Clinical microbiology and infection control

Objectives: Our objective was to examine whether or not women with symptoms of a urinary tract infection but with a negative culture (20%e30%) do have an infection. Methods: We performed quantitative PCR (qPCR) for Escherichia coli and Staphylococcus saprophyticus, on top of a standard culture, in urine samples from 220 women with dysuria and/or frequency and/or urgency and from 86 women without symptoms. For symptomatic women, qPCR was also carried out for four sexually transmitted agents. Results: In the symptomatic group, 80.9% (178/220) of the urine cultures were positive for any uropathogen and 95.9% (211/220) were E. coli qPCR-positive. For the control group, cultures for E. coli and E. coli qPCR were positive in, respectively, 10.5% (9/86) and 11.6% (10/86). In the symptomatic group, qPCR yielded 19 positive samples for S. saprophyticus qPCR, one positive sample for Mycoplasma genitalium and one for Trichomonas vaginalis. Conclusions: These findings suggest that almost all women with typical urinary complaints and a negative culture still have an infection with E. coli.

There are currently no standardized diagnostic tests available for the reliable detection of AmpC b-lactamases in Klebsiella spp., Escherichia coli, Proteus mirabilis and Salmonella spp. A study was designed to evaluate a confirmation disk test using cefotetan (CTT) and cefoxitin (FOX) with phenylboronic acid (PBA). It also investigated the most suitable screening concentrations of FOX, ceftriaxone (CRO) and ceftazidime (CAZ) for the detection of AmpC b-lactamases. A total of 126 control (consisting of 11 laboratory and 115 wellcharacterized clinical strains) and 29 840 non-repeat clinical isolates were included. FOX with PBA used in a confirmation test and CRO and CAZ as screening agents were found to be unreliable. FOX at ‡ 32 mg/L was the best screening agent and CTT with PBA was the best confirmation test. Of the clinical isolates 635 (2%) were found to be resistant to cefoxitin (MIC ‡ 32 ug/mL) and 332 (52%) were AmpC positive. E. coli was the most common organism with AmpC b-lactamases and was mostly present in urines from community patients. It is recommended that laboratories use FOX at 32 mg/L as a screening agent and perform a disk test with CTT and PBA to confirm the presence of an AmpC cephalosporinase in isolates of Klebsiella spp., E. coli, Salmonella spp. and P. mirabilis. This approach is convenient, practical and easy to incorporate into the workflow of a clinical laboratory. False-positive AmpC detection may occur with KPC-producing bacteria when inhibitor-based methods are used.

Antimicrobial resistance is of concern to global health security worldwide. We aimed to identify the prevalence, resistance patterns, and risk factors associated with Escherichia coli (E. coli) resistance from poultry farms in Kelantan, Terengganu, and Pahang states of east coast peninsular Malaysia. Between 8 February 2019 and 23 February 2020, a total of 371 samples (cloacal swabs = 259; faecal = 84; Sewage = 14, Tap water = 14) were collected. Characteristics of the sampled farms including management type, biosecurity, and history of disease were obtained using semistructured questionnaire. Presumptive E. coli isolates were identified based on colony morphology with subsequent biochemical and PCR confirmation. Susceptibility of isolates was tested against a panel of 12 antimicrobials and interpreted alongside risk factor data obtained from the surveys. We isolated 717 E. coli samples from poultry and environmental samples. Our findings revealed that cloacal (17.8%, 46/259), faecal (22.6%, 19/84), sewage (14.3%, 2/14) and tap water (7.1%, 1/14) were significantly (p < 0.003) resistant to at least three classes of antimicrobials. Resistance to tetracycline class were predominantly observed in faecal samples (69%, 58/84), followed by cloacal (64.1%, 166/259), sewage (35.7%, 5/14), and tap water (7.1%, 1/84), respectively. Sewage water (OR = 7.22, 95% CI = 0.95-151.21) had significant association with antimicrobial resistance (AMR) acquisition. Multivariate regression analysis identified that the risk factors including sewage samples (OR = 7.43, 95% CI = 0.96-156.87) and farm size are leading drivers of E. coli antimicrobial resistance in the participating states of east coast peninsular Malaysia. We observed that the resistance patterns of E. coli isolates against 12 panel antimicrobials are generally similar in all selected states of east coast peninsular Malaysia. The highest prevalence of resistance was recorded in tetracycline (91.2%), oxytetracycline (89.1%), sulfamethoxazole/trimethoprim (73.1%), doxycycline (63%), and sulfamethoxazole (63%). A close association between different risk factors and the high prevalence of antimicrobial-resistant E. coli strains reflects increased exposure to resistant bacteria and suggests a concern over rising misuse of veterinary antimicrobials that may contribute to the future threat of emergence of multidrug-resistant pathogen isolates. Public health interventions to limit antimicrobial resistance need to be tailored to local poultry farm practices that affect bacterial transmission.

