Detection Of Bacterial Antigens Research Articles

Application of Nanobiosensors in Detection of Pathogenic Bacteria: An Update

Bacterial infections remain a critical public health concern worldwide, necessitating the development of efficient and sensitive diagnostic tools. Nanobiosensors, comprising nanomaterials, offer a novel approach to bacterial pathogen detection. The present review aimed to explore the current research and applications of nanobiosensors for bacterial pathogen detection. Recent discoveries in nanotechnology have facilitated the development of nanobiosensors with remarkable sensitivity and specificity. These nanoscale sensors are designed to detect specific bacterial pathogens through various mechanisms, including aptamers, antibodies, and molecular recognition elements. Furthermore, miniaturization and integration with microfluidic systems have enabled the rapid and point-of-care detection of bacterial infections. Incorporating nanomaterials such as carbon nanotubes, quantum dots, and graphene into biosensing platforms has significantly enhanced their performance, leading to ultrasensitive detection of bacterial antigens and nucleic acids. Additionally, using nanobiosensors with advanced analytical techniques, such as electrochemical, optical, and piezoelectric methods, has expanded the possibilities for accurate and real-time monitoring of bacterial pathogens. Nanobiosensors represent a promising frontier in the battle against bacterial infections. Their exceptional sensitivity, rapid response times, and potential for multiplexed detection make them invaluable tools for the early diagnosis and monitoring of bacterial pathogens. Developing cost-effective and portable nanobiosensors for resource-limited settings becomes increasingly possible as nanotechnology advances.

KLF2 Regulates NLRP3 Inflammasome Priming and Activation in Murine Myeloid Cells

Introduction: Krüppel-like factor-2 (KLF2) is a critical nodal transcription factor and a tonic repressor of myeloid cell activation. Loss of KLF2 is associated with a pro-inflammatory and pro-thrombotic state. Mice deficient in myeloid-KLF2 (MK2KO) experience significantly higher mortality from lipopolysaccharide (LPS), and have increased serum and tissue levels of pro-inflammatory cytokines such as IL-1β. IL-1β is a product of NLRP3 inflammasome priming and activation, which has been implicated in numerous inflammatory and immune-mediated diseases. Neutrophils are the first responders of the innate immune system and are responsible for bacterial antigen detection. We thus aimed to elucidate the role of KLF2 in the regulation of NLRP3 inflammasome activation and subsequent IL-1β release in the neutrophil compartment. Methods: 8-week old adult MK2KO ( Lyz2Cre/ Klf2fl/fl) and control ( Lyz2Cre) mice were used for obtaining neutrophils from the bone marrow. Neutrophils were plated in RPMI-1640 media and stimulated with 10 ng/mL of LPS for 1 hour (h) and 2 h, followed by nigericin for 30 minutes. LPS acts as a priming agent for NLRP3 inflammasome assembly, and nigericin is a potassium ionophore which enables the activation step and subsequent IL-1β release. Supernatant was used for IL-1β ELISA and cell lysate was used for protein extraction and Western blotting to quantify NLRP3 protein expression. GraphPad Prism was used for statistical analyses. Results: Neutrophils from both MK2KO and Cre mice have significantly increased production of IL-1β when stimulated with LPS followed by nigericin (Figure 1), and loss of KLF2 leads to significantly increased IL-1B release from neutrophils. MK2KO neutrophils also have significantly more NLRP3 protein expression as compared to Cre neutrophils at baseline, and in response to LPS and nigericin (Figure 2). This increase in NLRP3 protein expression was time dependent and dose-dependent (Figure 2). Conclusion: KLF2 negatively regulates the transcriptional expression of NLRP3 and loss of KLF2 leads to increased NLRP3 inflammasome priming, at baseline and in response to LPS. Loss of KLF2 also affects the second step of the NLRP3 inflammasome pathway - the activation step, leading to increased IL-1β release in neutrophils in adult mice. Ongoing studies are examining the mechanisms underlying KLF2 control of NLRP3 transcription and other aspects of inflammasome activation. Inflammasome activation has been implicated in sepsis-related mortality due to the pro-inflammatory cytokine storm, and thus modulating KLF2 might help in developing novel targets to reduce this burden of mortality.

