Evaluation of multiplex real-time PCR for identifying dermatophytes in clinical samples-A multicentre study.

The gold-standard method for dermatophyte identification involves direct microscopy and culture, which have inherent shortcomings. Only few molecular methods have been standardised for routine clinical work. This study aimed to develop and test a platform for identifying the most common dermatophytes in Israel using multiplex real-time polymerase chain reaction (RT-PCR). Specific primers were designed for the multiplex system (LightCycler 480) according to known cultures and validated by reference isolates. The dermatophyte detection rate was compared to smear and culture in 223 clinical samples obtained from a tertiary medical centre. Inconsistencies between methods were evaluated by sequencing. The RT-PCR was further evaluated in 200 community-based samples obtained from a health maintenance organisation and 103 military-personnel-based samples analysed at a central laboratory. In hospital-based clinical samples, complete concordance between methods was observed in 190 samples (85%; Kappa = 0.69). In most cases of non-concordance, sequencing was consistent with RT-PCR results. RT-PCR correctly identified all smear- and culture-positive cases in community and military-personnel samples. The results were available within 4 hours. The multiplex RT-PCR platform is a rapid and efficient method for identifying dermatophyte species in clinical samples and may serve as a first step in the diagnostic algorithm of superficial fungal infections.

Antimicrobial Peptides against Multidrug-Resistant Pseudomonas aeruginosa Biofilm from Cystic Fibrosis Patients.

Lung infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients and is mainly dominated by Pseudomonas aeruginosa. Treatment of CF-associated lung infections is problematic because the drugs are vulnerable to multidrug-resistant pathogens, many of which are major biofilm producers like P. aeruginosa. Antimicrobial peptides (AMPs) are essential components in all life forms and exhibit antimicrobial activity. Here we investigated a series of AMPs (d,l-K6L9), each composed of six lysines and nine leucines but differing in their sequence composed of l- and d-amino acids. The d,l-K6L9 peptides showed antimicrobial and antibiofilm activities against P. aeruginosa from CF patients. Furthermore, the data revealed that the d,l-K6L9 peptides are stable and resistant to degradation by CF sputum proteases and maintain their activity in a CF sputum environment. Additionally, the d,l-K6L9 peptides do not induce bacterial resistance. Overall, these findings should assist in the future development of alternative treatments against resistant bacterial biofilms.

Persistent abdominal symptoms in returning travellers: clinical and molecular findings.

BACKGROUND: Persistent abdominal symptoms (PAS) are the leading cause of post-travel morbidity although there is a paucity of evidence concerning the aetiology of this condition. Recently molecular methods for protozoa detection in stool have been introduced. Herein, we describe the clinical aspects and the prevalence of gastrointestinal protozoa in returning travellers with PAS. METHODS: From 2017 to 2019, clinical information and stool specimens from returning travellers with PAS were analysed for the presence of parasites using the Allplex-GI-Parasite-assay. Stool findings from symptomatic patients without a travel history were used as a comparator. RESULTS: During the 2-year study, 203 stool specimens from returning travellers were analysed. The median duration of symptoms before seeking care was 6 months, the most common symptoms were fatigue (79.2%), abdominal pain (75.7%) and loose stool (70.8%).Most of travellers had returned from Asia (57.6%), mainly from the Indian-subcontinent and only 52.6% were backpackers. Altogether, 36.9% samples were positive for protozoa, with Blastocystis hominis being the most common (26.6%) in samples, followed by Dientamoeba fragilis (18.7%), Giardia lamblia (3.0%) and Cryptosporidium spp (0.5%). The former two were dominant in all regions. In all cases but one, G. lamblia was acquired, but one were acquired in the Indian subcontinent (odds ratios 16.9; 95% confidence intervals: 1.9-148.3). Entamoeba histolytica was not detected. The demographic characterization of the 1359 non-travellers was comparable with the travellers. Among them D. fragilis was the most common followed by B. hominis, which was significantly less frequent compared among the travellers (16.7% vs 26.6%, P < 0.001). Average Cycle threshold values for each stool parasites were comparable between the two groups. CONCLUSION: Among returning travellers with PAS, more than one-third were positive for gastrointestinal protozoa. A low rate of giardia was found and no E. histolytica while B. hominis followed by D. fragilis were the dominant findings. Further studies are required to better understand the role of these protozoa in PAS.

Containment of a country-wide outbreak of carbapenem-resistant Klebsiella pneumoniae in Israeli hospitals via a nationally implemented intervention.