In patients with community-onset acute pyelonephritis (CO-APN), assessing the risk factors for poor clinical response after 72 h of antibiotic treatment (early clinical failure) is important. The objectives of this study were to define those risk factors, and to assess whether early clinical failure influences mortality and treatment outcomes. We prospectively collected the clinical and microbiological data of women with CO-APN in South Korea from March 2010 to February 2012. The numbers of cases in the early clinical success and early clinical failure groups were 840 (79.1%) and 222 (20.9%), respectively. Final clinical failure and mortality were higher in the early clinical failure group than in the early clinical success group (14.9% vs 2.3%, p <0.001; 6.8% vs 0.1%, p 0.001, respectively). In a multiple logistic regression model, the risk factors for early clinical failure among the total 1062 patients were diabetes mellitus (OR 1.5; 95% CI 1.1-2.1), chronic liver diseases (OR 3.3; 95% CI 1.6-6.7), malignancy (OR 2.2; 95% CI 1.1-4.4), Pitt score ≥2 (OR 2.5; 95% CI 1.6-3.8), presence of azotaemia (OR 1.8; 95% CI 1.2-2.7), white blood cell count ≥20 000/mm 3 (OR 2.5; 95% CI 1.6-4.0), serum C-reactive protein level ≥20 mg/dL (OR 1.7; 95% CI 1.2-2.4), and history of antibiotic usage within the previous year (OR 1.5; 95% CI 1.1-2.2). Analysing the subgroup of 743 patients with CO-APN due to Enterobacteriaceae, fluoroquinolone resistance of the uropathogen was another factor associated with early clinical failure (OR 1.7; 95% CI 1.1-2.5). Simple variables of underlying diseases, previous antibiotic usage and initial laboratory test outcomes can be used to decide on the direction of treatment in CO-APN.

Objective To detect the incidence of drug-resistant Plasmodium falciparum malaria infection in immigrants and travellers in non-endemic Kuwait. Methods Over a period of 3 years, July 1995 to September 1998, 1352 malaria patients were enrolled in the study. Of these, 1293 were immigrants from countries where malaria is endemic and 59 were non-immune travellers with a recent history of travel to these countries. The in vitro drug sensitivity was determined in 892 patients. Results In all, 892 of 1352 (66.0%) P. falciparum isolates were successfully cultured in vitro for drug sensitivity and 419 (47.0%) isolates showed in vitro resistance to chloroquine or mefloquine. Fifty-six (13.4%) isolates were resistant to both drugs. Chloroquine resistance was observed in >70% of the isolates from Africa and India followed by Pakistan (39.9%) and Bangladesh (35.9%). The resistance to mefloquine ranged from 26.2% in isolates from Sri Lanka to 47.5% in isolates from African countries. The study highlights the important trend in drug resistance in P. falciparum malaria in immigrants from south-east Asian and African countries.

We evaluated the sensitivity of surveillance cultures for carbapenem-resistant Acinetobacter baumannii (CRAB) in patients and in their environment. Patients with a CRAB positive clinical culture were sampled within 7 days; the buccal mucosa and rectum were sampled using swabs, and skin was sampled using pre-moistened sterile sponges. Sponges were also used to sample the surrounding environment. Specimens were inoculated onto CHROMagar MDR Acinetobacter plates both directly and after overnight enrichment. CRAB load was scored semi-quantitatively and composite scores for patient colonization and environmental contamination were calculated. Thirty-four patients were included. Screening sensitivity was 28/34 (82%) for buccal mucosa, 30/34 (88%) for skin, and 25/34 (74%) for rectum. Combined sensitivity was 32/34 (94%). Among patients with CRAB-positive respiratory cultures, sensitivity for buccal mucosa was 20/20 (100%). Direct inoculation had excellent sensitivity: 25/28 (89%) for all ...

Knowledge of beta-lactam resistance rates in Streptococcus mitis group bacteria is especially important in neutropenic haematological patients as adequate empirical antibiotic treatment (often including agents like meropenem [1]) is paramount. EUCAST guidelines allow one to infer viridans group streptococci (VGS) beta-lactam susceptibility from benzylpenicillin susceptibility; penicillin-resistant isolates should be tested for individual agents. Few studies reported the association between penicillin resistance and broad-spectrum beta-lactam resistance in VGS [2,3], and data from Northern European countries is lacking. Here, we first describe the prevalence of beta-lactam resistance in S. mitis/oralis in The Netherlands. Second, we describe the association between penicillin MIC and broad-spectrum beta-lactam resistance. The Dutch national surveillance system for antimicrobial resistance (ISIS-AR) [4], covering 44 out of 56 Dutch clinical microbiology laboratories in 2017, was searched for susceptibility results of S. mitis/oralis isolates for (benzyl)penicillin, amoxicillin/ampicillin, cefotaxime/ceftriaxone, and meropenem from 2013 through 2017.

Correct detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control and antibiotic choice. We performed a study to determine the cost-effectiveness of phenotypical testing, which can be inaccurate, and genotypical tests, which are considered to be more reliable but also more expensive. All patients that had been in isolation in the Amphia hospital because of the detection of ESBL according to the ESBL Etest were included in the survey. All strains were retested using the double disk confirmation test (DDCT) and a genotypical method. This was a commercially available microarray (Check-Points). Discordant results were confirmed by PCR and sequencing. In total 174 patients were included. In 24 of 174 (14%) patients, ESBL carriage could not be confirmed with the microarray. This was verified with PCR and sequencing. The mean duration of isolation was 15 days, adding up to a total number of isolation days of 2571. False-positive results according to the microarray resulted in a total of 279 days of unnecessary isolation for the Etest and 151 days for the DDCT. Using Etest to detect the presence of ESBL results in a false-positive outcome in 14% of the cases. This results in unnecessary isolation of patients, which can be omitted by using a genotypic method.