Comparative Analysis of Gram Stain, Culture and Bacterial Antigen Detection in Cerebrospinal Fluid Samples for Laboratory Diagnosis of Acute Bacterial Meningitis in Pediatric Population in A Tertiary Care Hospital

Acute bacterial meningitis (ABM) is a life threatening infection in children, associated with long term complications and high mortality rate.1,2 Gram staining and culture are routinely used for diagnosis of ABM. Antigen detection by latex agglutination can provide prompt results thereby facilitating early initiation of empirical antibiotic treatment. To estimate the proportion of Laboratory confirmed cases among children admitted with clinical suspicion of acute bacterial meningitis in a tertiary care hospital. To compare and analyse the diagnostic efficacy of Culture, Gram stain and antigen detection by Latex agglutination in Cerebrospinal fluid (CSF) samples for laboratory detection of Acute bacterial meningitis. CSF samples from pediatric patients with clinical suspicion of ABM were analysed by Gram stain, culture and Antigen detection by Latex agglutination method. Results were recorded and analysed. Of the 50 clinically suspected cases, 13(26%) were confirmed as Acute bacterial meningitis by laboratory investigations. Among the organisms identified, Streptococcus pneumoniae was the most common isolate in 5(38.46%) cases followed by Neisseria meningitidis, Klebsiella pneumoniae and Acinetobacter baumannii in 2(15.38%) cases each and Escherichia coli and Group B Streptococcus in 1(7.69%) case each. Among the confirmed cases, 7(53%) samples showed culture positivity while Gram stain identified 8(61.53%)cases. Latex agglutination test showed positivity in 9(69.23%) cases. In life threatening infections like acute bacterial meningitis, where early diagnosis and prompt treatment is of utmost importance, Latex agglutination test can provide results within minutes facilitating early initiation of empirical therapy, making it an effective adjunct to gram stain and culture.

Localization of Pasteurella multocida antigens in the brains of pigs naturally infected with Pasteurellosis revealing a newer aspect of pathogenesis

Pasteurella multocida is an economically important respiratory pathogen of pigs confronting swine industry worldwide. Despite extensive research over the decades, its pathogenesis is still poorly understood. Recent reports have demonstrated the nervous system affection as a newer aspect of pathogenesis by Pasteurella multocida type B:2 in Haemorrhagic Septicemia, but there are no reports of the involvement of nervous system by P. multocida in pigs. Therefore, the study was aimed to explore the neurovirulence of Pasteurella multocida in naturally infected pigs. A total of 15 brains were collected from the natural cases of pig mortality suggestive of Pasteurellosis. Grossly, the leptomeninges were markedly congested and brains were oedematously swollen. Histologically, there was moderate to severe fibrinohaemorrhagic and mononuclear cells exudates present in the leptomeningeal tissue and cerebrospinal spaces. Similar vascular inflammatory lesions (perivascular and perineuronal) along with gliosis, neuronal degeneration and necrosis were noted in various subanatomical sites of the brain (cerebrum, cerebellum, brainstem and spinal cord). The culture and biochemical tests showed the presence of P. multocida within the brain tissue. P. multocida type specific antibody staining in the brain tissues revealed intense distribution of antigens in the inflammatory exudates of meningeal vessels, neurons, glial cells and endothelial cells of the blood vessels contributing its association with neuropathological lesions. Pasteurella multocida specific PCR amplification of capsular polysaccharide gene yielded 460 bp and multiplex PCR showed the involvement of capsular serogroups A &D. All the isolates showed the presence of 10 genes for virulence factors. The disease confirmation of both serotypes was proven by Koch's postulates using Swiss albino mice. Further, histopathological brain lesions along with the immunohistochemical detection of bacterial antigens were corroborated with natural cases of P. multocida as described above. To the best of our knowledge, we first time report the neuroinvasion of P. multocida in naturally infected pigs.

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG.

