Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance

Highly Sensitive Detection of Staphylococcus aureus Directly from Patient Blood

Background: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing – polymerase chain reaction (PCR) platform as a model diagnostic system. Methodology/Principal Findings: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced limit of detection (LOD) of 5 genomic copies per reaction or 10 colony forming units (CFU) per ml blood over 50 copies per reaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance of the bacteria in different components of the blood (white blood cells (WBCs), plasma or whole blood), using the above assays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in maximum PCR positives with sodA assay (90 % positive) as opposed to cell-associated bacteria (in WBCs) (71 % samples positive) or free bacterial DNA in plasma (62.5 % samples positive). Both the assays were further tested for direct detection of S. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of culture-positive patients (sensitivity 89%, 95 % CI 0.75–0.96) and 0/59 negative controls with the sodA assay (specificit

Tuberculosis diagnostics and biomarkers: needs, challenges, recent advances, and opportunities

Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostics

New Trends in Impedimetric Biosensors for the Detection of Foodborne Pathogenic Bacteria

The development of a rapid, sensitive, specific method for the foodborne pathogenic bacteria detection is of great importance to ensure food safety and security. In recent years impedimetric biosensors which integrate biological recognition technology and impedance have gained widespread application in the field of bacteria detection. This paper presents an overview on the progress and application of impedimetric biosensors for detection of foodborne pathogenic bacteria, particularly the new trends in the past few years, including the new specific bio-recognition elements such as bacteriophage and lectin, the use of nanomaterials and microfluidics techniques. The applications of these new materials or techniques have provided unprecedented opportunities for the development of high-performance impedance bacteria biosensors. The significant developments of impedimetric biosensors for bacteria detection in the last five years have been reviewed according to the classification of with or without specific bio-recognition element. In addition, some microfluidics systems, which were used in the construction of impedimetric biosensors to improve analytical performance, are introduced in this review

Xpert MTB/RIF and Xpert Ultra assays for screening for pulmonary tuberculosis and rifampicin resistance in adults, irrespective of signs or symptoms