Objectives: Antibacterial resistance is emerging in patients undergoing haematopoietic stem cell transplantation (HSCT), and most data on the epidemiology of bloodstream infections (BSI)-causing pathogens come from retrospective single-centre studies. This study sought to investigate trends in the epidemiology of BSI in HSCT patients from a prospective multicentre cohort. Methods: We investigated changes in the incidence of causative organisms of BSI during neutropenia among adult HSCT patients for 2002e2014. The data were collected from a prospective cohort for infection surveillance in 20 haematologic cancer centres in Germany, Austria and Switzerland (ONKO-KISS). Results: A total of 2388 of 15 181 HSCT patients with neutropenia (1471 allogeneic (61.6%) and 917 autologous (38.4%) HSCT) developed BSI (incidence 15.8% per year). The incidence of Gram-negative BSI increased over time both in patients after allogeneic HSCT (allo-HSCT) and autologous HSCT (auto-HSCT). BSI caused by Escherichia coli in allo-HSCT patients increased from 1.1% in 2002 to 3.8% in 2014 (3/279 vs. 31/810 patients, p <0.001), and the incidence of BSI caused by enterococci increased from 1.8% to 3.3% (5 vs. 27 patients, p <0.001). In contrast, the incidence of BSI due to coagulase-negative staphylococci decreased in allo-HSCT patients from 8.2% to 5.1%, (23 vs. 40 patients, p <0.001) and in auto-HSCT patients from 7.7% to 2.0% (13/167 vs. 30/540 patients; p ¼ 0.028 for period 2002e2011). No significant trends were observed for the incidence of BSI due to methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci or extended-spectrum b-lactamase-producing Enterobacteriaceae. The BSI case fatality remained unchanged over the study period (total of 477 fatalities, 3.1%). Conclusions: The incidence of Gram-negative BSI significantly increased over time in this vulnerable patient population, providing evidence for reevaluating empiric therapy for neutropenic fever in HSCT patients.

This prospective study determined the antibiotic susceptibility of 164 isolates of Escherichia coli from the urine of 164 patients (112 female, 52 male; mean age of 54.12 years) with community-acquired urinary tract infection (UTI). Half of the isolates were from uncomplicated UTI and half from complicated UTI (52 males and 34 females). Overall, 57.3% of isolates were resistant to ampicillin, 25% to co-trimoxazole, 20.1% to nalidixic acid, 14% to norfloxacin and ciprofloxacin, and 0% to fosfomycin and nitrofurantoin. Of the 82 isolates from complicated UTI, 16 (19.5%) were resistant to norfloxacin and ciprofloxacin, compared with seven (8.5%) from uncomplicated UTI (p 0.043). Isolates from patients aged >50 years were significantly more resistant than those from patients aged <50 years for nalidixic acid (p 0.007) and the fluoroquinolones tested (p 0.015). Resistance to fluoroquinolones was 25% (13 ⁄ 52) in males and 9% (10 ⁄ 112) in females (p 0.006). For patients with and without previous antimicrobial therapy, there was a significant difference only for resistance to nalidixic acid (p < 0.001) and the fluoroquinolones (p 0.011). There were adequate susceptibility rates to fosfomycin, nitrofurantoin and the fluoroquinolones for empirical use in the treatment of acute uncomplicated UTI. In order to interpret cumulative susceptibility data from the primary healthcare setting, it is necessary to take into account the type of UTI (uncomplicated vs. complicated), previous antimicrobial therapy, and the sex and age of each patient.

Carbapenem-resistant Acinetobacter baumannii (CR-Ab) ranked third, with a frequency of 24.8%, among 202 strains of multidrug-resistant bacteria isolated from clinical samples in the main hospital of New Caledonia in 2004. All CR-Ab isolates were analysed by isoelectric focusing, conjugation, pulsed-field gel electrophoresis and PCR for the presence of carbapenemase genes. Fifty CR-Ab isolates produced carbapenemase OXA-23. The isolates belonged to a single clone presenting several subtypes, suggesting an endemic situation. This study further illustrates the widespread prevalence of carbapenemase OXA-23-producing CR-Ab isolates in the South Pacific.

Solid organ transplant recipients (SOTR) are at risk of serious influenza-related complications. The impact of respiratory co-infection in SOTR with 2009 pandemic influenza A(H1N1) is unknown. A multicentre prospective study of consecutive cases of pandemic influenza A(H1N1) in SOTR was carried out to assess the clinical characteristics and outcome and the risk factors for co-infection. Overall, 51 patients were included. Median time from transplant was 3.7 years, 5.9% of the cases occurred perioperatively and 7.8% were hospitalacquired. Pneumonia was diagnosed in 15 (29.4%) patients. Ten cases were severe (19.6%): 13.7% were admitted to intensive care units, 5.9% suffered septic shock, 5.9% developed acute graft rejection and 7.8% died. Co-infection was detected in 15 patients (29.4%): eight viral, six bacterial and one fungal. Viral co-infection did not affect the outcome. Patients with non-viral co-infection had a worse outcome: longer hospital stay (26.2 ± 20.7 vs. 5.5 ± 10.2) and higher rate of severe diseases (85.7% vs. 2.3%) and mortality (42.8% vs. ). Independent risk factors for non-viral co-infection were: diabetes mellitus and septic shock. Other factors associated with severe influenza were: delayed antiviral therapy, diabetes mellitus, time since transplantation <90 days and pneumonia. In conclusion, pandemic influenza A can cause significant direct and indirect effects in SOTR, especially in the early post-transplant period, and should be treated early. Clinicians should be aware of the possibility of non-viral co-infection, mainly in diabetic patients and severe cases. An effort should be made to prevent influenza with immunization of the patient and the environment.

The relevance of microorganisms in preterm birth is still under discussion. Using a diagnostic fluorescence in situ hybridization probe panel, we visualized Staphylococcus aureus and Streptococcus mitis group in two cases of acute chorioamnionitis. This technique provides spatial resolution and quantity of bacteria, clarifying the epidemiology and pathogenic pathways of acute chorioamnionitis.

