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1.  The use of dried cerebrospinal fluid filter paper spots as a substrate for PCR diagnosis of the aetiology of bacterial meningitis in the Lao PDR 
Clinical Microbiology and Infection  2013;19(10):E466-E472.
We investigated whether dried cerebrospinal fluid (CSF) conserved on filter paper can be used as a substrate for accurate PCR diagnosis of important causes of bacterial meningitis in the Lao PDR. Using mock CSF, we investigated and optimized filter paper varieties, paper punch sizes, elution volumes and quantities of DNA template to achieve sensitive and reliable detection of bacterial DNA from filter paper specimens. FTA Elute Micro Card™ (Whatman, Maidstone, UK) was the most sensitive, consistent and practical variety of filter paper. Following optimization, the lower limit of detection for Streptococcus pneumoniae from dried mock CSF spots was 14 genomic equivalents (GE)/μL (interquartile range 5.5 GE/μL) or 230 (IQR 65) colony forming units/mL. A prospective clinical evaluation for S. pneumoniae, S. suis and Neisseria meningitidis was performed. Culture and PCR performed on fresh liquid CSF from patients admitted with a clinical diagnosis of meningitis (n = 73) were compared with results derived from dried CSF spots. Four of five fresh PCR-positive CSF samples also tested PCR positive from dried CSF spots, with one patient under the limit of detection. In a retrospective study of S. pneumoniae samples (n = 20), the median (IQR; range) CSF S. pneumoniae bacterial load was 1.1 × 104 GE/μL (1.2 × 105; 1 to 6.1 × 106 DNA GE/μL). Utilizing the optimized methodology, we estimate an extrapolated sensitivity of 90%, based on the range of CSF genome counts found in Laos. Dried CSF filter paper spots could potentially help us to better understand the epidemiology of bacterial meningitis in resource-poor settings and guide empirical treatments and vaccination policies.
PMCID: PMC4285853  PMID: 23738720
Bacterial meningitis; cerebrospinal fluid; filter paper; Lao PDR; Streptococcus pneumoniae
2.  Deletion in the gene BruAb2_0168 of Brucella abortus strains: diagnostic challenges 
Clinical Microbiology and Infection  2014;20(9):O550-O553.
Three Brucella abortus strains were isolated from joint hygromas from cows in northern Togo. Two deletions in the 5′ side of the gene BruAb2_0168 were identified. As this gene is used for species identification, these deletions have consequences for diagnostic procedures. Multiple locus variable number of tandem repeat (VNTR) analysis was therefore performed for species identification. The strains showed unique VNTR profiles, providing some of the first genotypic data from West Africa. More molecular and epidemiological data are needed from the region, in order to better understand transmission patterns and develop suitable diagnostic assays.
PMCID: PMC4235395  PMID: 24450581
Brucella; cattle; diagnostics; genotyping; Togo
3.  Lipooligosaccharide locus class of Campylobacter jejuni: sialylation is not needed for invasive infection 
Campylobacter jejuni is a highly diverse enteropathogen that is commonly detected worldwide. It can sometimes cause bacteraemia, but the bacterial characteristics facilitating bloodstream infection are not known. A total of 73 C. jejuni isolates, consecutively collected from blood-borne infections during a 10-year period all over Finland and for which detailed clinical information of the patients were available, were included. We screened the isolates by PCR for the lipooligosaccharide (LOS) locus class and for the presence of the putative virulence genes ceuE, ciaB, fucP, and virB11. The isolates were also tested for γ-glutamyl transpeptidase production. The results were analysed with respect to the clinical characteristics of the patients, and the multilocus sequence types (MLSTs) and serum resistance of the isolates. LOS locus classes A, B, and C, which carry genes for sialylation of LOS, were detected in only 23% of the isolates. These isolates were not more resistant to human serum than those with the genes of non-sialylated LOS locus classes, but were significantly more prevalent among patients with underlying diseases (p 0.02). The fucose permease gene fucP was quite uncommon, but was associated with the isolates with the potential to sialylate LOS (p <0.0001). LOS locus classes and some of the putative virulence factors were associated with MLST clonal complexes. Although some of the bacterial characteristics studied here have been suggested to be important for the invasiveness of C. jejuni, they did not explain why the clinical isolates in the present study were able to cause bacteraemia.
