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The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.

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PURPOSE: Vancomycin-resistant Enterococcus (VRE) faecium (VREfm) are highly resistant bacteria emerging worldwide and rarely studied using molecular tools in Algeria since their first report in 2006. The aim of the study was to investigate healthcare-associated infections (HAIs) involving the first VRE in Batna University Hospital, Algeria, and characterize isolates using molecular tools. PATIENTS AND METHODS: Medical charts were reviewed for patients with VREfm. van genes were detected by multiplex polymerase chain reaction (PCR), and strains were characterized by automated repetitive sequence-based PCR (rep-PCR), multiplex rep-PCR, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). RESULTS: During a 6-month period, VREfm infections occurred in four patients hospitalized in three wards. The four isolates were E. faecium vanA belonging to the hospital-adapted clonal complex 17. PCR-based methods did not discriminate the isolates but MLST and PFGE delineated a subgroup of three VREfm of identical pulsotype and sequence type (ST) 80 (yet identified for five isolates in the international PubMLST database) while the fourth isolate was of ST789 (not previously identified for a VREfm) and displayed an unrelated pulsotype. The three genotypically related isolates were recovered in patients who underwent surgery in the same department, suggesting an outbreak for which the source and route of transmission remained unidentified. CONCLUSION: This first molecular epidemiology study of VRE in Algeria was useful in delimiting an outbreak involving three of the four HAI cases and revealed rarely encountered genotypes. Considering the threat and burden of VRE infections worldwide, particularly in the USA, and the late emergence in Algeria, our study supports the urgent need for improved and early adequate infection control measures to avoid VRE spread in North African hospitals.

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PURPOSE: Microbiological diagnosis (MD) of infections remains insufficient. The resulting empirical antimicrobial therapy leads to multidrug resistance and inappropriate treatments. We therefore evaluated the cost-effectiveness of direct molecular detection of pathogens in blood for patients with severe sepsis (SES), febrile neutropenia (FN) and suspected infective endocarditis (SIE). METHODS: Patients were enrolled in a multicentre, open-label, cluster-randomised crossover trial conducted during two consecutive periods, randomly assigned as control period (CP; standard diagnostic workup) or intervention period (IP; additional testing with LightCycler®SeptiFast). Multilevel models used to account for clustering were stratified by clinical setting (SES, FN, SIE). RESULTS: A total of 1416 patients (907 SES, 440 FN, 69 SIE) were evaluated for the primary endpoint (rate of blood MD). For SES patients, the MD rate was higher during IP than during CP [42.6% (198/465) vs. 28.1% (125/442), odds ratio (OR) 1.89, 95% confidence interval (CI) 1.43-2.50; P < 0.001], with an absolute increase of 14.5% (95% CI 8.4-20.7). A trend towards an association was observed for SIE [35.4% (17/48) vs. 9.5% (2/21); OR 6.22 (0.98-39.6)], but not for FN [32.1% (70/218) vs. 30.2% (67/222), P = 0.66]. Overall, turn-around time was shorter during IP than during CP (22.9 vs. 49.5 h, P < 0.001) and hospital costs were similar (median, mean ± SD: IP €14,826, €18,118 ± 17,775; CP €17,828, €18,653 ± 15,966). Bootstrap analysis of the incremental cost-effectiveness ratio showed weak dominance of intervention in SES patients. CONCLUSION: Addition of molecular detection to standard care improves MD and thus efficiency of healthcare resource usage in patients with SES. ClinicalTrials.gov registration number: NCT00709358.