BACKGROUND: During 2006, Israeli hospitals faced a clonal outbreak of carbapenem-resistant Klebsiella pneumoniae that was not controlled by local measures. A nationwide intervention was launched to contain the outbreak and to introduce a strategy to control future dissemination of antibiotic-resistant bacteria in hospitals. METHODS: In March 2007, the Ministry of Health issued guidelines mandating physical separation of hospitalized carriers of carbapenem-resistant Enterobacteriaceae (CRE) and dedicated staffing and appointed a professional task force charged with containment. The task force paid site visits at acute-care hospitals, evaluated infection-control policies and laboratory methods, supervised adherence to the guidelines via daily census reports on carriers and their conditions of isolation, provided daily feedback on performance to hospital directors, and intervened additionally when necessary. The initial intervention period was 1 April 2007-31 May 2008. The primary outcome measure was incidence of clinically diagnosed nosocomial CRE cases. RESULTS: By 31 March 2007, 1275 patients were affected in 27 hospitals (175 cases per 1 million population). Prior to the intervention, the monthly incidence of nosocomial CRE was 55.5 cases per 100,000 patient-days. With the intervention, the continuous increase in the incidence of CRE acquisition was halted, and by May 2008, the number of new monthly cases was reduced to 11.7 cases per 100,000 patient-days (P<.001). There was a direct correlation between compliance with isolation guidelines and success in containment of transmission (P=.02). Compliance neutralized the effect of carrier prevalence on new incidence (P=.03). CONCLUSIONS: A centrally coordinated intervention succeeded in containing a nationwide CRE outbreak after local measures failed. The intervention demonstrates the importance of strategic planning and national oversight in combating antimicrobial resistance.

High prevalence of methicillin-resistant Staphylococcus aureus among residents and staff of long-term care facilities, involving joint and parallel evolution.

Six long-term care facilities were surveyed for methicillin-resistant Staphylcoccus aureus (MRSA). Among 191 residents, 14% were carriers; 1 strain predominated (ST5-SCCmec II). Among 132 staff members, 11% were positive; 2 strains predominated (ST5-SCCmec II, ST8-SCCmec IV). All strains were Panton-Valentine leukocidin-negative. The epidemiology of MRSA among residents and staff involved joint and parallel evolution.

Rapid detection of blaKPC carbapenemase genes by internally controlled real-time PCR assay using bactec blood culture bottles.

Rapid detection of drug-resistant bacteria in clinical samples plays an instrumental role in patients' infection management and in implementing effective infection control policies. In the study described in this report, we validated a multiplex TaqMan real-time quantitative PCR (qPCR) assay for the detection of bla(KPC) genes and the human RNase P gene in Bactec blood culture bottles. The MagNA Pure LC (version 2.0) instrument was utilized to extract nucleic acids from the inoculated broth, while bovine serum albumin (BSA) was utilized as the PCR inhibitor reliever. The multiplex assay, which was specific for the detection of bla(KPC) genes, had a limit of detection of 19 CFU per reaction mixture with human blood-spiked Bactec bottles. Of the 323 Bactec blood culture sets evaluated, the same 55 (17%) blood cultures positive for carbapenem-resistant bacteria by culture were also positive by the validated qPCR assay. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the qPCR assay compared to the results of culture were all 100%. bla(KPC) genes were also detected from the same Bactec bottle broth after manual extraction with a QIAamp DNA minikit; however, there was an average 3-threshold-cycle delay in the qPCR readings. With the limited therapeutic options available, the accurate and rapid detection of bla(KPC)-possessing bacteria by the described bla(KPC)/RNase P assay will be a crucial first step in ensuring optimal clinical outcomes and infection control.

The effect of recurrent povidone-iodine usage on conjunctival flora in diabetic patients undergoing intravitreal injections.

PURPOSE: The purpose was to evaluate the change in the microbiological profile of diabetic patients undergoing intravitreal injections for diabetic macular edema. METHODS: Patients were included in this prospective study when referred for the first time for intravitreal injection to treat diabetic macular edema. For each patient, conjunctival cultures were taken from the lower fornix of each eye prior to the povidone-iodine application and the intravitreal injection. An additional culture was taken from the treated eye 20 min after the injection. The same culture protocol was used for the two following injections of these patients. A later conjunctival culture was also taken a month after the last injection. RESULTS: Twenty-one eyes of 21 patients were included. The mean duration of diabetes was 13.7 ± 7.9 years. Prior to the first intravitreal injection, 33% of cultures were positive. Prior to the third intravitreal injection, 26% of cultures were positive (p = 0.63), and 1 month after the last injection, 18% of cultures were positive (p = 0.495). The mean HbA1C was 8.1% ± 1.7%. HbA1C of patients with positive cultures was 8.0% ± 1.1% at the first intravitreal injection and 8.2% ± 1.0% at the third intravitreal injection. This was compared with HBA1C in eyes with negative cultures: 7.4% ± 1.2% (p = 0.45) and 7.1% ± 1.0% (p = 0.14), respectively. CONCLUSION: Repeated intravitreal injection for diabetic macular edema with application of povidone-iodine 5% in diabetic patients did not lead to a significant change in the percentage of positive conjunctival cultures. Patients with higher HbA1C had a slight, non-statistically significant trend for positive cultures.

