Preparing for laboratory automation and consolidation-Establishing the validity of pediatric-like low-volume urine samples in boric-acid containing tubes.

Microbiological services consolidation has increased the usage of preservative-containing urine tubes, potentially inhibiting pathogens in low-volume pediatric urine samples, yielding false-negative results. Our study demonstrates comparable growth with 1 ml versus the recommended 3 ml urine, following different shipping intervals. We advocate for regulators to consider similar large-scale validations, ensuring results' consistency.

Optimizing the Yield of In-and-Out Urinary Catheter Cultures by Applying a "Midstream-Like" Approach in Pediatric Patients.

Despite appropriate disinfection, sample contamination during in-and-out urinary catheterization is not uncommon, yielding false-positive and "mixed-culture" interpretations. We implemented a "midstream-like" catheterization technique, and cultured both first- and second-voided urine fractions. Second-fraction cultures exhibited less contaminants and "mixed-culture" interpretations and were better aligned with pyuria, thereby enhancing diagnostic accuracy and minimizing the risk of clinical misdiagnosis and unwarranted antibiotic use.

Respiratory cryptosporidiosis detected by commercial multiplex-PCR in immunosuppressed pediatric patients.

We report the off-label use of a commercial gastrointestinal protozoa multiplex-PCR panel for bronchoalveolar lavage samples, detecting respiratory cryptosporidiosis in 2 immunocompromised pediatric patients. We suggest that implying this readily available assay in cases in which systemic cryptosporidiosis is suspected, may widen our understanding regarding this rarely reported syndrome.

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.

Viral co-pathogens in COVID-19 acute respiratory syndrome - what did we learn from the first year of pandemic?

OBJECTIVE: This study aimed to describe the distribution of respiratory pathogens and the occurrence of co-pathogens during the first year of the COVID-19 pandemic. METHODS: We used a multiplex polymerase chain reaction (PCR) panel targeting 23 microorganisms to analyze the oro-pharyngeal samples of patients admitted to our hospital with acute respiratory infection (ARI) between March 1, 2020, and February 28, 2021. We matched 40 to 50 patients who were SARS-CoV-2 positive and SARS-CoV-2 negative per month for age and sex. RESULTS: A total of 939 patients with multiplex PCR test results were included in the study. Respiratory pathogens where detected in only 8/476 (1.6%) patients with COVID-19 versus 87/463 (18.7%) patients with non-COVID-19 ARI patients. Diversity and rates of pathogens vastly differed from previous years but showed seasonal variance. CONCLUSION: Patients with SARS-CoV-2 infection presenting with ARI during the first year of the COVID-19 pandemic demonstrated paucity of respiratory co-pathogens.

Rapid Detection of Carbapenemase-producing Enterobacteriaceae From Blood Culture Bottles of Known CPE Carriers: Real-world Experience.

We used a rapid antigen test for the detection of carbapenemases directly from positive blood culture bottles of pediatric hemato-oncologic patients, known carriers of carbapenemase-producing enterobacteriaceae. Resistance mechanism was detected within 15 minutes of observing Gram-negative bacilli from a positive bottle, leading to treatment modification. This simple-to-use, inexpensive assay shortens the interval between empiric to tailored antimicrobial therapy.

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.

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.

Molecular features of heterogeneous vancomycin-intermediate Staphylococcus aureus strains isolated from bacteremic patients.

BACKGROUND: Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) bacteremia is an emerging infection. Our objective was to determine the molecular features of hVISA strains isolated from bacteremic patients and to compare them to methicillin resistant S. aureus (MRSA) and methicillin sensitive S. aureus (MSSA) blood isolates. RESULTS: We assessed phenotypic and genomic changes of hVISA (n = 24), MRSA (n = 16) and MSSA (n = 17) isolates by PCR to determine staphylococcal chromosomal cassette (SCCmec) types, Panton-Valentine leukocidin (PVL) and the accessory gene regulator (agr) loci. Biofilm formation was quantified. Genetic relatedness was assessed by PFGE. PFGE analysis of isolates was diverse suggesting multiple sources of infection. 50% of hVISA isolates carried SCCmec type I, 21% type II; 25% type V; in 4% the SCCmec type could not be identified. Among MRSA isolates, 44% were SCCmec type I, 12.5% type II, 25% type V, 12.5% were non-typable, and 6% were SCCmec type IVd. Only one hVISA isolate and two MSSA isolates carried the PVL. Biofilm formation and agr patterns were diverse. CONCLUSION: hVISA isolates were diverse in all parameters tested. A considerable number of hVISA and MRSA strains carried the SCCmec type V cassette, which was not related to community acquisition.

Clinical features of heteroresistant vancomycin-intermediate Staphylococcus aureus bacteremia versus those of methicillin-resistant S. aureus bacteremia.

BACKGROUND: Heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) infections are emerging, but their clinical significance remains unclear. Our objective was to compare patients who had hVISA bacteremia with patients who had methicillin-resistant S. aureus (MRSA) bacteremia. METHODS: A total of 27 case patients with hVISA bacteremia were compared with 223 control patients with MRSA bacteremia. Medical records of all patients were reviewed, and factors independently associated with infection-related mortality were assessed by logistic regression. RESULTS: Patients with hVISA bacteremia were not significantly different from those with MRSA bacteremia with respect to age, comorbidities, duration of hospital stay, and infection-attributable mortality. However, the median duration of bacteremia among patients with hVISA was significantly longer than that among patients with MRSA (12 vs. 2 days; P = .005), and patients with hVISA had a greater prevalence of complications, such as endocarditis (18.5% vs. 3.6%; P = .007) and osteomyelitis (25.9% vs. 7.2%, respectively; P = .006). Rifampin resistance emerged more frequently among hVISA isolates than among MRSA isolates (44% vs. 5.9%; P < .001). Factors independently associated with infection-related mortality in all patients were age, Charlson comorbidity index, female sex, and being bedridden. CONCLUSIONS: hVISA bacteremia was significantly associated with prolonged bacteremia duration, greater rates of complications, and emergence of rifampin resistance, compared with MRSA bacteremia. However, no significant difference in mortality existed between patients with hVISA bacteremia and those with MRSA bacteremia.

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.