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OBJECTIVES: Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics. METHODS: MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli). Time-kill assays with LPC-058 were performed on four MDR/XDR strains, including Escherichia coli producing CTX-M-15 ESBL and Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii producing KPC-2, VIM-1 and OXA-23 carbapenemases, respectively. RESULTS: LPC-058 was the most potent antibiotic and displayed the broadest spectrum of antimicrobial activity, with MIC90 values for Enterobacteriaceae, P. aeruginosa, Burkholderia cepacia and A. baumannii of 0.12, 0.5, 1 and 1 mg/L, respectively. LPC-058 was bactericidal at 1× or 2× MIC against CTX-M-15, KPC-2 and VIM-1 carbapenemase-producing strains and bacteriostatic at ≤4× MIC against OXA-23 carbapenemase-producing A. baumannii. Combinations of LPC-058 with ß-lactams, amikacin and ciprofloxacin were synergistic against these strains, albeit in a species-dependent manner. LPC-058's high efficacy was attributed to the presence of the difluoromethyl-allo-threonyl head group and a linear biphenyl-diacetylene tail group. CONCLUSIONS: These in vitro data highlight the therapeutic potential of the new LpxC inhibitor LPC-058 against MDR/XDR strains and set the stage for subsequent in vivo studies.

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We evaluated the DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in 55 urine samples. Crossing threshold (Ct) values were highly similar after 3 to 14 days at room temperature (+0.002, P = 0.99). Consequently, it does not seem necessary to transfer urine specimens into a transport medium in less than 24 hours as recommended by manufacturers.

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BACKGROUND: In infective endocarditis (IE), blood cultures are negative in 2.5-31% of cases because of a previously prescribed antimicrobial treatment. Molecular methods may represent an alternative to conventional microbiological techniques to identify the causative agent. The aim of this prospective study was to evaluate the performance of a new primer pair (341F/785R) for 16S rDNA amplification in heart valves compared with primers 91E/13BS already used for the diagnosis of IE. The primer pair 341F/785R was previously selected in silico to allow 16S rDNA amplification for a large coverage of bacterial species. RESULTS: A total of 74 patients suspected of having IE were included in this study. IE was diagnosed in 55 of these patients using the modified Duke criteria, which was the gold standard here. 91E/13BS primers were more sensitive than 341F/785R primers: 38/55 (69.1%) samples were positive using 91E/13BS primers against 28/55 (50.9%) with 341F/785R (p = 0.013). When at least one of the two molecular methods was positive, the sensitivity and specificity of 16S rDNA amplification was 72.7% and 94.7%, respectively. CONCLUSION: Even if the new primer pair 341F/785R seemed promising in silico, it was less sensitive for 16S rDNA amplification in heart valves than the 91E/13BS pair already used. This study underlines a lack of standardization for 16S rDNA amplification for clinical samples.

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BACKGROUND: Staphylococci, especially coagulase-negative staphylococci (CoNS) represent the most frequent micro-organism associated with osteoarticular infections (OAIs), especially those involving orthopedic devices. The antibiotic susceptibility profile of the bacteria mostly responsible for OAIs is therefore crucial information for choosing the appropriate antibiotic regimen administered during the removal procedure until the first results of the conventional culture. METHODS: The antibiotic susceptibility profile of staphylococci isolated from OAIs in a referent center for osteoarticular infection was studied over a 10-y period to adapt antibiotic protocols to the ecology. RESULTS: From 2002 to 2011, the resistance of Staphylococcus aureus to methicillin and rifampicin decreased (27.9% versus 20.6% and 13% versus 1%, respectively); the resistance to fluoroquinolones (FQ) was stable (24% on average), and all the isolates were susceptible to glycopeptides. For CoNS, the resistance to methicillin, rifampicin, and FQ increased (30.4% versus 43.9%, 13% versus 18.5%, and 20.3% versus 34.1%, respectively) over the same period. Resistance of the CoNS to vancomycin was observed in 2011 for the first time (2.3%), and 3.8% were resistant to teicoplanin in 2002 compared with 22% in 2011, with 3.5% resistant to linezolid in 2011. CONCLUSION: The sensibility of bacteria over 10 y remained stable, except for CoNS. The increase of the resistances for CoNS led us to exclude teicoplanin from the first-line empiric antibiotic treatment, to avoid linezolid, and to prefer vancomycin or daptomycin.

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We report a case of nosocomial mediastinitis and sternal osteitis due to M. hominis after open-heart surgery in an immuno-competent patient. This infection has been diagnosed by incubating the culture media for an extended period of time, and sequencing 16S rDNA directly from the clinical samples.