PMCID: PMC4235400  PMID: 24102802
Bacteraemia; blood; Campylobacter; infection; invasive; lipooligosaccharide; MLST; serum resistance
4.  Mycobacterium avium genotype is associated with the therapeutic response to lung infection 
Factors that can interfere with the successful treatment of Mycobacterium avium lung infection have been inadequately studied. To identify a potent predictor of therapeutic responses of M. avium lung infection, we analyzed variable number tandem repeats (VNTR) at 16 minisatellite loci of M. avium clinical isolates. Associations between the VNTR profiling data and a therapeutic response were evaluated in 59 subjects with M. avium lung infection. M. avium lung infection of 30 subjects in whom clarithromycin-containing regimens produced microbiological and radiographic improvement was defined as responsive disease, while that of the remaining 29 subjects was defined as refractory disease. In phylogenetic analysis using the genotypic distance aggregated from 16-dimensional VNTR data, 59 M. avium isolates were divided into three clusters, which showed a nearly significant association with therapeutic responses (p 0.06). We then subjected the raw 16-dimensional VNTR data directly to principal component analysis, and identified the genetic features that were significantly associated with the therapeutic response (p <0.05). By further analysis of logistic regression with a stepwise variable-selection, we constructed the highest likelihood multivariate model, adjusted for age, to predict a therapeutic response, using VNTR data from only four minisatellite loci. In conclusion, we identified four mycobacterial minisatellite loci that together were associated with the therapeutic response of M. avium lung infections.
PMCID: PMC4231998  PMID: 23829301
Logistic regression analysis; Mycobacterium avium; principal component analysis; therapeutic response; variable number tandem repeats
6.  The Cultivable Human Oral Gluten-Degrading Microbiome and its Potential Implications in Celiac Disease and Gluten Sensitivity 
Celiac disease is characterized by intestinal inflammation caused by gluten, proteins which are widely contained in the Western diet. Mammalian digestive enzymes are only partly capable of cleaving gluten, and fragments remain that induce toxic responses in celiac patients. We found that the oral microbiome is a novel and rich source of gluten degrading enzymes. Here we report on the isolation and characterization of the cultivable resident oral microbes that are capable of cleaving gluten, with special emphasis on its immunogenic domains. Bacteria were obtained by a selective culturing approach and enzyme activities were characterised by: 1) Hydrolysis of paranitroanilide-derivatised gliadin-derived tripeptide substrates; 2) Gliadin degradation in-gel (gliadin zymography); 3) Gliadin degradation in solution; 4) Proteolysis of the highly immunogenic α-gliadin-derived 33-mer. For select strains pH activity profiles were determined. The culturing strategy yielded 87 aerobic and 63 anaerobic strains. Species with activity in at least two of the four assays were typed as: Rothia mucilaginosa HOT-681, Rothia aeria HOT-188, Actinomyces odontolyticus HOT-701, Streptococcus mitis HOT-677, Streptococcus sp. HOT-071, Neisseria mucosa HOT-682 and Capnocytophaga sputigena HOT-775, with Rothia species being active in all four assays. Cleavage specificities and substrate preferences differed among the strains identified. The approximate molecular weights of the enzymes were ~75 kD (Rothia spp.), ~60 kD (A. odontolyticus) and ~150 kD (Streptococcus spp.). In conclusion, this study identified new gluten-degrading microorganisms in the upper gastro-intestinal tract. A cocktail of the most active oral bacteria, or their isolated enzymes, may offer promising new treatment modalities for celiac disease.