Effect of amniotic membrane transplantation on the healing of bacterial keratitis.

PURPOSE: To study, with the use of an animal model, the efficacy of amniotic membrane (AM) transplantation as adjunctive treatment in corneal healing after bacterial keratitis. METHODS: Staphylococcus aureus keratitis was induced in 47 rats by injection of bacteria into the corneal stroma. Treatment was started 48 hours later with one of three randomly assigned protocols: cefazolin drops (50 mg/mL) and AM transplantation (n = 16); nonpreserved 0.9% saline drops and AM transplantation (n = 15); or cefazolin without AM transplantation (n = 16). Cefazolin and saline drops were administered every 30 minutes for 6 hours, then hourly for 6 hours. AM was transplanted 24 hours after termination of cefazolin or saline treatment. Results were clinically assessed 7 days after AM transplantation or at the corresponding time in the nontransplanted animals. The rats were then killed, and their corneas were removed for bacterial counts or histopathologic examination. RESULTS: The best clinical results were observed in the group treated with cefazolin and AM transplantation, manifested by the least corneal haze and neovascularization (P = 0.007 and P = 0.014, respectively) and minimal bacterial counts (28 colony-forming units [CFU]/mL compared with 160 CFU/mL and 240 CFU/mL, respectively). Histopathologic examination showed that the central corneal vessels from rats treated with cefazolin and AM were smaller and less congested than those from the other two groups. CONCLUSIONS: AM transplantation is a useful adjunctive treatment after bacterial keratitis in this rat model. The transplanted AM improved the healing process, resulting in decreased corneal haze and less neovascularization.

Sink traps as the source of transmission of OXA-48-producing Serratia marcescens in an intensive care unit.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) outbreaks are mostly attributed to patient-to-patient transmission via healthcare workers. OBJECTIVE: We describe successful containment of a prolonged OXA-48-producing S. marcescens outbreak after recognizing the sink traps as the source of transmission. METHODS: The Sheba Medical Center intensive care unit (ICU), contains 16 single-bed, semi-closed rooms. Active CPE surveillance includes twice-weekly rectal screening of all patients. A case was defined as a patient detected with OXA-48 CPE >72 hours after admission. A root-cause analysis was used to investigate the outbreak. All samples were inoculated on chrom-agar CRE, and carbapenemase genes were detected using commercial molecular Xpert-Carba-R. Environmental and patient S. marcescens isolates were characterized using PFGE. RESULTS: From January 2016 to May 2017, 32 OXA-48 CPE cases were detected, and 81% of these were S. marcescens. A single clone was the cause of all but the first 2 cases. The common factor in all cases was the use of relatively large amounts of tap water. The outbreak clone was detected in 2 sink outlets and 16 sink traps. In addition to routine strict infection control measures, measures taken to contain the outbreak included (1) various sink decontamination efforts, which eliminated the bacteria from the sink drains only temporarily and (2) educational intervention that engaged the ICU team and lead to high adherence to 'sink-contamination prevention guidelines.' No additional cases were detected for 12 months. CONCLUSIONS: Despite persistence of the outbreak clones in the environmental reservoir for 1 year, the outbreak was rapidly and successfully contained. Addressing sink traps as hidden reservoirs played a major role in the intervention.

Fecal microbial characterization of hospitalized patients with suspected infectious diarrhea shows significant dysbiosis.

Hospitalized patients are at increased risk for acquiring healthcare-associated infections (HAIs) and inadequate nutrition. The human intestinal microbiota plays vital functions in nutrient supply and protection from pathogens, yet characterization of the microbiota of hospitalized patients is lacking. We used 16S rRNA amplicon sequencing to characterize the global pattern of microbial composition of fecal samples from 196 hospitalized patients with suspected infectious diarrhea in comparison to healthy, non-hospitalized subjects (n = 881), and to traditional culture results. We show that hospitalized patients have a significant rise in α-diversity (richness within sample) from birth to <4 years of age, which continues up to the second decade of life. Additionally, we noted a profoundly significant increase in taxa from Proteobacteria phylum in comparison to healthy subjects. Finally, although more than 60% of hospitalized samples had a greater than 10% abundance of Proteobacteria, there were only 19/196 (10%) positive cultures for Campylobacter, Salmonella, or Shigella entero-pathogens in traditional culturing methods. As hospitalized patients have increased risk for HAIs and inadequate nutrition, our data support the consideration of nutritional and/or microbial modification in this population.