Background Tuberculosis is a leading cause of infectious disease‐related death and is one of the top 10 causes of death worldwide. The World Health Organization (WHO) recommends the use of specific rapid molecular tests, including Xpert MTB/RIF or Xpert Ultra, as initial diagnostic tests for the detection of tuberculosis and rifampicin resistance in people with signs and symptoms of tuberculosis. However, the WHO estimates that nearly one‐third of all active tuberculosis cases go undiagnosed and unreported. We were interested in whether a single test, Xpert MTB/RIF or Xpert Ultra, could be useful as a screening test to close this diagnostic gap and improve tuberculosis case detection. Objectives To estimate the accuracy of Xpert MTB/RIF and Xpert Ultra for screening for pulmonary tuberculosis in adults, irrespective of signs or symptoms of pulmonary tuberculosis in high‐risk groups and in the general population. Screening "irrespective of signs or symptoms" refers to screening of people who have not been assessed for the presence of tuberculosis symptoms (e.g. cough). To estimate the accuracy of Xpert MTB/RIF and Xpert Ultra for detecting rifampicin resistance in adults screened for tuberculosis, irrespective of signs and symptoms of pulmonary tuberculosis in high‐risk groups and in the general population. Search methods We searched 12 databases including the Cochrane Infectious Diseases Group Specialized Register, MEDLINE and Embase, on 19 March 2020 without language restrictions. We also reviewed reference lists of included articles and related Cochrane Reviews, and contacted researchers in the field to identify additional studies. Selection criteria Cross‐sectional and cohort studies in which adults (15 years and older) in high‐risk groups (e.g. people living with HIV, household contacts of people with tuberculosis) or in the general population were screened for pulmonary tuberculosis using Xpert MTB/RIF or Xpert Ultra. For tuberculosis detection, the reference standard was culture. For rifampicin resistance detection, the reference standards were culture‐based drug susceptibility testing and line probe assays. Data collection and analysis Two review authors independently extracted data using a standardized form and assessed risk of bias and applicability using QUADAS‐2. We used a bivariate random‐effects model to estimate pooled sensitivity and specificity with 95% credible intervals (CrIs) separately for tuberculosis detection and rifampicin resistance detection. We estimated all models using a Bayesian approach. For tuberculosis detection, we first estimated screening accuracy in distinct high‐risk groups, including people living with HIV, household contacts, people residing in prisons, and miners, and then in several high‐risk groups combined. Main results We included a total of 21 studies: 18 studies (13,114 participants) evaluated Xpert MTB/RIF as a screening test for pulmonary tuberculosis and one study (571 participants) evaluated both Xpert MTB/RIF and Xpert Ultra. Three studies (159 participants) evaluated Xpert MTB/RIF for rifampicin resistance. Fifteen studies (75%) were conducted in high tuberculosis burden and 16 (80%) in high TB/HIV‐burden countries. We judged most studies to have low risk of bias in all four QUADAS‐2 domains and low concern for applicability. Xpert MTB/RIF and Xpert Ultra as screening tests for pulmonary tuberculosis In people living with HIV (12 studies), Xpert MTB/RIF pooled sensitivity and specificity (95% CrI) were 61.8% (53.6 to 69.9) (602 participants; moderate‐certainty evidence) and 98.8% (98.0 to 99.4) (4173 participants; high‐certainty evidence). Of 1000 people where 50 have tuberculosis on culture, 40 would be Xpert MTB/RIF‐positive; of these, 9 (22%) would not have tuberculosis (false‐positives); and 960 would be Xpert MTB/RIF‐negative; of these, 19 (2%) would have tuberculosis (false‐negatives). In people living with HIV (1 study), Xpert Ultra sensitivity and specificity (95% CI) were 69% (57 to 80) (68 participants; very low‐certainty evidence) and 98% (97 to 99) (503 participants; moderate‐certainty evidence). Of 1000 people where 50 have tuberculosis on culture, 53 would be Xpert Ultra‐positive; of these, 19 (36%) would not have tuberculosis (false‐positives); and 947 would be Xpert Ultra‐negative; of these, 16 (2%) would have tuberculosis (false‐negatives). In non‐hospitalized people in high‐risk groups (5 studies), Xpert MTB/RIF pooled sensitivity and specificity were 69.4% (47.7 to 86.2) (337 participants, low‐certainty evidence) and 98.8% (97.2 to 99.5) (8619 participants, moderate‐certainty evidence). Of 1000 people where 10 have tuberculosis on culture, 19 would be Xpert MTB/RIF‐positive; of these, 12 (63%) would not have tuberculosis (false‐positives); and 981 would be Xpert MTB/RIF‐negative; of these, 3 (0%) would have tuberculosis (false‐negatives). We did not identify any studies using Xpert MTB/RIF or Xpert Ultra for screening in the general population. Xpert MTB/RIF as a screening test for rifampicin resistance Xpert MTB/RIF sensitivity was 81% and 100% (2 studies, 20 participants; very low‐certainty evidence), and specificity was 94% to 100%, (3 studies, 139 participants; moderate‐certainty evidence). Authors' conclusions Of the high‐risks groups evaluated, Xpert MTB/RIF applied as a screening test was accurate for tuberculosis in high tuberculosis burden settings. Sensitivity and specificity were similar in people living with HIV and non‐hospitalized people in high‐risk groups. In people living with HIV, Xpert Ultra sensitivity was slightly higher than that of Xpert MTB/RIF and specificity similar. As there was only one study of Xpert Ultra in this analysis, results should be interpreted with caution. There were no studies that evaluated the tests in people with diabetes mellitus and other groups considered at high‐risk for tuberculosis, or in the general population

Xpert MTB/RIF Ultra and Xpert MTB/RIF assays for extrapulmonary tuberculosis and rifampicin resistance in adults.

