This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% ( = 101) of the XDR isolates, distantly followed by ST244 ( = 16), ST253 ( = 12), ST235 ( = 8), and ST111 ( = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital ( = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the β-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in β-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

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The objective of this study was to assess the antimicrobial resistance of enteroaggregative Escherichia coli (EAEC) and enterotoxigenic E. coli (ETEC) strains causing traveler’s diarrhea (TD) and to investigate the molecular characterization of antimicrobial resistance genes to third-generation cephalosporins, cephamycins, and quinolones. Overall, 39 EAEC and 43 ETEC clinical isolates were studied. The susceptibilities of EAEC and ETEC against ampicillin, amoxicillin-clavulanic acid, cefotaxime, imipenem, chloramphenicol, tetracycline, co-trimoxazole, nalidixic acid, ciprofloxacin, azithromycin, and rifaximin were determined. All genes encoding resistance determinants were detected by PCR or PCR plus DNA sequencing. The epidemiology of selected EAEC and ETEC strains was studied using multilocus sequence typing (MLST). The resistance to quinolones of EAEC and ETEC strains causing TD has significantly increased over the last decades, and high percentages have been found especially in patients traveling to India and sub-Saharan Africa. Sequence type 38 (ST38) and ST131, carrying the blaCTX-M-15 and blaCTX-M-27 genes, respectively, are highly prevalent among extended-spectrum β-lactamase (ESBL)-producing EAEC and ETEC strains. The cephamycinase ACT-20 is described in the present study for the first time in EAEC and ETEC strains causing TD in patients who had traveled to Central America. The percentages of resistance to azithromycin in EAEC and ETEC isolates from patients to Southeast Asia/India and Africa are above 25%. Meanwhile, rifaximin is still active against EAEC and ETEC, with the prevalence of resistant strains not being high. In conclusion, fluoroquinolones should no longer be considered the drugs of choice for the prevention or treatment in TD for travelers traveling to India and Africa. Azithromycin and rifaximin are still a good alternative to treat TD caused by EAEC or ETEC.

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IntroductionDiarrhea is a frequent complication in hematologic patients, being an infectious cause frequently suspected. Rapid and accurate detection of gastrointestinal pathogens is vital in immunocompromised hosts. The aim of this study was to compare routine diagnostic methods versus a multiplex polymerase chain reaction (PCR) assay for the diagnosis of infectious diarrhea in immunocompromised hematologic patients.Material and methodsWe conducted a prospective observational study from March 2015 to January 2016 to compare conventional methods for the diagnosis of infectious diarrhea with FIlmArray GI Panel (BioFire-bioMérieux, France). Samples from adult immunocompromised hematologic patients with acute diarrhea were collected. In cases with discordant results, a second multiplex assay was performed (Allplex, Seegene, Korea). The result was considered positive or negative when the same result was obtained by at least two of the methods.ResultsA total of 95 samples were obtained from 95 patients (median age of 52 years (46–64)). Sixty-one (64%) episodes were hospital-acquired and 34 (36%) were community-acquired diarrhea. Twenty-five (26%) patients had a positive microbiological result, being Clostridium difficile the most frequent pathogen, followed by Campylobacter spp and norovirus. The concordance between FilmArray methods was good (k = 0.79). The FilmArray GI panel showed a sensitivity of 95%, a specificity of 100% for positive results. The time required to obtain results was markedly reduced with the use of multiplex PCR methods.ConclusionsMultiplex molecular panels provide a rapid and sensitive tool for the diagnosis of infectious diarrhea, thereby allowing more timely clinical decisions in immunocompromised hematologic patients.

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Background Dengue virus (DENV) is the most important arbovirus worldwide, causing infections in endemic countries and returning travellers from these areas. Rapid diagnostic tests are needed to improve patient management and monitor local transmission. The detection of DENV non-structural protein 1 (NS1) is a useful tool for the diagnosis, but the currently available methods can be time consuming or lack sensitivity. The objective of our study was to evaluate a new rapid and semi-quantitative microfluidic DENV NS1 immuno-magnetic agglutination assay based on aggregation of magnetic nanoparticles detected by an electronic reader (Virotrack Dengue Acute and Blubox, Blusense diagnostics, Copenhagen, Denmark). Methodology/Principal findings A panel of 135 serum samples from travelers returning from dengue endemic countries was analyzed (74 DENV positive samples including the four DENV serotypes, 26 Zika virus positive samples, 25 chikungunya virus positive samples, 5 malaria positive samples and 5 negative samples). Samples were tested by three different antigen detection methods: SD Dengue NS1 Ag ELISA, SD BIOLINE Dengue Duo and ViroTrack Dengue Acute. The sensitivity observed for SD Dengue NS1 Ag ELISA, ViroTrack Dengue Acute and SD BIOLINE Dengue Duo was 97.2%, 91.1% and 68.1%, respectively. All methods showed high specificity (98.4% for ViroTrack Dengue Acute and 100% for both SD Dengue NS1 Ag ELISA and SD BIOLINE Dengue Duo). SD Dengue NS1 Ag ELISA and ViroTrack Dengue Acute only failed to detect samples positive for DENV-2. Conclusions/Significance ViroTrack Dengue Acute is a sensitive and specific assay for DENV NS1 detection. It provides faster results than the ELISA method and a better performance than the rapid

Malaria, arbovirus infection and travelers' diarrhea are among the most common etiologies of fever after a stay in the tropics. Because the initial symptoms of these diseases often overlap, the differential diagnostic remains a challenge. The aim of this study was to establish the effectiveness of platelet and leukocyte counts in the differential diagnosis of fever in the returning traveler. Between 2013 and 2016, patients with a clinical suspicion of malaria, who had thick blood smears performed were retrospectively included. The microbiological etiology of each episode was established based on molecular detection in the case of arbovirus infection, the detection of pathogens in stool samples for diarrhea and other gastrointestinal symptoms and the thick and thin blood smear results for malaria. A total of 1,218 episodes were included. Malaria, arbovirus infection, and diarrhea and other gastrointestinal symptoms caused 102 (8.4%), 68 (5.6%), and 72 (5.9%) episodes, respectively. The median platelet counts in malaria episodes were 89 × 10 9 /L and thrombocytopenia (< 150,000 × 10 9 platelets/L) yielded a 98% negative predictive value to predict malaria. The median leukocyte counts in arbovirus infection episodes were 3.19 × 10 9 /L and leucopenia (< 4 × 10 9 leukocytes/L) yielded a 97.9% negative predictive value to predict arbovirus infections. Platelet and leukocyte counts were not significantly altered in episodes caused by diarrhea and other gastrointestinal symptoms. Initial platelet and leukocyte counts might be useful for the clinical differential diagnosis of fever in the returning traveler. Although these results are insufficient to establish a diagnosis, they should be considered in the initial clinical assessment.