Population Screening Using Sewage Reveals Pan-Resistant Bacteria in Hospital and Community Samples.

The presence of pan-resistant bacteria worldwide possesses a threat to global health. It is difficult to evaluate the extent of carriage of resistant bacteria in the population. Sewage sampling is a possible way to monitor populations. We evaluated the presence of pan-resistant bacteria in Israeli sewage collected from all over Israel, by modifying the pour plate method for heterotrophic plate count technique using commercial selective agar plates. This method enables convenient and fast sewage sampling and detection. We found that sewage in Israel contains multiple pan-resistant bacteria including carbapenemase resistant Enterobacteriacae carrying blaKPC and blaNDM-1, MRSA and VRE. blaKPC carrying Klebsiella pneumonia and Enterobacter cloacae were the most common Enterobacteriacae drug resistant bacteria found in the sewage locations we sampled. Klebsiella pneumonia, Enterobacter spp., Escherichia coli and Citrobacter spp. were the 4 main CRE isolated from Israeli sewage and also from clinical samples in our clinical microbiology laboratory. Hospitals and Community sewage had similar percentage of positive samplings for blaKPC and blaNDM-1. VRE was found to be more abundant in sewage in Israel than MRSA but there were more locations positive for MRSA and VRE bacteria in Hospital sewage than in the Community. Therefore, our upgrade of the pour plate method for heterotrophic plate count technique using commercial selective agar plates can be a useful tool for routine screening and monitoring of the population for pan-resistant bacteria using sewage.

Genetic and Phenotypic Characterization of a Salmonella enterica serovar Enteritidis Emerging Strain with Superior Intra-macrophage Replication Phenotype.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the ubiquitous Salmonella serovars worldwide and a major cause of food-born outbreaks, which are often associated with poultry and poultry derivatives. Here we report a nation-wide S. Enteritidis clonal outbreak that occurred in Israel during the last third of 2015. Pulsed field gel electrophoresis and whole genome sequencing identified genetically related strains that were circulating in Israel as early as 2008. Global comparison linked this outbreak strain to several clinical and marine environmental isolates that were previously isolated in California and Canada, indicating that similar strains are prevalent outside of Israel. Phenotypic comparison between the 2015 outbreak strain and other clinical and reference S. Enteritidis strains showed only limited intra-serovar phenotypic variation in growth in rich medium, invasion into Caco-2 cells, uptake by J774.1A macrophages, and host cell cytotoxicity. In contrast, significant phenotypic variation was shown among different S. Enteritidis isolates when biofilm-formation, motility, invasion into HeLa cells and uptake by THP-1 human macrophages were studied. Interestingly, the 2015 outbreak clone was found to possess superior intra-macrophage replication ability within both murine and human macrophages in comparison to the other S. Enteritidis strains studied. This phenotype is likely to play a role in the virulence and host-pathogen interactions of this emerging clone.

Molecular detection of antibiotic resistance genes from positive blood cultures.

Rapid detection of the bacterial causative agent causing sepsis must be coupled with rapid identification of the antibiotic resistant mechanism that the pathogen might possess. Real-time PCR (qPCR)-based assays have been extensively utilized in the clinical microbiology field as diagnostic tools for the rapid detection of specific nucleic acid (NA) targets. In this chapter, we will discuss the technical aspects of using an internally controlled qPCR assay for the rapid detection of Klebsiella pneumoniae carbapenemase gene (bla KPC) in positive Bactec blood culture bottles. The multiplex qPCR (bla KPC/RNase P) utilizes specific primers and probes for the detection of the bacterial carbapenem resistance mechanism, bla KPC gene, and the internal control RNase P. The internal control of the qPCR assay is vital for detecting any inhibitors that are well known to be present in the blood culture bottles. Rapid detection of the antibiotic resistant mechanism present in the bacterial pathogen causing sepsis can help in better managing patients' infection.

Carbapenem-resistant Klebsiella pneumoniae in post-acute-care facilities in Israel.