BACKGROUND: Xpert MTB/RIF Ultra (Xpert Ultra) and Xpert MTB/RIF are World Health Organization (WHO)-recommended rapid nucleic acid amplification tests (NAATs) widely used for simultaneous detection of Mycobacterium tuberculosis complex and rifampicin resistance in sputum. To extend our previous review on extrapulmonary tuberculosis (Kohli 2018), we performed this update to inform updated WHO policy (WHO Consolidated Guidelines (Module 3) 2020). OBJECTIVES: To estimate diagnostic accuracy of Xpert Ultra and Xpert MTB/RIF for extrapulmonary tuberculosis and rifampicin resistance in adults with presumptive extrapulmonary tuberculosis. SEARCH METHODS: Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, Web of Science, Latin American Caribbean Health Sciences Literature, Scopus, ClinicalTrials.gov, the WHO International Clinical Trials Registry Platform, the International Standard Randomized Controlled Trial Number Registry, and ProQuest, 2 August 2019 and 28 January 2020 (Xpert Ultra studies), without language restriction. SELECTION CRITERIA: Cross-sectional and cohort studies using non-respiratory specimens. Forms of extrapulmonary tuberculosis: tuberculous meningitis and pleural, lymph node, bone or joint, genitourinary, peritoneal, pericardial, disseminated tuberculosis. Reference standards were culture and a study-defined composite reference standard (tuberculosis detection); phenotypic drug susceptibility testing and line probe assays (rifampicin resistance detection). DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed risk of bias and applicability using QUADAS-2. For tuberculosis detection, we performed separate analyses by specimen type and reference standard using the bivariate model to estimate pooled sensitivity and specificity with 95% credible intervals (CrIs). We applied a latent class meta-analysis model to three forms of extrapulmonary tuberculosis. We assessed certainty of evidence using GRADE. MAIN RESULTS: 69 studies: 67 evaluated Xpert MTB/RIF and 11 evaluated Xpert Ultra, of which nine evaluated both tests. Most studies were conducted in China, India, South Africa, and Uganda. Overall, risk of bias was low for patient selection, index test, and flow and timing domains, and low (49%) or unclear (43%) for the reference standard domain. Applicability for the patient selection domain was unclear for most studies because we were unsure of the clinical settings. Cerebrospinal fluid Xpert Ultra (6 studies) Xpert Ultra pooled sensitivity and specificity (95% CrI) against culture were 89.4% (79.1 to 95.6) (89 participants; low-certainty evidence) and 91.2% (83.2 to 95.7) (386 participants; moderate-certainty evidence). Of 1000 people where 100 have tuberculous meningitis, 168 would be Xpert Ultra-positive: of these, 79 (47%) would not have tuberculosis (false-positives) and 832 would be Xpert Ultra-negative: of these, 11 (1%) would have tuberculosis (false-negatives). Xpert MTB/RIF (30 studies) Xpert MTB/RIF pooled sensitivity and specificity against culture were 71.1% (62.8 to 79.1) (571 participants; moderate-certainty evidence) and 96.9% (95.4 to 98.0) (2824 participants; high-certainty evidence). Of 1000 people where 100 have tuberculous meningitis, 99 would be Xpert MTB/RIF-positive: of these, 28 (28%) would not have tuberculosis; and 901 would be Xpert MTB/RIF-negative: of these, 29 (3%) would have tuberculosis. Pleural fluid Xpert Ultra (4 studies) Xpert Ultra pooled sensitivity and specificity against culture were 75.0% (58.0 to 86.4) (158 participants; very low-certainty evidence) and 87.0% (63.1 to 97.9) (240 