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Ruminococcus gnavus is an anaerobic Gram positive coccus that can be found in the gastrointestinal tract of animals and humans. We report a case of septic arthritis caused by R. gnavus that was identified by mass spectrometry and confirmed by 16S rRNA sequencing.

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We report the first case of postneurosurgical meningitis due to Psychrobacter sanguinis, identified only by 16S rRNA analysis. Psychrobacter spp. usually live in deep sea environments and cold habitats. Despite a strict questioning of the patient and the medical staff, we did not find the source of this bacterium.

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BACKGROUND: Tuberculosis (TB) is one of the major public health problems in Congo. However, data concerning Mycobacterium tuberculosis drug resistance are lacking because of the insufficient processing capacity. So, the aim of this study was to investigate for the first time the resistance patterns and the strain lineages of a sample of M. tuberculosis complex (MTBC) isolates collected in the two main cities of Congo. METHODS: Over a 9-day period, 114 smear-positive sputa isolated from 114 patients attending centers for the diagnosis and treatment of TB in Brazzaville and Pointe Noire were collected for culture and drug susceptibility testing (DST). Detection of mutations conferring drug resistance was performed by using line probe assays (GenoType MTBDRplus and MTBDRsl) and DNA sequencing. Strain lineages were determined by MIRU-VNTR genotyping. RESULTS: Of the 114 sputa, 46 were culture positive for MTBC. Twenty-one (46%) were resistant to one or more first-line antiTB drugs. Of these, 15 (71%) were multidrug resistant (MDR). The most prevalent mutations involved in rifampin and isoniazid resistance, D516V (60%) in rpoB and S315T (87%) in katG respectively, were well detected by MTBDRplus assay. All the 15 MDR strains were susceptible to fluoroquinolone and injectable second-line drug. No mutation was detected in the rrs locus involved in resistance to amikacin and capreomycin by both the MTBDRsl assay and DNA sequencing. By contrast, 9 MDR strains belonging to the same cluster related to T-family were identified as being falsely resistant to fluoroquinolone by the MTBDRsl assay due to the presence of a double substitution T80A-A90G in GyrA. CONCLUSIONS: Taken together, these data revealed a possible spread of a particular MDR clone in Congo, misidentified as fluoroquinolone resistant by MTBDRsl assay. Thus, this test cannot replace gold-standard culture method and should be interpreted carefully in view of the patient's native land.

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BACKGROUND: Helicobacter pylori (Hp), which is one of the causative agents in human gastric adenocarcinoma, is known to interact with mucous gel and alter mucin gene expression. The aim of this work was to study, using an in vitro model of cell infection, the effects of urease, flagellin, and CagA virulence factors on the regulation of the four 11p15 mucin genes (MUC2, MUC5AC, MUC5B, and MUC6). METHODS: KATO-III and AGS gastric cancer cells were infected for 1, 3 or 6 h with Hp wild-type strains (ATCC 43504, N6, and SS1) or corresponding isogenic mutants deficient for urease subunit B, flagellin subunit A, and CagA. mRNA levels of MUC2, MUC5B, MUC5AC and MUC6 were assessed by RT-PCR, and functional activity of their promoters was measured by transient transfection assays. RESULTS: Infection of KATO-III cells with Hp wild-type strains resulted in an early (at 1 h) transient expression of MUC2, MUC5AC, and MUC6 mRNA concomitant with those of interleukin (IL)-1ß, IL-8, and TNF-α cytokines. In these cells, the UreB(-) isogenic mutant induced strong activation of MUC5AC expression, and UreB-responsive elements were located in the -486/-1 region of the promoter. FlaA(-) and CagA(-) mutants had no effect on mucin gene mRNA levels in KATO-III cells. In AGS cells, Hp-responsive elements were identified in all promoters, and overexpression of NF-κB induced upregulation of MUC5AC promoter activity when infected with the UreB(-) isogenic mutant. CONCLUSION: These results indicate that Hp infection of gastric cancer cells alters 11p15 mucin gene transcription and that MUC5AC downregulation is mediated by urease virulence factor.

