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.

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.

Diagnostic yield of a PCR assay in focal complications of brucellosis.

In order to evaluate the diagnostic yield of a PCR assay for patients with focal complications of brucellosis, we studied by PCR and by conventional microbiological techniques 34 nonblood samples from 32 patients with different focal forms of brucellosis. The samples from patients with brucellosis were paired to an equal number of control samples from the same locations of patients whose illnesses had different etiologies. Thirty-three of the 34 nonblood samples (97%) from the brucellosis patients were positive by PCR, whereas Brucella spp. were isolated from only 29.4% of the conventional cultures. For 11.4% of the patients, the confirmatory serological tests were either negative or showed titers below the diagnostic range. Two patients (6.2%) from the control group, both with tuberculous vertebral osteomyelitis, had a positive PCR result. The brucella PCR of blood from these two patients was also positive, and the two strains of Mycobacterium tuberculosis isolated were analyzed by the brucella PCR, with no evidence of amplification. These results show that the PCR assay is far more sensitive than conventional cultures, and this, coupled with its speed and reduction in risk to laboratory workers, makes this technique a very useful tool for the diagnosis of focal complications of brucellosis.

Liver abscess due to Salmonella enteritidis in a returned traveler with HIV infection: case report and review of the literature

Bacteremia due to non-typhi Salmonella is more frequent in patients infected with the human immunodeficiency virus (HIV). However, focal complications have been rarely described. We report a case of liver abscess due to Salmonella enteritidis in an HIV-infected patient who recently returned to Sao Paulo, Brazil, from a trip in the Caribbean. A good clinical and radiological response was seen with both percutaneous catheter drainage and antibiotic treatment. To our knowledge, this is the first culture proven case of non-typhi Salmonellaliver abscess in an HIV-infected patient in Brazil.

Neurologic cytomegalovirus complications in patients with AIDS: retrospective review of 13 cases and review of the literature

Neurological disorders caused by Cytomegalovirus (CMV) in patients with Acquired Immunodeficiency Syndrome (AIDS) are rarely reported in the Highly Active Antiretroviral Therapy (HAART) period. The objective of this study was to describe the main clinical and laboratory features of patients with CMV-related neurological complications in HIV-infected patients admitted to a referral center in São Paulo, Brazil. CMV disease requires the identification of the virus in the cerebrospinal fluid (CSF) using Polymerase Chain Reaction (PCR). Thirteen cases were identified between January, 2004 and December, 2008. The median age of patients was 38 years and nine (69%) were men. At admission all patients were aware of their HIV status and only four (31%) patients were on HAART. Patients who were not on antiretroviral therapy before admission received HAART while inpatients. CMV disease was the first AIDS-defining illness in eight (62%) patients. The neurologic syndromes identified were diffuse encephalitis (n = 7; 62%), polyradiculopathy (n = 7; 54%), focal encephalitis (rhombencephalitis) (n = 1; 8%), and ventriculo-encephalitis (n = 1; 8%). Seven (54%) patients presented extra-neural CMV disease and four (31%) had retinitis. The median of CD4+ T-cell count was 13 cells/µL (range: 1-124 cells/µL). Overall in-hospital mortality was 38%. Eight patients used ganciclovir or foscarnet (in-hospital mortality: 50%) and five patients used ganciclovir and foscarnet (in-hospital mortality: 20%). None of the patients fulfilled the diagnosis criteria of immune reconstitution inflammatory syndrome. Four patients were lost to follow-up, and three patients presented immune recovery and discontinued secondary prophylaxis. Although infrequent, distinct neurological syndromes caused by CMV continue to cause high mortality among AIDS patients. Survival depends upon the use of effective antiviral therapy against CMV and the early introduction of HAART.

Initial assessment of safety and clinical feasibility of irreversible electroporation in the focal treatment of prostate cancer.