participants; very low-certainty evidence). Of 1000 people where 100 have pleural tuberculosis, 192 would be Xpert Ultra-positive: of these, 117 (61%) would not have tuberculosis; and 808 would be Xpert Ultra-negative: of these, 25 (3%) would have tuberculosis. Xpert MTB/RIF (25 studies) Xpert MTB/RIF pooled sensitivity and specificity against culture were 49.5% (39.8 to 59.9) (644 participants; low-certainty evidence) and 98.9% (97.6 to 99.7) (2421 participants; high-certainty evidence). Of 1000 people where 100 have pleural tuberculosis, 60 would be Xpert MTB/RIF-positive: of these, 10 (17%) would not have tuberculosis; and 940 would be Xpert MTB/RIF-negative: of these, 50 (5%) would have tuberculosis. Lymph node aspirate Xpert Ultra (1 study) Xpert Ultra sensitivity and specificity (95% confidence interval) against composite reference standard were 70% (51 to 85) (30 participants; very low-certainty evidence) and 100% (92 to 100) (43 participants; low-certainty evidence). Of 1000 people where 100 have lymph node tuberculosis, 70 would be Xpert Ultra-positive and 0 (0%) would not have tuberculosis; 930 would be Xpert Ultra-negative and 30 (3%) would have tuberculosis. Xpert MTB/RIF (4 studies) Xpert MTB/RIF pooled sensitivity and specificity against composite reference standard were 81.6% (61.9 to 93.3) (377 participants; low-certainty evidence) and 96.4% (91.3 to 98.6) (302 participants; low-certainty evidence). Of 1000 people where 100 have lymph node tuberculosis, 118 would be Xpert MTB/RIF-positive and 37 (31%) would not have tuberculosis; 882 would be Xpert MTB/RIF-negative and 19 (2%) would have tuberculosis. In lymph node aspirate, Xpert MTB/RIF pooled specificity against culture was 86.2% (78.0 to 92.3), lower than that against a composite reference standard. Using the latent class model, Xpert MTB/RIF pooled specificity was 99.5% (99.1 to 99.7), similar to that observed with a composite reference standard. Rifampicin resistance Xpert Ultra (4 studies) Xpert Ultra pooled sensitivity and specificity were 100.0% (95.1 to 100.0), (24 participants; low-certainty evidence) and 100.0% (99.0 to 100.0) (105 participants; moderate-certainty evidence). Of 1000 people where 100 have rifampicin resistance, 100 would be Xpert Ultra-positive (resistant): of these, zero (0%) would not have rifampicin resistance; and 900 would be Xpert Ultra-negative (susceptible): of these, zero (0%) would have rifampicin resistance. Xpert MTB/RIF (19 studies) Xpert MTB/RIF pooled sensitivity and specificity were 96.5% (91.9 to 98.8) (148 participants; high-certainty evidence) and 99.1% (98.0 to 99.7) (822 participants; high-certainty evidence). Of 1000 people where 100 have rifampicin resistance, 105 would be Xpert MTB/RIF-positive (resistant): of these, 8 (8%) would not have rifampicin resistance; and 895 would be Xpert MTB/RIF-negative (susceptible): of these, 3 (0.3%) would have rifampicin resistance. AUTHORS' CONCLUSIONS: Xpert Ultra and Xpert MTB/RIF may be helpful in diagnosing extrapulmonary tuberculosis. Sensitivity varies across different extrapulmonary specimens: while for most specimens specificity is high, the tests rarely yield a positive result for people without tuberculosis. For tuberculous meningitis, Xpert Ultra had higher sensitivity and lower specificity than Xpert MTB/RIF against culture. Xpert Ultra and Xpert MTB/RIF had similar sensitivity and specificity for rifampicin resistance. Future research should acknowledge the concern associated with culture as a reference standard in paucibacillary specimens and consider ways to address this limitation

