Genetic Diversity of the Flavohemoprotein Gene of Giardia lamblia: Evidence for High Allelic Heterozygosity and Copy Number Variation

Purpose: The flavohemoprotein (gFlHb) in Giardia plays an important role in managing nitrosative and oxidative stress, and potentially also in virulence and nitroimidazole drug tolerance. The aim of this study was to analyze the genetic diversity of gFlHb in Giardia assemblages A and B clinical isolates. Methods: gFlHb genes from 20 cultured clinical Giardia isolates were subjected to PCR amplification and cloning, followed by Sanger sequencing. Sequences of all cloned PCR fragments from each isolate were analyzed for single nucleotide variants (SNVs) and compared to genomic Illumina sequence data. Identical clone sequences were sorted into alleles, and diversity was further analyzed. The number of gFlHb gene copies was assessed by mining PacBio de novo assembled genomes in eight isolates. Homology models for assessment of SNV’s potential impact on protein function were created using Phyre2. Results: A variable copy number of the gFlHb gene, between two and six copies, depending on isolate, was found. A total of 37 distinct sequences, representing different alleles of the gFlHb gene, were identified in AII isolates, and 41 were identified in B isolates. In some isolates, up to 12 different alleles were found. The total allelic diversity was high for both assemblages (> 0.9) and was coupled with a nucleotide diversity of < 0.01. The genetic variation (SNVs per CDS length) was 4.8% in sub-assemblage AII and 5.4% in assemblage B. The number of non-synonymous (ns) SNVs was high in gFIHb of both assemblages, 1.6% in A and 3.0% in B, respectively. Some of the identified nsSNV are predicted to alter protein structure and possibly function. Conclusion: In this study, we present evidence that gFlHb, a putative protective enzyme against oxidative and nitrosative stress in Giardia, is a variable copy number gene with high allelic diversity. The genetic variability of gFlHb may contribute metabolic adaptability against metronidazole toxicity.Peer Reviewe

Surveillance of antimicrobial resistance at a tertiary hospital in Tanzania

BACKGROUND: Antimicrobial resistance is particularly harmful to infectious disease management in low-income countries since expensive second-line drugs are not readily available. The objective of this study was to implement and evaluate a computerized system for surveillance of antimicrobial resistance at a tertiary hospital in Tanzania. METHODS: A computerized surveillance system for antimicrobial susceptibility (WHONET) was implemented at the national referral hospital in Tanzania in 1998. The antimicrobial susceptibilities of all clinical bacterial isolates received during an 18 months' period were recorded and analyzed. RESULTS: The surveillance system was successfully implemented at the hospital. This activity increased the focus on antimicrobial resistance issues and on laboratory quality assurance issues. The study identified specific nosocomial problems in the hospital and led to the initiation of other prospective studies on prevalence and antimicrobial susceptibility of bacterial infections. Furthermore, the study provided useful data on antimicrobial patterns in bacterial isolates from the hospital. Gram-negative bacteria displayed high rates of resistance to common inexpensive antibiotics such as ampicillin, tetracycline and trimethoprim-sulfamethoxazole, leaving fluoroquinolones as the only reliable oral drugs against common Gram-negative bacilli. Gentamicin and third generation cephalosporins remain useful for parenteral therapy. CONCLUSION: The surveillance system is a low-cost tool to generate valuable information on antimicrobial resistance, which can be used to prepare locally applicable recommendations on antimicrobial use. The system pinpoints relevant nosocomial problems and can be used to efficiently plan further research. The surveillance system also functions as a quality assurance tool, bringing attention to methodological issues in identification and susceptibility testing

Antimicrobial resistance predicts death in Tanzanian children with bloodstream infections: a prospective cohort study