BACKGROUND: To evaluate the safety and clinical feasibility of focal irreversible electroporation (IRE) of the prostate. METHODS: We assessed the toxicity profile and functional outcomes of consecutive patients undergoing focal IRE for localised prostate cancer in two centres. Eligibility was assessed by multi-parametric magnetic resonance imaging (mpMRI) and targeted and/or template biopsy. IRE was delivered under transrectal ultrasound guidance with two to six electrodes positioned transperineally within the cancer lesion. Complications were recorded and scored accordingly to the NCI Common Terminology Criteria for Adverse Events; the functional outcome was physician reported in all patients with at least 6 months follow-up. A contrast-enhanced MRI 1 week after the procedure was carried out to assess treatment effect with a further mpMRI at 6 months to rule out evidence of residual visible cancer. RESULTS: Overall, 34 patients with a mean age of 65 years (s.d.=±6) and a median PSA of 6.1 ng ml(-1) (interquartile range (IQR)= 4.3-7.7) were included. Nine (26%), 24 (71%) and 1 (3%) men had low, intermediate and high risk disease, respectively (D'Amico criteria). After a median follow-up of 6 months (range 1-24), 12 grade 1 and 10 grade 2 complications occurred. No patient had grade >/= 3 complication. From a functional point of view, 100% (24/24) patients were continent and potency was preserved in 95% (19/20) men potent before treatment. The volume of ablation was a median 12 ml (IQR=5.6-14.5 ml) with the median PSA after 6 months of 3.4 ng ml(-1) (IQR=1.9-4.8 ng ml(-1)). MpMRI showed suspicious residual disease in six patients, of whom four (17%) underwent another form of local treatment. CONCLUSIONS: Focal IRE has a low toxicity profile with encouraging genito-urinary functional outcomes. Further prospective development studies are needed to confirm the functional outcomes and to explore the oncological potential.

Preliminary Experience in Treatment of Papillary and Macular Retinoblastoma: Evaluation of Local Control and Local Complications After Treatment with Linear Accelerator-Based Stereotactic Radiotherapy with Micromultileaf Collimator as Second-Line or Salvage Treatment after chemotherapy.

PURPOSE: To determine the local control and complication rates for children with papillary and/or macular retinoblastoma progressing after chemotherapy and undergoing stereotactic radiotherapy (SRT) with a micromultileaf collimator. METHODS AND MATERIALS: Between 2004 and 2008, 11 children (15 eyes) with macular and/or papillary retinoblastoma were treated with SRT. The mean age was 19 months (range, 2-111). Of the 15 eyes, 7, 6, and 2 were classified as International Classification of Intraocular Retinoblastoma Group B, C, and E, respectively. The delivered dose of SRT was 50.4 Gy in 28 fractions using a dedicated micromultileaf collimator linear accelerator. RESULTS: The median follow-up was 20 months (range, 13-39). Local control was achieved in 13 eyes (87%). The actuarial 1- and 2-year local control rates were both 82%. SRT was well tolerated. Late adverse events were reported in 4 patients. Of the 4 patients, 2 had developed focal microangiopathy 20 months after SRT; 1 had developed a transient recurrence of retinal detachment; and 1 had developed bilateral cataracts. No optic neuropathy was observed. CONCLUSIONS: Linear accelerator-based SRT for papillary and/or macular retinoblastoma in children resulted in excellent tumor control rates with acceptable toxicity. Additional research regarding SRT and its intrinsic organ-at-risk sparing capability is justified in the framework of prospective trials.

Stimulus-induced, sleep-bound, focal seizures: a case report.

STUDY OBJECTIVES: In nocturnal frontal lobe epilepsy (NFLE), seizures occur almost exclusively during NREM sleep. Why precisely these seizures are sleep-bound remains unknown. Studies of patients with nonlesional familial forms of NFLE have suggested the arousal system may play a major role in their pathogenesis. We report the case of a patient with pharmaco-resistant, probably cryptogenic form of non-familial NFLE and strictly sleep-bound seizures that could be elicited by alerting stimuli and were associated with ictal bilateral thalamic and right orbital-insular hyperperfusion on SPECT imaging. DESIGN: Case report. SETTING: University Hospital Zurich. PATIENTS OR PARTICIPANTS: One patient with pharmaco-resistant epilepsy. CONCLUSION: This case shows that the arousal system plays a fundamental role also in cryptogenic non-familial forms of NFLE.

Multicontrast MRI Quantification of Focal Inflammation and Degeneration in Multiple Sclerosis.