Oriented immobilization of antibodies on a sensor surface is critical for enhancing both the antigen-binding capacity and the sensitivity of immunosensors. In this study, we describe a strategy to adsorb immunoglobulin G (IgG) anti-Brucella antibodies onto a silicon surface, oriented by protein A obtained from Staphylococcus aureus (SpA). X-ray photoelectron spectroscopy and atomic force microscopy were used to characterize topographically, morphologically, and chemical changes of the sensor functionalization. The activity of the biosensor was assessed by confocal microscopy, scanning electronic microscopy, and bacteria capture assays (BCA). According to the BCA, the efficiency of Brucella abortus detection with the SpA-IgG anti Brucella biosensor was three-fold higher than that of the random orientated IgG anti Brucella biosensor. The limit of detection was 1 × 106 CFU/ml. These data show that the orientation of antibodies immobilization is crucial to developing immunosensors for bacterial antigen detection as Brucella spp and improve its sensibility level. Functionalization with protein A increases Brucella detection by an antibody-coated surface. Functionalized silicon surface for Brucella detection was characterized by atomic force microscopy, X-ray photoelectron spectroscopy and confocal microscopy.

The structure of the C-terminal domain of the nucleoprotein from the Bundibugyo strain of the Ebola virus in complex with a pan-specific synthetic Fab.

The vast majority of platforms for the detection of viral or bacterial antigens rely on immunoassays, typically ELISA or sandwich ELISA, that are contingent on the availability of suitable monoclonal antibodies (mAbs). This is a major bottleneck, since the generation and production of mAbs is time-consuming and expensive. Synthetic antibody fragments (sFabs) generated by phage-display selection offer an alternative with many advantages over Fabs obtained from natural antibodies using hybridoma technology. Unlike mAbs, sFabs are generated using phage display, allowing selection for binding to specific strains or for pan-specificity, for identification of structural epitopes or unique protein conformations and even for complexes. Further, they can easily be produced in Escherichia coli in large quantities and engineered for purposes of detection technologies and other applications. Here, the use of phage-display selection to generate a pan-specific Fab (MJ20), based on a Herceptin Fab scaffold, with the ability to bind selectively and with high affinity to the C-terminal domains of the nucleoproteins (NPs) from all five known strains of the Ebola virus is reported. The high-resolution crystal structure of the complex of MJ20 with the antigen from the Bundibugyo strain of the Ebola virus reveals the basis for pan-specificity and illustrates how the phage-display technology can be used to manufacture suitable Fabs for use in diagnostic or therapeutic applications.

Novel aptamer-linked nanoconjugate approach for detection of waterborne bacterial pathogens: an update

The lack of microbiologically safe water in underdeveloped nations is the prime cause of infectious disease outbreaks. The need for the specific identification and detection of microorganisms encourages the development of advanced, rapid, sensitive and highly specific methods for the monitoring of pathogens and management of potential risk to human health. The rapid molecular assays based on detection of specific molecular signatures offer advantages over conventional methods in terms of specificity and sensitivity but require complex instrumentation and skilled personnel. Nanotechnology is an emerging area and provides a robust approach for the identification of pathogenic microorganism utilizing the peculiar properties of nanomaterials, i.e. small size (1–100 nm) and large surface area. This emerging technology promises to fulfill the urgent need of a novel strategy to enhance the bacterial identification and quantitation in the environment. In this context, the peculiar properties of gold nanoparticles, their plasmonic shifts, and changes in magnetic properties have been utilized for the simple and cost-effective detection of bacterial nucleic acids, antigens and toxins with quite improved sensitivity. One of the promising leads to develop an advance detection method might be the coupling of nucleic acid aptamers (capable of interacting specifically with bacteria, protozoa, and viruses) with nanomaterials. Such aptamer-nano conjugate can be used for the specific recognition of infectious agents in different environmental matrices. This review summarizes the application of nanotechnology in the area of pathogen detection and discusses the prospects of coupling nucleic acid aptamers with nanoparticles for the specific detection of targeted pathogens.

SIV Infection-Mediated Changes in Gastrointestinal Bacterial Microbiome and Virome Are Associated with Immunodeficiency and Prevented by Vaccination.

AIDS caused by simian immunodeficiency virus (SIV) infection is associated with gastrointestinal disease, systemic immune activation, and, in cross-sectional studies, changes in the enteric virome. Here we performed a longitudinal study of a vaccine cohort to define the natural history of changes in the fecal metagenome in SIV-infected monkeys. Matched rhesus macaques were either uninfected or intrarectally challenged with SIV, with a subset receiving the Ad26 vaccine, an adenovirus vector expressing the viral Env/Gag/Pol antigens. Progression of SIV infection to AIDS was associated with increased detection of potentially pathogenic viruses and bacterial enteropathogens. Specifically, adenoviruses were associated with an increased incidence of gastrointestinal disease and AIDS-related mortality. Viral and bacterial enteropathogens were largely absent from animals protected by the vaccine. These data suggest that the SIV-associated gastrointestinal disease is associated with the presence of both viral and bacterial enteropathogens and that protection against SIV infection by vaccination prevents enteropathogen emergence.