Bloodstream infection is a common cause of hospitalization, morbidity and death in children. The impact of antimicrobial resistance and HIV infection on outcome is not firmly established. We assessed the incidence of bloodstream infection and risk factors for fatal outcome in a prospective cohort study of 1828 consecutive admissions of children aged zero to seven years with signs of systemic infection. Blood was obtained for culture, malaria microscopy, HIV antibody test and, when necessary, HIV PCR. We recorded data on clinical features, underlying diseases, antimicrobial drug use and patients' outcome. The incidence of laboratory-confirmed bloodstream infection was 13.9% (255/1828) of admissions, despite two thirds of the study population having received antimicrobial therapy prior to blood culture. The most frequent isolates were klebsiella, salmonellae, Escherichia coli, enterococci and Staphylococcus aureus. Furthermore, 21.6% had malaria and 16.8% HIV infection. One third (34.9%) of the children with laboratory-confirmed bloodstream infection died. The mortality rate from Gram-negative bloodstream infection (43.5%) was more than double that of malaria (20.2%) and Gram-positive bloodstream infection (16.7%). Significant risk factors for death by logistic regression modeling were inappropriate treatment due to antimicrobial resistance, HIV infection, other underlying infectious diseases, malnutrition and bloodstream infection caused by Enterobacteriaceae, other Gram-negatives and candida. Bloodstream infection was less common than malaria, but caused more deaths. The frequent use of antimicrobials prior to blood culture may have hampered the detection of organisms susceptible to commonly used antimicrobials, including pneumococci, and thus the study probably underestimates the incidence of bloodstream infection. The finding that antimicrobial resistance, HIV-infection and malnutrition predict fatal outcome calls for renewed efforts to curb the further emergence of resistance, improve HIV care and nutrition for children

Differences between normal and demineralized dentine pretreated with silver fluoride and potassium iodide after an in vitro challenge by Streptococcus mutans

The document attached has been archived with permission from the Australian Dental Association (8 March 2008). An external link to the publisher’s copy is included.Background: The application of diamine silver fluoride (Ag(NH3)2F) and potassium iodide (KI) to demineralized dentine has been shown to inhibit the growth of Streptococcus mutans. The purpose of this study was to observe the differences between demineralized and non-demineralized dentine treated with AgF/KI. Methods: Thirty-five dentine discs were bonded to the bases of 5mL polycarbonate screw top vials which were filled with nutrient medium, sterilized and placed into the overflow from a continuous culture of S. mutans. Samples were divided as follows: 10 samples of demineralized dentine; 10 samples of demineralized dentine treated with AgF/KI; 5 samples of non-demineralized dentine; and 10 samples of non-demineralized dentine treated with AgF/KI. Following two weeks connected to the Chemostat, an electron probe microanalysis (EPMA) of percentage weights and penetration depths of calcium, phosphorous silver and fluoride was conducted. Bacterial growth was monitored by taking optical density readings of the growth medium in each vial and outer surfaces of the specimens were examined by scanning electron microscopy (SEM). Results: AgF/KI treatment of demineralized and non-demineralized dentine prevented biofilm formation and reduced further demineralization by S. mutans. AgF/KI treatment of demineralized dentine was more effective in reducing dentine breakdown and the growth of S. mutans. Significantly higher levels of silver and fluoride were deposited within demineralized dentine. Conclusions: A topical treatment with AgF/KI on dentine reduced in vitro caries development and inhibited surface biofilm formation. Reduction of in vitro caries development and viability of S. mutans was more pronounced on the dentine samples that had been demineralized prior to the application of AgF/KI.GM Knight, JM McIntyre, GG Craig, Mulyani, PS Zilm and NJ Gull

A large community outbreak of waterborne giardiasis- delayed detection in a non-endemic urban area

BACKGROUND: Giardia is not endemic in Norway, and more than 90% of reported cases acquire the infection abroad. In late October 2004, an increase in laboratory confirmed cases of giardiasis was reported in the city of Bergen. An investigation was started to determine the source and extent of the outbreak in order to implement control measures. METHODS: Cases were identified through the laboratory conducting giardia diagnostics in the area. All laboratory-confirmed cases were mapped based on address of residence, and attack rates and relative risks were calculated for each water supply zone. A case control study was conducted among people living in the central area of Bergen using age- and sex matched controls randomly selected from the population register. RESULTS: The outbreak investigation showed that the outbreak started in late August and peaked in early October. A total of 1300 laboratory-confirmed cases were reported. Data from the Norwegian Prescription Database gave an estimate of 2500 cases treated for giardiasis probably linked to the outbreak. There was a predominance of women aged 20–29 years, with few children or elderly. The risk of infection for persons receiving water from the water supply serving Bergen city centre was significantly higher than for those receiving water from other supplies. Leaking sewage pipes combined with insufficient water treatment was the likely cause of the outbreak. CONCLUSION: Late detection contributed to the large public health impact of this outbreak. Passive surveillance of laboratory-confirmed cases is not sufficient for timely detection of outbreaks with non-endemic infections