Objectives: This study aimed at evaluating the reliability of self-reported recent antibiotic use, including ability to name the antibiotic, among pharmacy customers from the general population. Methods: This cross-sectional observational study of adults took place in pharmacies in northeastern France from January to April 2016. Participants were asked if they had used any antibiotics in the preceding 4 months and if so, to name them. Their reports were compared with an electronic pharmacy dispensing record listing all of the medication dispensed to them in France. The study included 653 individual customers from 15 pharmacies. Antibiotics had been dispensed 325 times, according to the records, but the patients reported only 237 courses of treatment. Agreement between self-report and the electronic record about whether or not they had used an antibiotic was 80% (k 0.60). Among the discordant responses, 57% (81/143) involved underreporting. Only 24% (79/325) of the patients could name the antibiotic for each course of treatment; 49% (160/325) could not. The multivariate analysis showed that patients who had purchased an antibiotic in the previous 30 days were 2.5 times more likely to know its name (p 0.01). Conclusions: Participants' self-reports were relatively reliable for recent use of an antibiotic, but not for its name. Because physicians cannot base prescription decisions on these self-reports only, an electronic pharmacy dispensing file shared among prescribers would be useful.

The objective of this study was to determine the molecular epidemiology of Salmonella typhi isolates from patients with typhoid fever in southeast Turkey and to determine the relationship between the clinical strains and environmental subtypes. Both demographic and clinical data were obtained by chart review and compared to the molecular subtyping results.

While performing molecular confirmation of phenotypically identified Candida tropicalis isolates, we re-identified a few isolates as Kodamaea ohmeri. This led us to the present epidemiological investigation of K. ohmeri fungaemia cases. All phenotypically identified C. tropicalis blood isolates during October 2008 through to December 2009 at our advanced paediatric centre were included for molecular identification by sequencing of the internal transcribed spacer and D1/D2 regions of rDNA. After identifying a large cluster K. ohmeri fungaemia cases, a case-control study was carried out retrospectively to analyse potential risk factors for K. ohmeri fungaemia. Molecular typing of the isolates was performed using a fluorescent amplified fragment length polymorphism (FAFLP) technique. The antifungal susceptibility testing was performed as per the M27-A3 protocol of CLSI. Thirty-eight (25.7%) of 148 phenotypically identified C. tropicalis isolates were confirmed as K. ohmeri by sequencing and FAFLP. By case-control analysis, piperacillin-tazobactam was significantly associated with the K. ohmeri fungaemia. The FAFLP analysis showed that all K. ohmeri isolates had >92% similarity. The azoles and echinocandins had good in vitro activity against K. ohmeri, though 86.8% of the isolates had MIC of 1 mg/L for amphotericin B. The response to antifungal therapy could be evaluated in 27 patients and 70.4% of patients recovered after antifungal therapy. The present study reports the largest cluster of K. ohmeri fungaemia from a single centre. The study also stresses the need for accurate identification of clinical yeast isolates.

Objective: TO determine the nature (clonal type and antibiotic resistance pattern) of methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from the largest teaching hospital in Portugal and to detect temporal trends in clonal types during three consecutive surveillance periods in 1992-93,1994-95 and 1996. Methods: MRSA strains were characterized by chromosomal Smal macrorestriction patterns using pulsed-field gel electrophoresis (PFGE) and by DNA fingerprints-applied to Clal digests-apable of probing two specific areas of the staphylococcal chromosome: (1) the vicinity of the mecA gene, and (2) the attachment site(s) and copy number of transposon Tn554. The combination of these methods can generate 'clonal types' useful for epidemiological tracking of MRSA strains. Results: During the 1992-93 collection period, 65% of MRSA strains carried the mecA polymorph I, Tn554 pattern E and PFGE pattern A (1::E::A)-a clonal type that was used to define the 'Iberian MRSA', which is widely spread throughout southern Europe. The representation of this clone decreased to 42% in 1994-95 and to 20% in 1996. At the same time, a second multiresistant MRSA strain carrying mecA polymorph XI, Tn554 type B and PFGE pattern B (XI::B::B)-a clonal type characteristic of the so-called 'Brazilian MRSA '-increased from 5% in 1992-93 to 36% in 1994-95 and 29% in 1996. Conclusions: Throughout the four years of surveillance, the Iberian and Brazilian MRSA types and their single subtype variants together have been responsible for the overwhelming majority (close to 90%) of all MRSA infections in the largest teaching hospital of Portugal. The mechanism of epidemicity of these two multiresistant international MRSA clones remains to be elucidated.

BackgroundThis study aims to estimate the prevalence of COVID-19 in the general population of Iran.MethodsThe target population was all Iranian people aged six years and older in the country. A stratified random sampling design was used to select 28,314 subjects from among the individuals registered in the electronic health record systems used in primary health care in Iran. Venous blood was taken from each participant and tested for the IgG antibody against COVID-19. The prevalence of COVID-19 was estimated at provincial and national levels after adjusting for the measurement error of the laboratory test, non-response bias, and sampling design.ResultsOf the 28,314 Iranians selected, 11,256 (39.75%) participated in the study. Of these, 5406 (48.0%) were male, and 6851 (60.9%) lived in urban areas. The mean (standard deviation) participant age was 35.89 (18.61) years. The adjusted prevalence of COVID-19 until August 20, 2020 was estimated as 14.2% (95% uncertainty interval: 13.3%, 15...