INTRODUCTION: Local microstructural pathology in multiple sclerosis patients might influence their clinical performance. This study applied multicontrast MRI to quantify inflammation and neurodegeneration in MS lesions. We explored the impact of MRI-based lesion pathology in cognition and disability. METHODS: 36 relapsing-remitting MS subjects and 18 healthy controls underwent neurological, cognitive, behavioural examinations and 3 T MRI including (i) fluid attenuated inversion recovery, double inversion recovery, and magnetization-prepared gradient echo for lesion count; (ii) T1, T2, and T2(*) relaxometry and magnetisation transfer imaging for lesion tissue characterization. Lesions were classified according to the extent of inflammation/neurodegeneration. A generalized linear model assessed the contribution of lesion groups to clinical performances. RESULTS: Four lesion groups were identified and characterized by (1) absence of significant alterations, (2) prevalent inflammation, (3) concomitant inflammation and microdegeneration, and (4) prevalent tissue loss. Groups 1, 3, 4 correlated with general disability (Adj-R (2) = 0.6; P = 0.0005), executive function (Adj-R (2) = 0.5; P = 0.004), verbal memory (Adj-R (2) = 0.4; P = 0.02), and attention (Adj-R (2) = 0.5; P = 0.002). CONCLUSION: Multicontrast MRI provides a new approach to infer in vivo histopathology of plaques. Our results support evidence that neurodegeneration is the major determinant of patients' disability and cognitive dysfunction.

Neurotoxicity of advanced glycation endproducts during focal stroke and neuroprotective effects of aminoguanidine.

Cerebral infarction (stroke) is a potentially disastrous complication of diabetes mellitus, principally because the extent of cortical loss is greater in diabetic patients than in nondiabetic patients. The etiology of this enhanced neurotoxicity is poorly understood. We hypothesized that advanced glycation endproducts (AGEs), which have previously been implicated in the development of other diabetic complications, might contribute to neurotoxicity and brain damage during ischemic stroke. Using a rat model of focal cerebral ischemia, we show that systemically administered AGE-modified bovine serum albumin (AGE-BSA) significantly increased cerebral infarct size. The neurotoxic effects of AGE-BSA administration were dose- and time-related and associated with a paradoxical increase in cerebral blood flow. Aminoguanidine, an inhibitor of AGE cross-linking, attenuated infarct volume in AGE-treated animals. We conclude that AGEs may contribute to the increased severity of stroke associated with diabetes and other conditions characterized by AGE accumulation.

Integrated Proteomics Identified Up-regulated Focal Adhesion-mediated Proteins In Human Squamous Cell Carcinoma In An Orthotopic Murine Model.

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.

Bleeding Complications In Dengue Are Not Associated With Significant Changes In The Modulators Of The Endothelial Barrier.

Bleeding complications in dengue may occur irrespective of the presence of plasma leakage. We compared plasma levels of modulators of the endothelial barrier among three dengue groups: bleedings without plasma leakage, dengue hemorrhagic fever, and non-complicated dengue. The aim was to evaluate whether the presence of subtle alterations in microvascular permeability could be detected in bleeding patients. Plasma levels of VEGF-A and its soluble receptors were not associated with the occurrence of bleeding in patients without plasma leakage. These results provide additional rationale for considering bleeding as a complication independent of endothelial barrier breakdown, as proposed by the 2009 WHO classification.

Focal Osteoporotic Bone Marrow Defect Mimicking A Mandibular Cystic Lesion.

An unusual presentation of a focal osteoporotic bone marrow defect (FOBMD) of the mandible mimicking a cystic lesion is documented. A definitive diagnosis could be established only on the basis of the histopathologic evaluation. A 66-year-old Brazilian woman was referred by her dentist for well-defined radiolucency of the mandibular molar region suggesting a cystic lesion of odontogenic origin. The computed tomography scan confirmed that the lesion did not affect the corticals. The biopsy confirmed the diagnosis of FOBMD. The diagnostic difficulty in the current case is obvious, because FOBMD, usually exhibiting an ill-defined radiolucency, is seldom suspected preoperatively when a differential diagnosis is considered for focal well-defined radiolucent areas in the jaws.

Use Of Electrocautery For Coagulation And Wound Complications In Caesarean Sections.

To evaluate the safety of electrocautery for coagulation during Caesarean sections. A randomized, controlled, clinical pilot study was performed at a university maternity hospital. After admission for delivery and decision to perform a C-section, volunteers were randomized to either the intervention group (use of electrocautery for coagulation) or nonintervention group. The women were examined at the time of postpartum discharge (day 3), at days 7 to 10, and again at days 30 to 40 for signs of infection, hematoma, seroma, or dehiscence. Data were analyzed using an intention-to-treat analysis, and risk ratios were calculated. No significant differences were found between the two groups. Only 2.8% of patients in the intervention group developed surgical wound complications during hospitalization. However, 7 to 10 days following discharge, these rates reached 23.0% and 15.4% in the intervention and nonintervention groups, respectively (RR = 1.50, 95% CI = 0.84-2.60). Further studies should confirm whether the use of electrocautery for coagulation does not increase the risk of surgical wound complications in patients undergoing Caesarean sections.