OBJECTIVE: To assess the prevalence of and risk factors for carbapenem-resistant Klebsiella pneumoniae (CRKP) carriage among patients in post-acute-care facilities (PACFs) in Israel. DESIGN, SETTING, AND PATIENTS: A cross-sectional prevalence survey was conducted in 12 PACFs. Rectal swab samples were obtained from 1,144 patients in 39 [corrected] wards. Risk factors for CRKP carriage were assessed among the cohort. Next, a nested, matched case-control study was conducted to define individual risk factors for colonization. Finally, the cohort of patients with a history of CRKP carriage was characterized to determine risk factors for continuous carriage. RESULTS: The prevalence of rectal carriage of CRKP among 1,004 patients without a history of CRKP carriage was 12.0%. Independent risk factors for CRKP carriage were prolonged length of stay (odds ratio [OR], 1.001; P < .001), sharing a room with a known carrier (OR, 3.09; P = .02), and increased prevalence of known carriers on the ward (OR, 1.02; P = .013). A policy of screening for carriage on admission was protective (OR, 0.41; P = .03). Risk factors identified in the nested case-control study were antibiotic exposure during the prior 3 months (OR, 1.66; P = .03) and colonization with other resistant pathogens (OR, 1.64; P = .03). Among 140 patients with a history of CRKP carriage, 47% were colonized. Independent risk factors for continued CRKP carriage were antibiotic exposure during the prior 3 months (OR, 3.05; P = .04), receipt of amoxicillin-clavulanate (OR, 4.18; P = .007), and screening within 90 days of the first culture growing CRKP (OR, 2.9; P = .012). CONCLUSIONS: We found a large reservoir of CRKP in PACFs. Infection-control policies and antibiotic exposure were associated with patient colonization.

Potential role of active surveillance in the control of a hospital-wide outbreak of carbapenem-resistant Klebsiella pneumoniae infection.

BACKGROUND: The recent emergence of carbapenem resistance among Enterobacteriaceae is a major threat for hospitalized patients, and effective strategies are needed. OBJECTIVE: To assess the effect of an intensified intervention, which included active surveillance, on the incidence of infection with carbapenem-resistant Klebsiella pneumoniae. SETTING: Sheba Medical Center, a 1,600-bed tertiary care teaching hospital in Tel Hashomer, Israel. DESIGN: Quasi-experimental study. METHODS: The medical records of all the patients who acquired a carbapenem-resistant K. pneumoniae infection during 2006 were reviewed. An intensified intervention was initiated in May 2007. In addition to contact precautions, active surveillance was initiated in high-risk units. The incidence of clinical carbapenem-resistant K. pneumoniae infection over time was measured, and interrupted time-series analysis was performed. RESULTS: The incidence of clinical carbapenem-resistant K. pneumoniae infection increased 6.42-fold from the first quarter of 2006 up to the initiation of the intervention. In 2006, of the 120 patients whose clinical microbiologic culture results were positive for carbapenem-resistant K. pneumoniae, 67 (56%) developed a nosocomial infection. During the intervention period, the rate of carbapenem-resistant K. pneumoniae rectal colonization was 9%. Of the 390 patients with carbapenem-resistant K. pneumoniae colonization or infection, 204 (52%) were identified by screening cultures. There were a total of 12,391 days of contact precautions, and of these, 4,713 (38%) were added as a result of active surveillance. After initiation of infection control measures, we observed a significant decrease in the incidence of carbapenem-resistant K. pneumoniae infection. CONCLUSIONS: The use of active surveillance and contact precautions, as part of a multifactorial intervention, may be an effective strategy to decrease rates of nosocomial transmission of carbapenem-resistant K. pneumoniae colonization or infection.

Prevalence and characteristics of heteroresistant vancomycin-intermediate Staphylococcus aureus bacteremia in a tertiary care center.

Infections with S. aureus with heterogeneous intermediate resistance to vancomycin (hVISA) are occurring more frequently. The detection of these infections, their prevalence, clinical characteristics, and significance are controversial. During 2003 and 2004, all blood culture isolates of methicillin-resistant Staphylococcus aureus (264 patients) at the Sheba Medical Center, Tel Hashomer, Israel, were assessed for hVISA by using the Etest macromethod. A total of 16 patients (6%) were positive for hVISA. Resistance to teicoplanin alone and to vancomycin alone using the Etest macromethod was found in 14 and 10 patients, respectively. Standard MICs to vancomycin were between 1 to 4 mg/ml. Most of these isolates (12 of 16 [75%]) would have been missed without specific testing. The median number of bacteremic days was 4. Seven patients had positive blood cultures for more than 5 days. Twelve patients died, and for eight of these the deaths were directly related to hVISA sepsis. We found that hVISA bacteremia was prevalent in our institution, and we suggest seeking hVISA in patients with persistent S. aureus bacteremia.