A recent Supplement to Clinical Microbiology and Infection entitled 'Infection control measures to limit the spread of C. difficile' pointed out that the incidence of C. difficile-associated diarrhoea (CDAD) has been increasing worldwide, and stressed the importance of research in the fields of epidemiology and infection control [1]. Since 2003, one of the main causes of the increasing prevalence of CDAD has been claimed to be the emergence of PCR ribotype 027/NAP1, which has caused epidemics in North America, the UK, the Netherlands, Belgium and France. The presence of PCR ribotype 027 in Austria, Japan, Ireland, Germany and Switzerland has also been reported recently [2,3]. The majority of publications have emphasized that the presence of this strain is usually associated with more severe symptoms and signs than those associated with the other more common toxinpositive strains [4,5]. Whereas PCR ribotype 027 was present in the population earlier, the majority of the historic strains were fluoroquinolone sensitive [6]. The overuse of antibiotics such as fluoroquinolones may lead to the selection and emergence of resistant strains, and may contribute to the spread of PCR ribotype 027, which is usually resistant to erythromycin. Here, the Eastern European spread of C. difficile PCR ribotype 027 is reported.

COVID-19 or SARS-COV-2 becomes the universal deadly health alarm from December 2019 to till date with the global cases record of 180,777,842 as well as India’s cases record of 30,134,455 till 25th June, 2021. As the country&#39;s health system tries to recover from corona crisis, another fungal threat comes in existence known as Mucormycosis or Black fungus Infection, especially affecting the Asian continents like India with the presents recorded updates of more than 10,000 infected cases. Mucormycosis is opportunity seeking fungal infection mainly affects immunocompromised population such as diabetic ketoacidosis patients and those whose immunity has been already hijacked via SARS-CoV-2 virus, creates the fungal entry roadmap from various routes like inhalation, ingestion or inoculation to rule over the body. In the present review, we enlightened the Covid-19 tuned mucormycosis Indian epidemic figures, causative aspects, molecular studies or pharmacological and non-pharmacological ...

ObjectiveTo assess the effect of hydroxychloroquine (HCQ), Tenofovir disoproxil fumarate/Emtricitabine (TDF/FTC), and their combination as pre-exposure prophylaxis on the risk of symptomatic COVID-19.MethodsEPICOS is a double-blind, placebo-controlled randomized trial conducted in 51 hospitals in Spain, Bolivia, and Venezuela. Healthcare workers with negative SARS-CoV-2 IgM/IgG test were randomly assigned to: daily TDF/FTC plus HCQ for 12 weeks, TDF/FTC plus HCQ placebo, HCQ plus TDF/FTC placebo and TDF/FTC placebo plus HCQ placebo. The primary outcome was laboratory-confirmed, symptomatic COVID-19. We also studied any (symptomatic or asymptomatic) COVID-19 infection. We compared group-specific 14-week risks via differences and ratios with 95% confidence intervals (CI).ResultsOf 1002 individuals screened, 926 (92.4%) were eligible; 64.2% recruited in Spain, 22.3% in Bolivia, and 13.6% in Venezuela. Median age was 38 years (range 18 - 68), 62.5% were female, 62.3% worked at inpatient...

Whole genome sequencing (WGS) has the potential to revolutionize the diagnosis of Mycobacterium tuberculosis (MTB) but the lack of bioinformatic expertise among clinical microbiologists is a barrier for adoption. Software products for analysis should be simple, free of charge, able to accept data directly from the sequencer (FASTQ files) and to provide the basic functionalities all-in-one. The main aim of this narrative review is to provide a practical guide for the clinical microbiologist, with little or no practical experience of WGS analysis, with a specific focus on software products tailored made for MTB analysis. With sequencing performed by an external provider, it is now feasible to implement WGS analysis in the routine clinical practice of any microbiology laboratory, with the potential to detect resistances weeks before traditional phenotypic culture methods, but the clinical microbiologist should be aware of some major limitations.

Erythromycin and clindamycin are major alternatives to βlactam antibiotics. Bacterial resistance to macrolide and lincosamide antibiotics is increasing worldwide. Erm genes express either constitutive macrolide, lincosamide streptogramin B resistance phenotype (cMLSB) or inducible phenotype (iMLSB) and msrA gene expresses (MSB) phenotype. The aim of this study was to determine the frequency of MLSB resistance phenotypes and genotypes and correlation between them in erythromycin-resistant staphylococci. In present study, 154 staphylococcal isolates were collected from various clinical specimens from Cairo University Specialized Pediatric Hospital in Cairo, from September 2012February 2014, and investigated for erythromycin resistance phenotypes by D-zone test and genotypes by multiplex PCR. Ninety 90/154 (58.4%) were erythromycin resistant. Erythromycin-resistant S. aureus (ERSA) were 20/69 (28.9%), while coagulase negative staphylococci (ERCoNS) were 70/85 (82.3%). Only 35.8% of met...

Brevibacterium has been reported as a rare cause of implanted-device infection. In two cases of recurrent Brevibacterium casei bacteraemia associated with infection of surgically implanted intravascular devices, relapse occurred 2 and 5 months, respectively, after completion of therapy with vancomycin via the infected catheter. A second intravenous antibiotic therapy course by the antibiotic-lock technique led to bacteriological cure in one patient. Molecular typing results demonstrated that the recurrent bacteraemia was caused by the same strain. Implanted-device removal may be necessary, in addition to appropriate antibiotics, for successful management of such infections.