PMCID: PMC3749263  PMID: 23714165
celiac disease; gliadin; oral bacteria; proteases; degradation
7.  The Economic Burden of Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) 
The economic impact of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) remains unclear. We developed an economic simulation model to quantify the costs associated with CA-MRSA infection from the societal and third-party payer perspectives. A single CA-MRSA case costs third-party payers $2,277 – $3,200 and society $7,070 – $20,489, depending on patient age. In the United States (US), CA-MRSA imposes an annual burden of $478 million - 2.2 billion on third-party payers and $1.4 billion - 13.8 billion on society, depending on the CA-MRSA definitions and incidences. The US jail system and Army may be experiencing annual total costs of $7 – 11 million ($6 – 10 million direct medical costs) and $15 – 36 million ($14 – 32 million), respectively. Hospitalization rates and mortality are important cost drivers. CA-MRSA confers a substantial economic burden to third-party payers and society, with CA-MRSA-attributable productivity losses being major contributors to the total societal economic burden. Although decreasing transmission and infection incidence would decrease costs, even if transmission were to continue at present levels, early identification and appropriate treatment of CA-MRSA infections before they progress could save considerable costs.
PMCID: PMC3463640  PMID: 22712729
Community; MRSA; Economics; Cost; CA-MRSA
8.  Longitudinal surveillance for meningitis by Acinetobacter in a large urban setting in Brazil 
The study aim was to describe the emergency of carbapenem resistance and clonal complexes (CC), defined by multilocus sequence typing (MLST), in Acinetobacter baumannii in a surveillance system for meningitis. Starting in 1996 at an urban setting of Brazil, surveillance detected meningitis by Acinetobacter sp for the first time in 2002. Until 2008, 35 isolates were saved. Carbapenem resistance emerged in 2006, reaching 70% of A. baumannii isolates in 2008, including one colistin-resistant. A. baumannii belonged to CC113/79 (University of Oxford/ Institute Pasteur schemes), CC235/162 and CC103/15. Dissemination of infections resistant to all antimicrobial agents may occur in the future.
PMCID: PMC3625502  PMID: 23398654
Acinetobacter baumannii; bacterial meningitis; carbapenem-resistance; multilocus sequence typing; clonal complexes
9.  Seasonal Variation in Escherichia coli Bloodstream Infection: A Population-Based Study 
Seasonal variation in the rates of infection with certain Gram-negative organisms has been previously examined in tertiary-care centers. We performed a population-based investigation to evaluate the seasonal variation in Escherichia coli bloodstream infection (BSI). We identified 461 unique patients in Olmsted County, Minnesota, from 1 January 1998 to 31 December 2007, with E. coli BSI. Incidence rates (IR) and IR ratios (IRR) were calculated using Rochester Epidemiology Project tools. Multivariable Poisson regression was used to examine the association between the IR of E. coli BSI and average temperature. The age- and gender-adjusted IR of E. coli BSI per 100,000 person-years was 50.2 (95 % confidence interval [CI]: 42.9-57.5) during the warmest 4 months (June through September) compared to 37.1 (95% CI: 32.7-41.5) during the remainder of the year, resulting in a 35% (95% CI: 12-66%) increase in IR during the warmest four months. The average temperature was predictive of increasing IR of E. coli BSI (p=0.004); there was a 7% (95% CI: 2-12%) increase in the IR for each 10-degree Fahrenheit (c. 5.5 °C) increase in average temperature. To our knowledge, this is the first study to demonstrate seasonal variation in E. coli BSI, with a higher IR during the warmest four months than during the remainder of the year.
PMCID: PMC3938910  PMID: 19845704
bacteraemia; epidemiology; seasonal variation; incidence; Escherichia coli
10.  Obesity and outcomes in patients hospitalized with pneumonia 
Studies suggest obesity is paradoxically associated with better outcomes for patients with pneumonia. Therefore, we examined the impact of obesity on short-term mortality in patients hospitalized with pneumonia. For 2 years clinical and radiographic data were prospectively collected on all consecutive adults admitted with pneumonia to six hospitals in Edmonton, Alberta, Canada. We identified 907 patients who also had body mass index (BMI, kg/m2) collected and categorized them as underweight (BMI < 18.5), normal (18.5 to <25), overweight (25 to <30) and obese (>30). Overall, 65% were >65 years, 52% were female, and 15% reported recent weight loss. Eighty-four (9%) were underweight, 358 (39%) normal, 228 (25%) overweight, and 237 (26%) obese. Two-thirds had severe pneumonia (63% PSI Class IV/V) and 79 (9%) patients died. In-hospital mortality was greatest among those that were underweight (12 [14%]) compared with normal (36 [10%]), overweight (21 [9%]) or obese (10 [4%], p <0.001 for trend). Compared with those of normal weight, obese patients had significantly lower rates of in-hospital mortality in multivariable logistic regression analyses: adjusted odds ratio (OR), 0.46; 95% CI, 0.22–0.97; p 0.04. However, compared with patients with normal weight, neither underweight (adjusted OR, 1.13; 95% CI, 0.54–2.4; p 0.7) nor overweight (adjusted OR, 0.94; 95% CI, 0.52–1.69; p 0.8) were associated with in-hospital mortality. In conclusion, in patients hospitalized with pneumonia, obesity was independently associated with lower short-term mortality, while neither being underweight nor overweight were. This suggests a protective influence of BMIs > 30 kg/m2 that requires better mechanistic understanding.