Detection of bacterial antigens and Alzheimer's disease-like pathology in the central nervous system of BALB/c mice following intranasal infection with a laboratory isolate of Chlamydia pneumoniae.

Pathology consistent with that observed in Alzheimer’s disease (AD) has previously been documented following intranasal infection of normal wild-type mice with Chlamydia pneumoniae (Cpn) isolated from an AD brain (96-41). In the current study, BALB/c mice were intranasally infected with a laboratory strain of Cpn, AR-39, and brain and olfactory bulbs were obtained at 1–4 months post-infection (pi). Immunohistochemistry for amyloid beta or Cpn antigens was performed on sections from brains of infected or mock-infected mice. Chlamydia-specific immunolabeling was identified in olfactory bulb tissues and in cerebrum of AR-39 infected mice. The Cpn specific labeling was most prominent at 1 month pi and the greatest burden of amyloid deposition was noted at 2 months pi, whereas both decreased at 3 and 4 months. Viable Cpn was recovered from olfactory bulbs of 3 of 3 experimentally infected mice at 1 and 3 months pi, and in 2 of 3 mice at 4 months pi. In contrast, in cortical tissues of infected mice at 1 and 4 months pi no viable organism was obtained. At 3 months pi, only 1 of 3 mice had a measurable burden of viable Cpn from the cortical tissues. Mock-infected mice (0 of 3) had no detectable Cpn in either olfactory bulbs or cortical tissues. These data indicate that the AR-39 isolate of Cpn establishes a limited infection predominantly in the olfactory bulbs of BALB/c mice. Although infection with the laboratory strain of Cpn promotes deposition of amyloid beta, this appears to resolve following reduction of the Cpn antigen burden over time. Our data suggest that infection with the AR-39 laboratory isolate of Cpn results in a different course of amyloid beta deposition and ultimate resolution than that observed following infection with the human AD-brain Cpn isolate, 96-41. These data further support that there may be differences, possibly in virulence factors, between Cpn isolates in the generation of sustainable AD pathology.

Helicobacter pylori infection and allergic diseases in pediatric age. A retrospective study

s / Digestive and Liver Disease 46 (2014) e85–e127 e119 HELICOBACTER PYLORI INFECTION AND ALLERGIC DISEASES IN PEDIATRIC AGE. A RETROSPECTIVE STUDY Angela Lanzafame ∗, Stefania Tomarchio, Martina Filippelli, Salvatore Leonardi, Elena Lionetti Unit of Pediatric Pneumo-Allergology and Cystic Fibrosis, University of Catania, Catania, Italy Objective: The recent spread of allergic diseases in industrialized countries has been associated with a reduced prevalence of infectious diseases. To evaluate the hypothesis that Helicobacter pylori (Hp) infection can protect from allergic diseases, we determined the prevalence of Hp infection and atopic diseases in a pediatric population. Methods: 95 patients [male: 49 (51.6%), median age: 8 years, range: 1–18 years] living in the province of Catania were enrolled. The status of atopy was evaluated by performing skin prick test and radioallergosorbent test for principal allergens; Hp status was assayed through the detection of bacterial antigens in diluted fecal samples. Results: Overall, 17 out of 95 children enrolled (17.9%) were positive forHp infection; 4.8% (2/42) of atopic patientswereHppositive, as compared to28.3% (15/53) of non-atopic (P<0.01,OR=0.1). Conclusions: As in other geographic areas with a high standard of living, even in the province of Catania the prevalence ofHp infection in the 1–18 aged population is low. Our study shows that the prevalence ofHp infection is significantly higher in non-atopic subjects, confirming a protective role of Hp infection versus atopic diseases. http://dx.doi.org/10.1016/j.dld.2014.07.143 EVALUATION OF MAGNETIC RESONANCE ENTEROGRAPHY IN PEDIATRIC CROHN DISEASE: A RETROSPECTIVE PILOT-STUDY Martina Vallorani1,∗, Alessandra Palpacelli 1, Matilde Rossi1, Simona Gatti 1, Giovanni Pieroni2, Veronica Albano1, Carlo Catassi1 1 Dipartimento di Pediatria, Ospedale Salesi/Universita Politecnica delle Marche, Ancona, Italy 2 Dipartimento di Radiologia, Ospedale Salesi/Universita Politecnica delle Marche, Ancona,