Multiresistant Enterobacteriaceae, like carbapenemase-producing strains, have their primary reservoir in medical institutions. They can also be found with increasing tendency in other reservoirs. One possible way for entrance of multiresistant Enterobacteriaceae into the environment is via waste water. The aim of the study was to screen isolates from a wastewater treatment plant for the presence of carbapenemase-producing Enterobacteriaceae. Three isolates harboured carbapenemase genes, one Klebsiella pneumoniae harboured KPC-2 and one K. pneumoniae and one Escherichia coli harboured OXA-48. This is the first report of carbapenemase-harbouring Enterobacteriaceae isolated outside medical institutions in Austria and the first detection of KPC-harbouring K. pneumonia MLST ST 1245.

sasX is a colonization-virulence factor that potentially underlies the success of methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 239 in Asia. We aimed to study the spread of sasX and the population structure of MRSA in two geographically distinct regions, Europe and India. MRSA (n = 128) from screening and clinical samples from tertiary care patients in 12 European countries (n = 119), and from India (n = 9) were multilocus-sequence-typed and screened for sasX and its carrier φSPβ-like prophage by PCR. Whole genome sequencing was performed on sasX-harbouring strains from India (n = 5) and Europe (n = 2) and on a selection non-harbouring sasX (n = 36) (2 × 150 bp, Miseq, Illumina). Reads were mapped to the ST239 reference strain, TW20. sasX and sesI, a sasX homologue native to Staphylococcus epidermidis, were detected in five of the nine Indian MRSA belonging to ST239 and to other sequence types of CC8. In contrast, sasX was restricted to two ST239 strains in E...

We surveyed European medical schools regarding teaching of prudent antibiotic prescribing in the undergraduate curriculum. We performed a cross-sectional survey in 13 European countries (Belgium, Croatia, Denmark, France, Germany, Italy, Netherlands, Norway, Serbia, Slovenia, Spain, Switzerland, United Kingdom) in 2013. Proportional sampling was used, resulting in the selection of two to four medical schools per country. A standardized questionnaire based on literature review and validated by a panel of experts was sent to lecturers in infectious diseases, medical microbiology and clinical pharmacology. In-depth interviews were conducted with four lecturers. Thirty-five of 37 medical schools were included in the study. Prudent antibiotic use principles were taught in all but one medical school, but only four of 13 countries had a national programme. Interactive teaching formats were used less frequently than passive formats. The teaching was mandatory for 53% of the courses and star...

There is increasing interest concerning the possible impact of anti-tumour necrosis factor (TNF)-a therapeutic agents on the emergence of infections. However, these agents do not seem to increase the incidence of adverse infectious events significantly. Published observations concern mostly infections of the urinary and upper respiratory tracts that develop in the setting of co-morbidities, such as anterior or concomitant immunosuppressive treatment. Infliximab appears to increase the risk of tuberculosis, but this effect has not been observed with other anti-TNF-a agents. To better characterise the adverse infectious effects associated with these agents, physicians should be encouraged to notify the microbiological data relating to all cases.

Background: A 65-year-old patient developed an unexplained and ultimately lethal metabolic acidosis under prolonged treatment with tigecycline. Tigecycline is known to have a selective inhibitory effect on eukaryotic mitochondrial translation. The underlying molecular mechanisms of the metabolic acidosis in this patient were explored. Methods: Oxidative phosphorylation system (OXPHOS) analysis, blue native polyacrylamide gel electrophoresis followed by in-gel activity staining in mitochondria, molecular analysis of mitochondrial DNA (mtDNA) for genomic rearrangements and sequencing of the rRNA genes was performed on the subject's skeletal muscle. Results: OXPHOS analysis revealed a combined deficiency of the complexes I, III, IV and V, with a preserved function of complex II (encoded by nuclear DNA), thus demonstrating a defective mtDNA translation. There were no known underlying mitochondrial genetic defects. The patient had a (m.1391T>A) variant within the 12SrRNA gene in heteroplasmy (50e60%). Conclusions: This patient developed an ultimately lethal mitochondrial toxicity while receiving prolonged treatment with tigecycline, which was caused by a defective translation of the mtDNA. Tigecycline is known to suppress eukaryotic mitochondrial DNA translation, but until now this effect has been considered to be clinically insignificant. The observations in this patient suggest a clinically significant mitochondrial toxicity of tigecycline in this patient, and warrant further investigation.

The molecular epidemiology of Helicobacter pylori in Africa is poorly documented. From January 2007 to December 2008, we investigated 187 patients with gastric symptoms in one of the main tertiary hospitals in Dakar, Senegal. One hundred and seventeen patients were culture-positive for H. pylori. Polymorphisms in vacA and cagA status were investigated by PCR; the 3¢-region of cagA was sequenced, and EPIYA motifs were identified. Bacterial heterogeneity within individuals was extensively assessed by using an approach based on vacA and cagA heterogeneity. Fourteen per cent of H. pylori-positive patients displayed evidence of mixed infection, which may affect disease outcome. Patients with multiple vacA alleles were excluded from subsequent analyses. Among the final study population of 105 patients, 29 had gastritis only, 61 had ulcerated lesions, and 15 had suspicion of neoplasia based on endoscopic findings. All cases of suspected neoplasia were histologically confirmed as gastric cancer (GC). The cagA gene was present in 73.3% of isolates. CagA proteins contained zero (3.7%), one (93.9%) or two (2.4%) EPIYA-C segments, and all were western CagA. Most of the isolates possessed presumed high-vacuolization isotypes (s1i1m1 (57.1%) or s1i1m2 (21.9%)). Despite the small number of cases, GC was associated with cagA (p 0.03), two EPIYA-C segments in the C-terminal region of CagA (p 0.03), and the s1 vacA allele (p 0.002). Multiple EPIYA-C segments were less frequent than reported in other countries, possibly contributing to the low incidence of GC in Senegal.