PMCID: PMC3594436  PMID: 22963453
Body mass index; community-acquired pneumonia; mortality; obesity; outcomes
In temperate regions, influenza typically arrives with the onset of colder weather. Seasonal waves travel over large spaces covering many climatic zones in a relatively short period of time. The precise mechanism for this striking seasonal pattern is still not well understood and the interplay of factors that influence the spread of infection and the emergence of new strains is largely unknown. The study of influenza seasonality has been fraught with problems. One of these is the ever shifting description of illness due to influenza and the use of both the historical definitions and new definitions based on actual isolation of the virus. The compilation of records describing influenza oscillations on a local and global scale is massive, but the value of these data is a function of the definitions used. In this review we argue that both observations of seasonality and deviation from the expected pattern stem from the nature of this disease. Heterogeneity in seasonal patterns may arrive from differences in behavior of specific strains, emergence of a novel strain or cross-protection from previously observed strains. Most likely the seasonal patterns emerge from interactions of individual factors behaving as coupled resonators. We emphasize that both seasonality and deviations from it may merely be a reflection of our inability to disentangle signal from noise, be it due to ambiguity in measurement and/or terminology. We conclude the review with suggestions for new promising and realistic directions with tangible consequences to model complex influenza dynamics in order to effectively control infection.
PMCID: PMC3442949  PMID: 22958213
Influenza; Seasonality
12.  The economic burden of Clostridium difficile 
Although Clostridium difficile (C. difficile) is the leading cause of infectious diarrhoea in hospitalized patients, the economic burden of this major nosocomial pathogen for hospitals, third-party payers and society remains unclear. We developed an economic computer simulation model to determine the costs attributable to healthcare-acquired C. difficile infection (CDI) from the hospital, third-party payer and societal perspectives. Sensitivity analyses explored the effects of varying the cost of hospitalization, C. difficile-attributable length of stay, and the probability of initial and secondary recurrences. The median cost of a case ranged from $9179 to $11 456 from the hospital perspective, $8932 to $11 679 from the third-party payor perspective, and $13 310 to $16 464 from the societal perspective. Most of the costs incurred were accrued during a patient’s primary CDI episode. Hospitals with an incidence of 4.1 CDI cases per 100 000 discharges would incur costs ≥$3.2 million (hospital perspective); an incidence of 10.5 would lead to costs ≥$30.6 million. Our model suggests that the annual US economic burden of CDI would be ≥$496 million (hospital perspective), ≥$547 million (third-party payer perspective) and ≥$796 million (societal perspective). Our results show that C. difficile infection is indeed costly, not only to third-party payers and the hospital, but to society as well. These results are consistent with current literature citing C. difficile as a costly disease.