Readiness of first-graders for the subject “Sport and health” following the new standard in health and physical activity education

Diplomdarba mērķis ir noskaidrot, vai pirmklasnieki ir pietiekami sagatavoti mācību priekšmetam “Sports un veselība”, un vai viņu sagatavotības līmenis ir atbilstošs jaunajam valsts pamatizglītības standartam. Diplomdarba ietvaros autore izvērtēja faktorus, kas ietekmē to, cik lielā mērā pirmklasnieks ir gatavs iesaistīties “Sports un veselība” stundās, un vai skolēni ir sagatavoti šim mācību priekšmetam atbilstoši jaunajam valsts pamatizglītības standartam. Faktori, kas tika analizēti, ir attiecīgais vecumposms un adaptācijas nozīmīgums. Iegūtie rezultāti tika salīdzināti starp bērniem, kas ir pirmsskolas pēdējā posmā un pirmklasniekiem. Tāpat tika veikta pirmsskolas un skolas sporta pedagogu anketēšana, kuri praktiski, saskaroties darbā ar pirmklasniekiem, spēj sniegt noderīgas atziņas un vērtējumu par bērnu gatavību iesaistīties sporta stundās. Diplomdarba ietvaros tika anketēti 53 pirmsskolas sporta skolotāji, tāpat tika anketētas 59 sākumskolas sporta skolotāji par vispārējiem novērojumiem praktiskajā darbā ar bērniem skolā. Par iegūtiem datiem izdarīti secinājumi, ka skolēni ir pietiekami sagatavoti sporta stundām, atbilstoši jaunā vienotā valsts pamatizglītības standarta veselības un fiziskās aktivitātes mācību jomā. Atslēgvārdi: pirmklasnieks, sporta stundas, sākumskola, sporta skolotājs, mācību standarts.The aim of the diploma thesis is to find out whether first-graders are sufficiently prepared for the subject "Sports and health" and whether their level of preparation corresponds to the new state basic education standard. As part of the diploma thesis, the author evaluated the factors that affect the extent to which a first-grader is ready to participate in "Sports and health" classes, and whether students are prepared for this subject in accordance with the new national basic education standard. The factors that were analyzed are the relevant age stage and the importance of adaptation. The obtained results were compared between children who are in the last stage of preschool and first graders. Also, questionnaires were conducted for preschool and school sports pedagogues, who are able to provide useful insights and evaluations about children's readiness to participate in sports lessons when they practically work with first-graders. As part of the diploma thesis, 53 preschool sports teachers were interviewed, and 59 school sports teachers were also interviewed about general observations in practical work with children at school. Based on the obtained data, conclusions have been drawn that the students are sufficiently prepared for sports lessons, in accordance with the new unified state basic education standard in the field of health and physical activity. Keywords: first grader, sports lessons, primary school, sports teacher, learning standard

Larval and early juvenile fishes associated with milkfish fry at Malandog, Hamtik, Antique

Contribution No. 135 of SEAFDEC Aquaculture Department.From 5 May 1981 to 7 November 1981 a total of 122,841 specimens of milkfish fry and early juvenile fishes was collected with the use of a milkfish fry sweeper. Of these, 66,361 or 54.02% consisted of early juvenile stages of 30 species of finfish and the rest were 56,480 milkfish fry of 45.98% of the total collection. The most abundant finfish excluding milkfish fry was Ambassis sp. comprising 59.43% of the total fish catch, followed by Elops machnata, 12.7%; Sillago sihama, 8.66% Gobiidae, 6.11%; Therapon jarbua, 5.49% Stolephorus sp., 2.06%; Chonoporus sp., 1.53%; Mullet, 0.97% and Scatophagus argus, 0.32%. The rest of the species combined comprised less than 1%. Water temperature, pH and dissolved oxygen (D.O.) showed inverse relationships with fish abundance at the early hours of the day (0800H-1200H) but incosistent correlations were noted during night time when these parameters were rather constant. Salinity did not show any correlation to fish abundance although more species were collected after a heavy downpour. Fish catch was higher during low tide than at high tide

A Review of Techniques in Microbiology: a Student Handbook

<strong>Review of</strong>: <em>Techniques in Microbiology: a Student Handbook </em>; John M. Lammert; (2007) . Pearson Education, Inc., Upper Saddle River, NJ. 225 pages