Objective: To review two recent cases in HIV-negative subjects in the light of literature reports (52 patients without HIV infection till 1994). Methods: Epidemiology (animal contacts, risk factors, year, country), clinical presentation, diagnostic methods (X-ray, tomography, microbiological techniques), therapeutic approach (antibiotics, surgery) and outcome were evaluated on the basis of clinical literature reports. Results: Tumors constituted an important predisposing factor and less frequently hepatobiliary pathology, rheumatologic diseases, iatrogenic causes, psychiatric pathology and trauma. Exposure to animals was reported by 55% of the patients. Pneumonia and pleurisy, without preferential localization, were detected in 50% of the patients. Etiologic diagnosis was usually obtained after an invasive collection. Combined medical therapy and surgery were required by 27.8% of the patients, and 16.7% of the patients died. In recent years the number of Rhodococcus egoicases has been rising also in HIV-negative patients. The infection is ubiquitous. Accurate diagnosis and the prompt selection of the most appropriate therapy depend on close cooperation between clinicians and microbiologists.

Clinical and microbiological characteristics of neutropenic enterocolitis in adults with blood cancer in the National cancer Institute of Bogota D.C. (Colombia) OBJECTIVES. This study describes the clinical, epidemiological and microbiological characteristics of adult patients with blood cancer and neutropenic enterocolitis treated in the Instituto Nacional de Cancerologia (National Cancer Institute) in Bogota, Colombia. METHODS. The clinical histories of 692 adult patients hospitalized in the Instituto Nacional de Cancerología between 1997 and 2001 with a diagnosis of leukemia or lymphoma were reviewed. Thirty-five of these cases met the criteria for probable or confirmed neutropenic enterocolitis. RESULTS. Twenty-two cases were confirmed and the remaining 13 were probable neutropenic enterocolitis. All patients were undergoing chemotherapy and all presented watery diarrhea and abdominal pain. In addition, 17% had melena and 25% severe vomiting. Eight of 26 stool cultures (30%) and 17 of 32 (58%) blood cultures were positive for potentially pathogenic microorganisms, particularly gram-negative bacilli. Three patients with probable neutropenic enterocolitis and ten confirmed cases died (37%); mortality was higher among patients who were managed surgically. CONCLUSION. Neutropenic colitis presents as a multifactorial syndrome in patients with blood cancer undergoing cytotoxic therapy with agents such as cytosine arabinoside, etoposide, vincristine, cyclophosphamide and corticoids. This highly lethal complication is partly due to infections caused by gram-negative bacilli.

Objectives: To evaluate the aetiological role of the main bacterial pathogens associated with acute otitis media (AOM) in children with AOM and spontaneous tympanic membrane perforation (STMP). Methods: Between 1 May 2015 and 30 April 2016, 177 children, aged 6 months to 7 years, with AOM complicated by STMP within 12 h were prospectively enrolled. Middle ear fluid (MEF) was tested by realtime PCR for Streptococcus pneumoniae, non-typeable Haemophilus influenzae, Streptococcus pyogenes, Moraxella catarrhalis and Staphylococcus aureus. Results: Among the 177 children with AOM and STMP, 92/100 (92.0%) of those with recurrent AOM and 13/77 (16.9%) without recurrent AOM had recurrent STMP (p <0.001). A single pathogen was identified in 70 (39.5%) MEF samples, whereas two, three and four bacteria were detected in 54 (30.5%), 20 (11.3%), and 7 (4.0%) cases, respectively. Non-typeable H. influenzae was the most common and was identified in 90 children (50.8%), followed by M. catarrhalis (62 cases, 35.0%) and S. pneumoniae (48 cases, 27.1%). Nontypeable H. influenzae was the most frequent pathogen in children with co-infections. Children with coinfections, including non-typeable H. influenzae, had significantly more frequent recurrent AOM (adjusted OR 6.609, 95% CI 1.243e39.096, p 0.029). Conclusions: Recurrent AOM episodes appear to be associated with an increased risk of AOM with STMP. In AOM with STMP, non-typeable H. influenzae is detected at a high frequency, especially in children with recurrent STMP and often in association with other pathogens.

Despite the potential for protection against a broad spectrum of pathogens, the availability of an increased number of effective vaccines could lead to a significant reduction in vaccination coverage as the result of issues with implementation of new vaccines within existing protocols. To overcome these problems, the development of combined vaccines has been promoted. The use of combined vaccines offers a number of potential benefits, including a reduction in the number of patient visits, reduced complications associated with multiple intramuscular injections, decreased costs of stocking and administering separate vaccines, and a lowering of the risk of delayed or missed vaccinations. The hexavalent vaccine includes antigens against diphtheria, tetanus, acellular pertussis (DTaP), hepatitis B (HBsAg), poliomyelitis (P1, P2, P3) and Haemophilus influenzae type B (Hib) infections. The primary goal of this review is to discuss the immunogenicity, efficacy, safety and tolerability of several hexavalent preparations that are either commercially available or still under development.

Time-to-positivity is useful in the diagnosis of catheter-related bacteraemia and as a predictor of an endovascular source in patients with Staphylococcus aureus bacteraemia. However, this parameter has been evaluated for only a limited number of microorganisms. In the present study, time-to-positivity was recorded for 1872 episodes of significant monomicrobial bacteraemia diagnosed at a teaching hospital during a 2-year period, and the associated microbial and clinical variables were investigated. According to multivariate analysis, Streptococcus pneumoniae, b-haemolytic streptococci, Escherichia coli, Klebsiella, Enterobacter, Citrobacter and Aeromonas were characterised by fast growth, with an endovascular source, shock, liver cirrhosis and neutropenia also predicting a short time-to-positivity. For patients not receiving appropriate antibiotics, detection of Gram-positive cocci in clusters within 14 h was predictive of Staph. aureus; a time-to-positivity of >21 h ruled out the possibility that a Grampositive organism in chains was a b-haemolytic streptococcus or Strep. pneumoniae, and a time-topositivity of £12 h meant that it was very unlikely that a Gram-negative bacillus was a non-fermenter. A time-to-positivity of £8 h was predictive of a non-urinary tract source in patients with E. coli bacteraemia, and detection of growth within 13 h predicted an endovascular source in those with Staph. aureus bacteraemia. In conclusion, time-to-positivity depended on the microorganism, original source and clinical variables involved. Although this measurement may provide some early clues concerning the microorganisms involved and the source of bacteraemia, its clinical impact remains to be defined.