Detection of potential microbial antigens by immuno-PCR (PCR-amplified immunoassay)

Immuno-PCR (PCR-amplified immunoassay; I-PCR) is a novel ultrasensitive method combining the versatility of ELISA with the sensitivity of nucleic acid amplification of PCR. The enormous exponential amplification power of PCR in an I-PCR assay leads to at least a 10(2)-10(4)-fold increase in sensitivity compared with an analogous ELISA. I-PCR has been used to detect many biological molecules such as proto-oncogenes, toxins, cytokines, hormones, and biomarkers for autoimmune and Alzheimer's diseases, as well as microbial antigens and antibodies, and it can be adapted as a novel diagnostic tool for various infectious and non-infectious diseases. Quantitative real-time I-PCR has the potential to become the most analytically sensitive method for the detection of proteins. The sensitivity and specificity of a real-time I-PCR assay can be enhanced further with the use of magnetic beads and nanoparticles. This review is primarily focused on the detection of potential viral, bacterial and parasitic antigens by I-PCR assay, thus enabling their application for immunological research and for early diagnosis of infectious diseases.

Study of bacterial meningitis in children below 5 years with comparative evaluation of gram staining, culture and bacterial antigen detection.

Bacterial meningitis is one of the most serious infections seen in infants and children, which is associated with acute complications and chronic morbidity. Infections of Central Nervous System (CNS) still dominate the scene of childhood neurological disorders in most of the developing tropical countries. To isolate, identify and determine the antibiotic susceptibility patterns of pathogens associated with bacterial meningitis. We also aimed to comparatively evaluate of Gram staining, culture and bacterial antigen detection in cerebrospinal fluid samples. Present comparative study included 100 CSF samples of children below the age of 5 years, who were clinically suspected meningitis cases. The samples were subjected to Gram staining, culture and Latex agglutination test (LAT). The organisms isolated in the study were characterized and antibiotic susceptibility test was done according to standard guidelines. It was done by using Gaussian test. Of the 100 cases, 24 were diagnosed as Acute bacterial meningitis (ABM) cases by. Gram staining, culture and latex agglutination test. 21 (87.5%) cases were culture positive, with 2 cases being positive for polymicrobial isolates. Gram staining was positive in 17 (70.53%) cases and LAT was positive in 18 (33.33%) cases. Streptococcus pneumoniae was the predominant organism which was isolated and it was sensitive to antibiotics. In the present study, male to female ratio was 1.27:1, which showed a male preponderance. With the combination of Gram staining, culture, and LAT, 100% sensitivity and specificity can be achieved (p < 0.001). Gram staining and LAT can detect 85% of cases of ABM. Bacterial meningitis is a medical emergency and making an early diagnosis and providing treatment early are life saving and they reduce chronic morbidity.

Neurocysticercosis in a 23-year-old Chinese man

Patient: Male, 23Final Diagnosis: NeurocysticerosisSymptoms: Diplopia • fever • headache • insomnia • neck stiffness • vomitingMedication: AlbendazoleClinical Procedure: —Specialty: NeurologyObjective:Challenging differential diagnosisBackground:Neurocysticercosis is a brain infection caused by the larval stage of the tapeworm Taenia (T.) solium. It is the most important parasitic disease of the human central nervous system and represents the most common cause of acquired epilepsy in developing countries.Case Report:Here, we report the case of a 23-year-old Chinese man who presented to the emergency department with a 7-day history of helmet headache radiating to the nuchal region and associated with vomiting, confusion, and fever. Cerebrospinal fluid (CSF) was clear, with increased pressure, lymphocytic pleocytosis, decreased glucose, and increased protein levels. Bacterial antigen detection test on CSF was negative, as were CSF bacterial and fungal cultures. Despite broad-spectrum antibiotic and antiviral therapy, the patient still complained of insomnia, diplopia, headache, neck stiffness, and pain in the sacral region. A second LP was performed and CSF had the same characteristics as the first LP. A brain and spinal cord MRI revealed widespread arachnoiditis and small septated cysts with CSF-like signal in the cisterna magna, within the fourth ventricle, and at the level of L3–L4. Cysticercus-specific immunoglobin G antibodies were detected by ELISA in the CSF. The patient received albendazole (15 mg/kg/day) and dexamethasone (5 mg/day) for 4 weeks, with progressive resolution of neurological symptoms.Conclusions:This case shows that, even if rare, neurocysticercosis may be responsible for meningeal symptoms and should be included in the differential diagnosis, especially in patients from endemic countries.