PMCID: PMC3763211  PMID: 21668576
Burden; Clostridium difficile; cost; economics; stochastic model
13.  Clinical Characteristics of Bacteremia Due to Extended-Spectrum β-Lactamase (ESBL)-Producing Enterobacteriaceae in the Era of CTX-M and KPC-type β-Lactamases 
A multicenter, case-control study was conducted to assess risk factors and patient outcomes from bacteremia due to Enterobacteriaceae producing extended-spectrum β-lactamases (ESBL) and Klebsiella pneumoniae carbapenemases (KPCs). One hundred five and 20 patients with bacteremia due to ESBL and KPC-producing organisms were matched to controls that had bacteremia with non-ESBL/KPC-producing organisms, respectively. Independent risk factors for ESBL production included admission from a nursing home (odds ratio [OR], 4.64; 95% confidence interval [CI], 2.64–8.16), chronic renal failure (OR, 2.09; 95% CI, 1.11–3.92), the presence of a gastrostomy tube (OR, 3.36; 95% CI, 1.38–8.18), length of hospital stay before infection (OR, 1.02; 95% CI, 1.01–1.03), transplant recipients (OR, 2.48; 95% CI, 1.24–4.95) and receipt of antibiotics with Gram-negative activity in the preceding 30 days (OR, 1.76; 95% CI, 1.00–3.08). 28-day crude mortality rates for patients infected with ESBL or KPC-producing organisms and controls were 29.1% (34/117) and 19.5% (53/272), respectively (OR 1.70; 95% CI 1.04–2.80). On multivariate analysis, inadequate empiric therapy (OR, 2.26; 95% CI, 1.18–4.34), onset of bacteremia while in ICU (OR, 2.74; 95% CI, 1.47–5.11), Apache II score (OR, 1.17; 95% CI, 1.12–1.23), and malignancy (OR, 2.66; 95% CI, 1.31–5.41) were independent risk factors for mortality. CTX-M was the most common ESBL type in E. coli, whereas SHV predominated in Klebsiella spp. and Enterobacter spp.
PMCID: PMC3252485  PMID: 21951551
Enterobacteriaceae; bacteremia; extended-spectrum β-lactamase (ESBL); Klebsiella pneumoniae carbapenemase (KPC); carbapenem
14.  Multidrug resistant (MDR) Klebsiella pneumoniae clinical isolates: a zone of high heterogeneity (HHZ) as a tool for epidemiological studies 
Clinical Microbiology and Infection  2012;18(7):E254-E258.
Comparison of genome-wide, high-resolution restriction maps of Klebsiella pneumoniae clinical isolates, including an NDM-1 producer, and in silico-generated restriction maps of sequenced genomes revealed a highly heterogeneous region we designated the “high heterogeneity zone” (HHZ). The HHZ consists of several regions including a “hot spot” prone to insertions and other rearrangements. The HHZ is a characteristic genomic area that can be used in the identification and tracking of outbreak-causing strains.
PMCID: PMC3377791  PMID: 22551038
Klebsiella pneumoniae; Genomic analysis; optical map; NDM-1; ICE
15.  Evidence for a link between locus R-R sequence type and outcome of infection with Entamoeba histolytica 
Clinical Microbiology and Infection  2012;18(7):E235-E237.
The Entamoeba histolytica infections result from asymptomatic colonization to variable disease outcomes. However, markers that may predict infection outcomes are not known. Here, we investigated sequence types of a non-coding tRNA-linked locus R-R to identify surrogate markers that may show association with infection outcomes. Among 112 clinical samples -21 asymptomatic, 20 diarrhea/dysentery, and 71 liver abscesses (LA), we identified 11 sequence types. Sequence type 5RR mostly associated with asymptomatic samples, and sequence type 10RR predominantly associated with the symptomatic (diarrhea/dysentery and LA) samples. This is the first report that identifies markers that may predict disease outcomes in E. histolytica.
PMCID: PMC3377832  PMID: 22448930
16.  Management of multidrug-resistant enterococcal infections 
Enterococci are organisms with a remarkable ability to adapt to the environment and acquire antibiotic resistance determinants. The evolution of antimicrobial resistance in these organisms poses enormous challenges for clinicians when faced with patients affected with severe infections. The increased prevalence and dissemination of multidrug-resistant Enterococcus faecium worldwide has resulted in a major decrease in therapeutic options because the majority of E. faecium isolates are now resistant to ampicillin and vancomycin, and exhibit high-level resistance to aminoglycosides, which are three of the traditionally most useful anti-enterococcal antibiotics. Newer antibiotics such as linezolid, daptomycin and tigecycline have good in vitro activity against enterococcal isolates, although their clinical use may be limited in certain clinical scenarios as a result of reduced rates of success, possible underdosing for enterococci and low serum levels, respectively, and also by the emergence of resistance. The experimental agent oritavancin may offer some hope for the treatment of vancomycin-resistant enterococci but clinical data are still lacking. Thus, optimal therapies for the treatment of multidrug-resistant enterococcal infections continue to be based on empirical observations and extrapolations from in vitro and animal data. Clinical studies evaluating new strategies, including combination therapies, to treat severe vancomycin-resistant E. faecium infections are urgently needed.