Transmission of respiratory syncytial virus (RSV) from children with lower respiratory tract infection (LRTI) at a paediatric intensivecare unit (PICU) was examined using a highly sensitive real-time PCR. Twenty-four children with RSV LRTI were admitted during the study period (total days of potential transmission: 239). Fortyeight RSV-negative patients were followed up for RSV acquisition every 5 days (total days of exposure: 683). No single RSV transmission was documented with this highly sensitive diagnostic method. Therefore, routine infection control measures of LRTI patients seem to be adequate to prevent RSV transmission at the PICU.

To determine vaccine coverage of invasive pneumococcal disease (IPD) in Saudi Arabian children aged 5 years and under, 350 IPD isolates were tested for antibiotic susceptibility. Of these 46%, 42% and 12% were penicillin-sensitive, pencillinintermediate, and penicillin-resistant, respectively. Rates of resistance to erythromycin and cefotaxime were 26% and 6%, respectively. The potential serotype coverage of the PCV7 vaccine in Saudi Arabia among children <5 years of age is 62%, and vaccine coverage significantly improved in children <2 years of age, to reach 83% against IPD isolates. PCV7 is expected to have a substantial impact on the burden of invasive and antibiotic-resistant pneumococcal disease in Saudi Arabia.

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Objective To express the views of a working party held to consider antibiotic resistance surveillance systems, their strengths and weaknesses, and their current and future applications. The participants, all of whom were experienced in this field, discussed the development of surveillance systems in relation to the increasing prevalence of resistance to antibacterial agents and the current interest in surveillance systems shown by many official bodies, in both the human and veterinary fields. The problems inherent in surveillance systems were considered together with the applications of different systems. The properties of good antibiotic resistance surveillance systems were defined. Surveillance systems vary widely from those with a narrow base, focusing on few organisms in one disease area, to those covering many diseases, many organisms (including normal flora) and many compounds. Whatever their design, they should be able to detect significant differences and shifts in susceptibility to various antibacterial agents, and the information derived from them should reach as many interested parties as possible in a timely manner. In using this information to decide strategies, criteria for action need to be determined by pragmatic consensus. Funding remains a major problem, with few large studies being supported by official bodies in spite of their professed enthusiasm for surveillance. In consequence, many current systems are funded by the pharmaceutical industry and are of necessity restricted in their focus. Conclusions Antibiotic resistance surveillance studies should and can be well planned and well executed. Many current systems suffer from well-recognized but uncorrected biases. Consortium funding will be necessary for large schemes to be successful. There is no 'ideal' surveillance system.

Objectives: Increasing use of improved culture techniques and sensitive nucleic acid amplification assays have resulted in recognition of Kingella kingae as an important cause of invasive infections in young children, especially in septic arthritis, osteomyelitis, bacteraemia, and endocarditis. In 2016, EUCAST established clinical MIC breakpoints for K. kingae (published in EUCAST Clinical Breakpoint Tables v 7.0, 2017). The present study was carried out to produce MIC-zone diameter correlations for K. kingae on an international collection of isolates, with the aim of suggesting zone diameter breakpoints corresponding to the clinical MIC breakpoints. Methods: Antimicrobial susceptibility testing was performed for 18 clinically relevant agents on a collection of 159 clinical isolates of K. kingae. Broth microdilution MIC determination and disk diffusion were performed according to EUCAST recommendations for fastidious organisms. Results: The correlation between MICs and zone diameters was good for all agents with EUCAST breakpoints for K. kingae. b-lactamase was detected in 41 isolates (26%) and these isolates were resistant to aminopenicillins. These isolates were also resistant to trimethoprim-sulfamethoxazole. Resistance to tetracyclines was detected in 8% of all isolates. All resistant isolates were correctly categorized for these agents with the proposed zone diameter breakpoints. One isolate, resistant to erythromycin but susceptible to other macrolides, was categorized as susceptible with erythromycin disk diffusion. No resistance was detected for the cephalosporins, carbapenems, and fluoroquinolones tested. Conclusion: Based on the results in this study, zone diameter breakpoints for K. kingae calibrated to EUCAST clinical MIC breakpoints were proposed and approved by EUCAST.

b-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of b-lactamase production among K. kingae isolates from different geographical origins and genetically characterized b-lactamase-producing strains. Seven hundred and seventy-eight isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for b-lactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, bla TEM and 16S rRNA sequencing. b-lactamase was identified in invasive strains from Iceland (n = 4/14, 28.6%), the USA (n = 3/15, 20.0%) and Israel (n = 2/ 190, 1.1%) and in carriage strains in the USA (n = 5/17, 29.4%) and Israel (n = 66/429, 15.4%). No French, Spanish or Canadian isolates were b-lactamase producers. Among b-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic b-lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/ MLST ST-6, but differed from the four genetically unrelated Israeli b-lactamase-producing clones. Representative strains of different genotypes produced the TEM-1 enzyme. K. kingae b-lactamase producers exhibit a clear clonal distribution and have dissimilar invasive potential. The presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of b-lactam resistance, and emphasizes the importance of routine testing of all K. kingae clinical isolates for b-lactamase production.