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INTRODUCTION: This report describes the first case, to the best of our knowledge, of pacemaker-induced endocarditis due to Gordonia bronchialis. PRESENTATION: Pacemaker-induced endocarditis due to G. bronchialis infection was determined in a 92-year old man. This Gram-positive bacillus failed to be identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology, whereas the taxon was indexed in the database. 16S rRNA and rpoB gene sequencing were required to determine the correct strain identity. CONCLUSION: Infections caused by G. bronchialis remain a rare phenomenon affecting immunocompromised patients and/or medical device carriers. Molecular tools may be necessary to ensure accurate identification.

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Nucleic acid amplification techniques (NAATs) represent a major advance in the diagnosis of Clostridium difficile (C. difficile) infection. This review analyzes the different options available for a molecular diagnosis of C. difficile infection, as well as the strengths and weaknesses of NAATs. The performances of seven commercials NAATs are compared (BD GeneOhm Cdiff, Illumigene C. difficile, Xpert C. difficile, BD Max Cdiff, Portrait Toxigenic C. difficile, ProGastro Cd, Seeplex Diarrhea ACE). The sensitivity and the rapidity of NAATs are excellent: additional efforts should focus on the discrimination between infection and colonization. Reporting the DNA load of toxigenic C. difficile in the stool sample may represent a solution. Diagnostic algorithms combining immunoassays and NAATs could also improve the specificity and reduce the global cost of this analysis.

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We evaluated retrospectively the yield of stool culture (SC) depending on the length of hospitalization, and we characterized the patients missed by the 3-day rejection rule. SC detects bacterial enteric pathogens (Campylobacter spp., Salmonella enterica, Yersinia spp., Shigella spp.). During this 5-year study period, 13,039 SCs were requested, and 376 were positive (2.9%). The yield of SC dropped from 11.7% before 3 days of hospitalization to 0.7% after 3 days in children and 4.3% to 0.3% in adults. Finally, only 13 clinically relevant cases (0.2% of SC prescribed after 3 days) were undiagnosed by strict application of the 3-day rule. In conclusion, rejection of SC prescribed after 3 days of hospitalization allows to reduce workload by 37.8% for children and 65.7% for adults, representing a cost of €12,500 ($16,250) per year in our hospital.

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The study through Monte Carlo simulation of ß-lactam pharmacokinetic/pharmacodynamic target attainment and determination of subsequent serum concentrations of piperacillin-tazobactam administered through continuous infusion to children treated for fever and neutropenia shows that 400 mg/kg/day has the highest probability of target attainment against Pseudomonas aeurginosa in our oncology ward compared with the standard regimen of 300 mg/kg/day.

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A polymerase chain reaction with an injection of the amplicons in an electrospray ionization mass spectrometry (PCR-ESI-MS) technique was evaluated for the diagnosis of bacterial and yeast pathogens on 13 cardiac valves with suspected endocarditis. At the moment of surgery, 3/13 PCR-ESI-MS results matched with microbiological documentation. Nine PCR-ESI-MS results correlated with Duke's criteria, leukocytes, C-reactive protein and blood cultures before surgery. The PCR-ESI-MS result of the last valve failed to confirm the blood culture result obtained fifteen days before. With speed and accuracy, this method may be useful to assert microbiological identification and adapt treatment.

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CHROMagar Yersinia (CAY) is a new chromogenic medium for the presumptive detection of virulent Yersinia enterocolitica in stools. Based on a comparative analysis of 1,494 consecutive stools from hospitalized patients, CAY was found to be just as sensitive as the reference medium (cefsulodin-irgasan-novobiocin agar) but was significantly more specific and had a very low false-positive rate. CAY reduces the workload (and thus costs) for stool analysis and can therefore be recommended for routine laboratory use.

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Pericarditis due to Listeria monocytogenes is a very uncommon and serious disease. We describe a case of fatal subacute pericarditis that was caused by L. monocytogenes in a 61-year-old woman with Hodgkin's disease who was diagnosed in 1975 and considered cured. In addition, we review the literature on this condition.

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