Clinical and bacteriological spectrum of community-acquired acute bacterial meningitis in adults at a tertiary care hospital in northern India

Objective : To evaluate the clinical and bacteriological spectrum of community-acquired acute bacterial meningitis (ABM) in an adult population at Lok Nayak Hospital, New Delhi, a tertiary care hospital in northern India. Materials and Methods: Cerebrospinal fluid (CSF) samples from 120 clinically suspected cases of pyogenic meningitis were processed for cell counts, differential counts, bacterial stain and culture, antibiotic susceptibility test, and bacterial antigen detection by latex agglutination test (LAT) in CSF, serum and urine. Results : CSF LAT was positive in 56/120 cases of ABM. Streptococcus pneumoniae was the predominant pathogen in 41 of these 120 cases i.e. 34.0% of all cases. Neisseria meningitidis accounted for 24 cases i.e. 20% of all cases. Staphylococcus aureus was isolated in one case. Bacterial cultures were positive in 22 (18.3%) cases only. Conclusions: Evidence for presence of microbial activity could be detected in 44.1% cases by LAT, whereas a direct microbiological confirmation could be documented in only 22 cases i.e. 18.3%. Streptococcus pneumoniae remains the major etiological agent of ABM in our study. Penicillin resistance was detected in 50% of cases among the isolates which came positive by culture. Neisseria meningitidis was detected in 24 cases. But since none were cultured, sensitivity patterns could not be correlated. LAT is a convenient and rapid test to support bacterial etiology in ABM. In remote situations, their use in serum or urine needs to be considered after evaluation in longer studies. Further research should focus on the preventable aspects of ABM, especially pneumococcal and meningococcal vaccines, to potentially protect susceptible groups.

Role of Immunological Test in Rapid Diagnosis of Acute Bacterial Meningitis

Introduction:Conventional diagnostic methods for bacterial meningitis are frequently not rapid or sensitive enough to guide initial antimicrobial therapy. As bacterial meningitis is a medical emergency, rapid diagnosis and prompt treatment remains the cornerstone in the management of patients. Therefore, the study was carried out for bacterial antigen detection by Latex particle agglutination test and compared with culture and Gram stain.Methods: The study, carried out for a period for one year, included 85 cerebrospinal fluid (CSF) samples of clinically suspected meningitis cases in children below 5 years. CSF samples were tested by Latex particle agglutination test and were compared with culture and Gram stain.Results: Of the 85 cases studied, there were 32 laboratory confirmed cases. Of these, 27 cases (84.37 %) were culture positive, 18 cases (56.25 %) were Gram stain positive, and 10 cases (31.25 %) were Latex particle agglutination test positive. Streptococcus pneumoniae was the most common organism identified by the three tests.Conclusion: Antigen detection by Latex particle agglutination is a useful adjunct to routine Gram stain and can detect non-viable bacteria in CSF, thus permitting the detection of bacterial antigen in patients pretreated with antibiotics when culture is negative.

Rapid diagnosis of bacteric meningitis

Gram stain and bacterial antigen detection are economic and rapid tests for the identification of the bacteria in cerebrospinal fluid (CSF) for the diagnosis of meningitis. Even if culture is the gold standard of these diagnosis, it requires long time (growth time). The purpose of our study was to compare in the laboratory diagnosis of meningitis real time PCR (Eurospital, Italy). with standard methods. Samples were collected in patients referred to L. Sacco Universital Hospital (range of age: 16 to 70 years) from September 2008 to July 2009. 9 out of 130 CFS samples investigated were positive in PCR but they produced different results to the other methods used.