PMCID: PMC3686902  PMID: 20569266
Antibiotics; enterococci; resistance; review; vancomycin
17.  Human milk oligosaccharide consumption by intestinal microbiota 
Human milk oligosaccharides (HMO) constitute the third most abundant class of molecules in breast milk. Since infants lack the enzymes required for milk glycan digestion, this group of carbohydrates passes undigested to the lower part of the intestinal tract, where they can be consumed by specific members of the infant gut microbiota. We review proposed mechanisms for the depletion and metabolism of HMO by two major bacterial genera within the infant intestinal microbiota, Bifidobacterium and Bacteroides
PMCID: PMC3671919  PMID: 22647041
Bacteroides; Bifidobacterium; human milk oligosaccharides; infant gut
18.  Post-diagnostic Kinetics of the (1→3)-β-D-Glucan Assay in Invasive Aspergillosis, Invasive Candidiasis, and Pneumocystis jirovecii Pneumonia 
The kinetics of serum (1→3)-β-D-glucan (BG) following the diagnosis of invasive fungal disease and administration of antifungal therapy are poorly characterized. It is unknown whether early BG changes have prognostic implications. We assessed the post-diagnostic kinetics of BG in patients with an initial serum BG ≥80 pg/mL and at least one additional post-diagnostic BG value in the setting of invasive aspergillosis (IA, n=69), invasive candidiasis (IC, n=40), or Pneumocystis jirovecii pneumonia (PCP, n=18), treated with antifungal therapy. Clinical failure of antifungal therapy and mortality were assessed at 6 and 12 weeks, and Cox modeling was used to assess the hazard of initial BG and change in BG at 1 or 2 weeks for these outcomes. In patients with ≥2 BG values, median initial BG was >500 pg/mL (IQR (interquartile range) 168, >500; range 80, >500) in IA, 136 pg/mL (IQR 88, >500; range 31, >500) in IC, and >500 pg/mL (IQR 235, >500; range 86, >500) in PCP. In patients with ≥2 BG values through one week after diagnosis, overall one-week decline in BG was 0 pg/mL (IQR 0, 53) in IA, 0 (IQR −65, 12) in IC, and 17 (IQR 0, 82) in PCP. Most patients with BG values through 6 and 12 weeks had persistent levels >80 pg/mL. Initial BG and the early trajectory of BG were not predictive of 6 or 12-week clinical failure or mortality. While BG eventually declines in patients with IA, IC, and PCP, it lacks prognostic value within a clinically meaningful time frame.
PMCID: PMC3618285  PMID: 22404638
beta-glucan; invasive aspergillosis; candidiasis; Pneumocystis jirovecii; invasive fungal disease; galactomannan; kinetics
19.  Evaluation of rK28 antigen for Serodiagnosis of Visceral Leishmaniasis in India 
Antibody detection is a safely applied method at wide scale in diagnosis of Visceral Leishmaniasis (VL). Towards further advancement of serodiagnosis, rK28 antigen has been recently introduced as a candidate for diagnosis of VL. We evaluated sensitivity and specificity of rK28 antigen in a micro-ELISA format in comparison to rk39 antigen. The test was conducted on 252 parasitologically confirmed VL cases, 103 endemic healthy controls, 95 non endemic healthy controls, 88 other infectious disease and 53 follow-up cases. Of 252 parasitologically confirm VL cases, 251 cases were reported positive by rK28 antigen yielding 99.6% sensitivity (95% CI, 0.97–0.99) which was similar to sensitivity of rK39 ELISA (99.6%) (95% CI, 0.97–0.99). Specificity of rK28 antigen in non-endemic and endemic healthy controls was 100% (95%CI 0.96–1) and 94.17% (95% CI, 0.88–0.97), respectively. In 88 different diseases, specificity was 95.45% (95% CI, 0.84–0.96). With rK39 antigen, specificity of non-endemic and endemic controls, and different diseases was 100% (95%CI 0.96–1) and 92.23% (95% CI 0.85–0.96), and 96.59% (95% CI 0.90–0.98) respectively. Our results show that rK39 and rK28 antigens have similar sensitivity and specificity and rK28 can also be used as a serodiagnostic tool in endemic population of Bihar.