Risk assessment for the spread of Serratia marcescens within dental-unit waterline systems using Vermamoeba vermiformis

Vermamoeba vermiformis is associated with the biofilm ecology of dental-unit waterlines (DUWLs). This study investigated whether V. vermiformis is able to act as a vector for potentially pathogenic bacteria and so aid their dispersal within DUWL systems. Clinical dental water was initially examined for Legionella species by inoculating it onto Legionella selective-medium plates. The molecular identity/profile of the glassy colonies obtained indicated none of these isolates were Legionella species. During this work bacterial colonies were identified as a non-pigmented Serratia marcescens. As the water was from a clinical DUWL which had been treated with Alpron™ this prompted the question as to whether S. marcescens had developed resistance to the biocide. Exposure to Alpron™ indicated that this dental biocide was effective, under laboratory conditions, against S. marcescens at up to 1x108 colony forming units/millilitre (cfu/ml). V. vermiformis was cultured for eight weeks on cells of S. marcescens and Escherichia coli. Subsequent electron microscopy showed that V. vermiformis grew equally well on S. marcescens and E. coli (p = 0.0001). Failure to detect the presence of S. marcescens within the encysted amoebae suggests that V. vermiformis is unlikely to act as a vector supporting the growth of this newly isolated, nosocomial bacterium

An automated in vitro model for the evaluation of ultrasound modalities measuring myocardial deformation

<p>Abstract</p> <p>Background</p> <p>Echocardiography is the method of choice when one wishes to examine myocardial function. Qualitative assessment of the 2D grey scale images obtained is subjective, and objective methods are required. Speckle Tracking Ultrasound is an emerging technology, offering an objective mean of quantifying left ventricular wall motion. However, before a new ultrasound technology can be adopted in the clinic, accuracy and reproducibility needs to be investigated.</p> <p>Aim</p> <p>It was hypothesized that the collection of ultrasound sample data from an in vitro model could be automated. The aim was to optimize an in vitro model to allow for efficient collection of sample data.</p> <p>Material & Methods</p> <p>A tissue-mimicking phantom was made from water, gelatin powder, psyllium fibers and a preservative. Sonomicrometry crystals were molded into the phantom. The solid phantom was mounted in a stable stand and cyclically compressed. Peak strain was then measured by Speckle Tracking Ultrasound and sonomicrometry.</p> <p>Results</p> <p>We succeeded in automating the acquisition and analysis of sample data. Sample data was collected at a rate of 200 measurement pairs in 30 minutes. We found good agreement between Speckle Tracking Ultrasound and sonomicrometry in the in vitro model. Best agreement was 0.83 ± 0.70%. Worst agreement was -1.13 ± 6.46%.</p> <p>Conclusions</p> <p>It has been shown possible to automate a model that can be used for evaluating the in vitro accuracy and precision of ultrasound modalities measuring deformation. Sonomicrometry and Speckle Tracking Ultrasound had acceptable agreement.</p

Feasibility and reference values of left atrial longitudinal strain imaging by two-dimensional speckle tracking

<p>Abstract</p> <p>Background</p> <p>The role of speckle tracking in the assessment of left atrial (LA) deformation dynamics is not established. We sought to determine the feasibility and reference ranges of LA longitudinal strain indices measured by speckle tracking in a population of normal subjects.</p> <p>Methods</p> <p>In 60 healthy individuals, peak atrial longitudinal strain (PALS) and time to peak longitudinal strain (TPLS) were measured using a 12-segment model for the left atrium. Values were obtained by averaging all segments (global PALS and TPLS) and by separately averaging segments measured in the two apical views (4- and 2-chamber average PALS and TPLS).</p> <p>Results</p> <p>Adequate tracking quality was achieved in 97% of segments analyzed. Inter and intra-observer variability coefficients of measurements ranged between 2.9% and 5.4%. Global PALS was 42.2 ± 6.1% (5–95° percentile range 32.2–53.2%), and global TPLS was 368 ± 30 ms (5–95° percentile range 323–430 ms). The 2-chamber average PALS was slightly higher than the 4-chamber average PALS (44.3 ± 6.0% vs 40.1 ± 7.9%, p < 0.0001), whereas no differences in TPLS were found (p = 0.93).</p> <p>Conclusion</p> <p>Speckle tracking is a feasible technique for the assessment of longitudinal myocardial LA deformation. Reference ranges of strain indices were reported.</p