La tuberculose primitive ORL extraganglionnaire

Objectives The aim of this sinusonasal study was to study several locations of ENT extranodal tuberculosis. Material and methods Our study focused on eight cases of tuberculosis in the cervicofacial region gathered in the ENT department of the Rabat Specialization Hospital (Morocco) between August 2004 and August 2006. They comprise two cases of rhinopharyngeal tuberculosis, two cases of tuberculosis, two cases of endobuccal tuberculosis (mandibular and lingual locations), and two cases of glandular tuberculosis (thyroid and parotid locations). Results These locations are rarely affected by tuberculosis. The clinical signs as well as radiology and endoscopy are not specific. The final diagnosis is based on histology. Bacillus cultures are necessary when caseum tuberculosis is not found, causing problems of differential diagnosis with granulomatous and malignant lesions. PCR provides quick bacterial antigen detection when cultures are negative. The treatment provides a quick and complete recovery while recurrences are infrequent. Conclusion ENT tuberculosis is rare if excluding the nodal locations. It should be mentioned as a differential diagnosis, especially in endemic areas. Most often, this is a surprise diagnosis.

Impact of Haemophilus influenzae type b conjugate vaccine on bacterial meningitis in the Dominican Republic

Widespread use of Haemophilus influenzae type b (Hib) vaccines has dramatically reduced the burden of Hib disease throughout the Americas. Few studies have evaluated the impact of Hib vaccination on non-culture-confirmed disease. This study analyzed trends in probable bacterial meningitis before and after the introduction of Hib vaccine in the Dominican Republic and estimated vaccine effectiveness against Hib meningitis. Meningitis cases among children < 5 years of age were identified from admission records of the main pediatric hospital in Santo Domingo during 1998-2004. Laboratory criteria were used to classify meningitis cases with probable bacterial etiology; confirmed cases had positive bacterial culture or antigen detection in cerebrospinal fluid. Cumulative incidence rates of confirmed and probable bacterial meningitis were calculated for children living in the National District. Confirmed cases of Hib meningitis were enrolled in a case-control study with age- and neighborhood-matched control children to calculate vaccine effectiveness. Before vaccine introduction, annual rates of meningitis with probable bacterial etiology were 49 cases per 100 000 children < 5 years old; Hib accounted for 60% of confirmed bacterial cases. During 2002-2004, after vaccine introduction, annual rates of probable bacterial meningitis were 65% lower at 16 cases per 100 000, and Hib accounted for 26% of confirmed cases. Rates of Hib meningitis and probable bacterial meningitis with no determined etiology declined by 13 and 17 cases per 100 000, respectively. Introduction of Hib vaccine substantially reduced the incidence of confirmed and probable bacterial meningitis in the Dominican Republic. The estimated impact of Hib vaccination was twice as great when non-culture-confirmed disease was included.

Sensitivity by combination: immuno-PCR and related technologies

The versatility of immunoassays for the detection of antigens can be combined with the signal amplification power of nucleic acid amplification techniques in a broad range of innovative detection strategies. This review summarizes the spectrum of both, DNA-modification techniques used for assay enhancement and the resulting key applications. In particular, it focuses on the highly sensitive immuno-PCR (IPCR) method. This technique is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR or related techniques for signal generation and read-out. Various strategies for the combination of antigen detection and nucleic acid amplification are discussed with regard to their laboratory analytic performance, including novel approaches to the conjugation of antibodies with DNA, and alternative pathways for signal amplification and detection. A critical assessment of advantages and drawbacks of these methods for a number of applications in clinical diagnostics and research is conducted. The examples include the detection of viral and bacterial antigens, tumor markers, toxins, pathogens, cytokines and other targets in different biological sample materials.

Homologous high-throughput expression and purification of highly conserved E coli proteins

BackgroundGenetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s) involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies.ResultsAs a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen) destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP), N-utilizing substance A (NusA), or glutathione S-transferase (GST) to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS). Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500 μg.ConclusionHere, we report a cost-efficient procedure to produce around 200 soluble recombinant E. coli proteins in large-scale, including removal of LPS by polymyxin B coated beads for subsequent use of the proteins in downstream immunological studies.