PMCID: PMC3189430  PMID: 21722262
Visceral Leishmaniasis; rk-28; serodiagnosis; ELISA
20.  In vitro selection of influenza B viruses with reduced sensitivity to neuraminidase inhibitors 
We and others have previously isolated influenza B viruses with reduced sensitivity to neuraminidase (NA) inhibitors (oseltamivir and zanamivir) from patients who were never exposed to these drugs. It was unclear whether the NA substitutions found in these influenza B isolates arose spontaneously or were due to selective pressure. Here, we obtained influenza B viruses with reduced neuraminidase inhibitor sensitivity by in vitro selection with NA inhibitors. We found that these viruses possessed the same NA substitutions as those previously found in viruses isolated from untreated patients. These results suggest that these NA substitutions were selected in patients who were treated with an NA inhibitor and the resistant variants were then transmitted to others.
PMCID: PMC2980859  PMID: 20636420
Influenza B virus; neuraminidase; NA inhibitor; drug-resistant mutation; in vitro selection
21.  Serological survey of antibodies to influenza A viruses on a group of people without a history of influenza vaccination 
A serological survey for antibodies to influenza viruses was performed in China on a group of people without a history of influenza vaccination. Using the hemagglutination inhibition (HI) assay, we found seropositivity rates for seasonal H3N2 to be significantly higher than for seasonal H1N1. Samples positive for antibodies to pandemic (H1N1) 2009 virus increased from 0.6% pre-outbreak to 4.5% (p<0.01) at one year post-outbreak. Interestingly, HI and neutralization tests showed that 1.4% of people in the group have antibodies recognizing H9N2 avian influenza viruses, suggesting that infection with this subtype may be more common than previously thought.
PMCID: PMC3166400  PMID: 21749549
influenza; prevalence; antibody; pandemic H1N1; H9N2
22.  The effects of steroids during sepsis depend on dose and severity of illness: an updated meta-analysis 
A previous meta-analysis determined that the effects of steroids during sepsis were dose-dependent; since then, additional trials have been published. The current analysis updates our previous analysis examining the effects of steroids during sepsis. A literature search from 2004 to 2008 identified seven randomized controlled trials in adult patients; these were added to 14 previously identified trials. The effects of steroids on mortality were highly variable among the 21 trials (p <0.001, I2 = 60%). In trials published before 1989, which involved short courses of high-dose steroids, steroids increased mortality (n = 8, I2 = 14%, OR of death 1.39 (95% CI 1.04–1.86), p 0.03). In trials published after 1997, which involved longer courses of lower-dose steroids, steroids consistently improved shock reversal (n = 7, I2 = 0%, OR of shock reversal 1.66 [95% CI 1.25–2.20), p <0.001), but demonstrated a more heterogeneous beneficial effect on mortality (n = 12, I2 = 25%, OR of death 0.64 (95% CI 0.45–0.93), p 0.02). An inverse linear relationship between severity of illness and the effects of steroids on mortality was identified across all trials (p 0.03) and within the subgroup of trials published after 1997 (p 0.03); steroids were harmful in less severely ill patient populations and beneficial in more severely ill patient populations. There was no effect of response to adrenocorticotrophic hormone (ACTH) stimulation testing concerning the effects of steroids and no increase in steroid-associated adverse events. Low-dose steroids appear to improve mortality rates in patients with septic shock who are at high risk of death; however, additional trials in this subpopulation are necessary to definitively determine the role of low-dose steroids during sepsis.
PMCID: PMC3383780  PMID: 19416302
Corticosteroids; glucocorticoids; meta-analysis; review; sepsis; septic shock; steroids
23.  Strain diversity, epistasis and the evolution of drug resistance in Mycobacterium tuberculosis 
Mycobacterium tuberculosis harbours little DNA sequence diversity compared to other bacteria. However, there is mounting evidence that strain-to-strain variation in this organism has been underestimated. Here we review our current understanding of the genetic diversity among M. tuberculosis clinical strains and discuss the relevance of this diversity for the ongoing global epidemics of drug-resistant tuberculosis. Based on findings in other bacteria, we propose that epistatic interactions between pre-existing differences in strain genetic background, acquired drug resistance-conferring mutations and compensatory changes could play a role in the emergence and spread of drug-resistant M. tuberculosis.
