCSP-31 membrane protein for diagnosis of human brucellosis.

Brucellosis remains an emerging and re-emerging zoonosis worldwide causing high human morbidity. It usually affects persons who are permanently exposed to fastidious microorganisms of the Brucella genus and has a nonspecific clinical picture. Thus, diagnosis of brucellosis can sometimes be difficult. Molecular techniques have recently been found very useful in the diagnosis of brucellosis together with its common and very diverse focal complications. We herein review all the lessons learned by our group concerning the molecular diagnosis of human brucellosis over the last twenty years. The results, initially using one-step conventional PCR, later PCR-ELISA and more recently real-time PCR, using both fluorescent intercalating reagents (SYBR-Green I) and specific probes (Taqman), have shown that these techniques are all much more sensitive than bacteriological methods and more specific than the usual serological techniques for the diagnosis of primary infection, the post-treatment control of the disease, early detection of relapse and the diagnosis of focal complications. Optimization of the technique and improvements introduced over the years show that molecular methods, currently accessible for most clinical laboratories, enable easy rapid diagnosis of brucellosis at the same time as they avoid any risk to laboratory personnel while handling live Brucella spp.

Comparative Study of a Real-Time PCR Assay Targeting senX3-regX3 versus Other Molecular Strategies Commonly Used in the Diagnosis of Tuberculosis.

BACKGROUND: Nucleic acid amplification tests are increasingly used for the rapid diagnosis of tuberculosis. We undertook a comparative study of the efficiency and diagnostic yield of a real-time PCR senX3-regX3 based assay versus the classical IS6110 target and the new commercial methods. METHODS: This single-blind prospective comparative study included 145 consecutive samples: 76 from patients with culture-confirmed tuberculosis (86.8% pulmonary and 13.2% extrapulmonary tuberculosis: 48.7% smear-positive and 51.3% smear-negative) and 69 control samples (24 from patients diagnosed with non-tuberculous mycobacteria infections and 45 from patients with suspected tuberculosis which was eventually ruled out). All samples were tested by two CE-marked assays (Xpert®MTB/RIF and AnyplexTM plus MTB/NTM) and two in-house assays targeting senX3-regX3 and the IS6110 gene. RESULTS: The detection limit ranged from 1.00E+01 fg for Anyplex, senX3-regX3 and IS6110 to 1.00E+04 fg for Xpert. All three Xpert, senX3-regX3 and IS6110 assays detected all 37 smear-positive cases. Conversely, Anyplex was positive in 34 (91.9%) smear-positive cases. In patients with smear-negative tuberculosis, differences were observed between the assays; Xpert detected 22 (56.41%) of the 39 smear-negative samples, Anyplex 24 (61.53%), senX3-regX3 28 (71.79%) and IS6110 35 (89.74%). Xpert and senX3-regX3 were negative in all control samples; however, the false positive rate was 8.7% and 13% for Anyplex and IS6110, respectively. The overall sensitivity was 77.6%, 85.7%, 77.3% and 94.7% and the specificity was 100%, 100%, 90.8% and 87.0% for the Xpert, senX3-regX3, Anyplex and IS6110 assays, respectively. CONCLUSION: Real-time PCR assays targeting IS6110 lack the desired specificity. The Xpert MTB/RIF and in-house senX3-regX3 assays are both sensitive and specific for the detection of MTBC in both pulmonary and extrapulmonary samples. Therefore, the real time PCR senX3-regX3 based assay could be a useful and complementary tool in the diagnosis of tuberculosis.

Comparative clinical study of different multiplex real time PCR strategies for the simultaneous differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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%). CONCLUSIONS/SIGNIFICANCE: 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.

Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

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.

Establishing the diagnosis of tuberculous vertebral osteomyelitis.

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.

Miliary pulmonary tuberculosis following intravesical BCG therapy: case report and literature review.

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.

Quantitative real-time polymerase chain reaction improves conventional microbiological diagnosis in an outbreak of brucellosis due to ingestion of unpasteurized goat cheese.

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.

Utilización de simuladores en técnicas endovasculares / No disponible

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Multiplex real-time polymerase chain reaction: a practical approach for rapid diagnosis of tuberculous and brucellar vertebral osteomyelitis.

STUDY DESIGN: Case-control study for assessing a diagnostic test. OBJECTIVE: 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). SUMMARY OF BACKGROUND DATA: 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. METHODS: 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. RESULTS: 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%). CONCLUSION: 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.

Rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis using a multiplex real-time PCR assay.

BACKGROUND: 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. METHODOLOGY: 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. CONCLUSIONS: 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.

PTPN22 C1858T polymorphism and human brucellosis.

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.

Pravastatin increases the expression of the tissue inhibitor of matrix metalloproteinase-1 and the oncogene Bax in human aortic abdominal aneurysms.

The effect of pravastatin on matrix metalloproteinase-9 (MMP-9) and the level of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 was studied in explants of human abdominal aortic aneurysm (AAA) obtained from 13 patients. The effect of pravastatin on the apoptotic status of human AAA explants was also examined. Total MMP-9 content did not differ in human AAA explants incubated in vitro in the presence or absence of pravastatin (10-6 mol/L) for 48 h. TIMP-1 levels were significantly increased in pravastatin-incubated AAA explants, but TIMP-2 production was not modified by pravastatin. Western blot experiments showed that, whereas Bax expression was increased in pravastatin-incubated AAA explants, the expression of Bcl-2 was not modified. On the other hand, the ratio of the expression of Bax to Bcl-2, an apoptotic index, was not modified by pravastatin. In the human AAA explants, the increase in Bax expression, but not the increase in TIMP-1 expression elicited by pravastatin, was reversed by L-mevalonate, a downstream HMG-CoA reductase metabolite, suggesting that the expression of Bax and TIMP-1 followed HMG-CoA reductase-dependent and -independent pathways, respectively. In conclusion, pravastatin increases both TIMP-1 and Bax expression in human AAA explants without changes in either MMP-9 activity or the apoptotic status.

Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis.

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.

Rapid diagnosis of Brucella epididymo-orchitis by real-time polymerase chain reaction assay in urine samples.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Comparison between LightCycler Real-Time Polymerase Chain Reaction (PCR) assay with serum and PCR-enzyme-linked immunosorbent assay with whole blood samples for the diagnosis of human brucellosis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

PCR-DIG ELISA with biotinylated primers is unsuitable for use in whole blood samples from patients with brucellosis.

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.

Inducible nitric oxide synthase promoter polymorphism in human brucellosis.

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.

Development and evaluation of a PCR-enzyme-linked immunosorbent assay for diagnosis of human brucellosis.

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.