PMCID: PMC3122159  PMID: 21682802
24.  Mycobacterium avium subsp hominissuis biofilm is composed of distinct phenotypes and influenced by the presence of antimicrobials 
Mycobacterium avium subsp hominissuis, hereafter referred to as M. avium, forms biofilm, a property that, in mice, is associated with lung infection via aerosol. As M. avium might co-inhabit the respiratory tract with other pathogens, treatment of the co-pathogen-associated infections, such as in bronchiectasis, would expose M. avium to therapeutic compounds which may have their origin in other organisms sharing the natural environments. Incubation of M. avium with two compounds produced by environmental organisms, streptomycin and tetracycline in vitro at sub-inhibitory concentrations increased biofilm formation in a number of M. avium strains, although exposure to ampicillin, moxifloxacin, rifampin, and TMP/SMX had no effect on biofilm. No selection of genotypically resistant clones was observed. While bacteria incubation in presence of streptomycin upregulates the expression of biofilm-associated genes, the response to the antibiotics had no association with a regulation of a regulator (LysR) linked to the formation of biofilm in M. avium.
Biofilms are made of planktonic and sessile bacteria. While planktonic M. avium is susceptible to clarithromycin and ethambutol (clinically used antimicrobials), sessile bacteria are at least 3- to 4-fold more resistant to antibiotics. The sessile phenotype, though, is reversible, and no selection of resistant clones was observed. Mice infected through the airway with both phenotypes were infected with a similar number of bacteria, demonstrating no phenotype advantage.
M. avium biofilm formation is enhanced by commonly used compounds and, in the sessile bacterial phenotype, is resistant to clarithromycin and ethambutol, in a reversible manner.
PMCID: PMC2978799  PMID: 20636426
M. avium; biofilm; gene expression; antibiotics
25.  Temporal Trends in Enterobacter Species Bloodstream Infection: A Population-Based Study, 1998-2007 
Enterobacter species are the fourth most common cause of gram-negative bloodstream infection (BSI). We examined temporal changes and seasonal variation in the incidence rate of Enterobacter spp. BSI, estimated 28-day and 1-year mortality, and determined in vitro antimicrobial resistance rates of Enterobacter spp. bloodstream isolates in Olmsted County, Minnesota, from 1/1/1998 to 12/31/2007. Multivariable Poisson regression was used to examine temporal changes and seasonal variation in incidence rate and Kaplan-Meier method to estimate 28-day and 1-year mortality. The median age of patients with Enterobacter spp. BSI was 58 years and 53% were female. The overall age- and gender-adjusted incidence rate of Enterobacter spp. BSI was 3.3/100,000 person-years (95% confidence interval [CI]: 2.3-4.4). There was a linear trend of increasing incidence rate from 0.8 (95% CI: 0-1.9) to 6.2 (95% CI: 3.0-9.3) per 100,000 person-years between 1998 and 2007 (p=0.002). There was no significant difference in the incidence rate of Enterobacter spp. BSI during the warmest four months compared to the remainder of the year (incidence rate ratio 1.06 [95% CI: 0.47-2.01]). The overall 28-day and 1-year mortality rates of Enterobacter spp. BSI were 21% (95% CI: 8-34%) and 38% (95% CI: 22-53%), respectively. Up to 13% of Enterobacter spp. bloodstream isolates were resistant to third-generation cephalosporins. To our knowledge, this is the first population-based study to describe the epidemiology and outcome of Enterobacter spp. BSI. The increase in incidence rate of Enterobacter spp. BSI over the past decade, coupled with its associated antimicrobial resistance, dictate more investigation of this syndrome.
PMCID: PMC2972367  PMID: 20518795
bacteremia; epidemiology; mortality; seasonal variation; incidence; Enterobacter; antimicrobial resistance

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