An Application of Imipenem Discs or P. aeruginosa ATCC 27853 Reference Strain Increases Sensitivity of Carbapenem Inactivation Method for Non-Fermenting Gram-Negative Bacteria

Non-fermenting Gram-negative rods are one of the most commonly isolated bacteria from human infections. These microorganisms are typically opportunistic pathogens that pose a serious threat to public health due to possibility of transmission in the human population. Resistance to beta-lactams, due to carbapenemases synthesis, is one of the most important antimicrobial resistance mechanisms amongst them. The aim of this study was to evaluate the usefulness of the Carbapenem Inactivation Method (CIM), and its modifications, for the detection of carbapenemase activity amongst non-fermenting Gram-negative rods. This research involved 81 strains of Gram-negative rods. Of the tested strains, 55 (67.9%) synthesized carbapenemases. For non-fermenting rods, 100% sensitivity and specificity was obtained in the version of the CIM test using imipenem discs and E. coli ATCC 25922 strain. The CIM test allows for differentiation of carbapenems resistance mechanisms resulting from carbapenemase synthesis from other resistance types. It is a reliable diagnostic method for the detection of carbapenemase activity amongst non-fermenting Gram-negative rods. Application of imipenem discs and P. aeruginosa ATCC 27853 reference strain increases CIM results sensitivity, while imipenem discs and E. coli ATCC 25922 strain use maintains full precision of the test for non-fermenting rods

The Evaluation of Eazyplex® SuperBug CRE Assay Usefulness for the Detection of ESBLs and Carbapenemases Genes Directly from Urine Samples and Positive Blood Cultures

Increasing antimicrobial resistance of Gram-negative rods is an important diagnostic, clinical and epidemiological problem of modern medicine. Therefore, it is important to detect multi-drug resistant strains as early on as possible. This study aimed to evaluate Eazyplex® SuperBug CRE assay usefulness for beta-lactamase gene detection among Gram-negative rods, directly from urine samples and positive blood cultures. The Eazyplex® SuperBug CRE assay is based on a loop-mediated isothermal amplification of genetic material and allows for the detection of a selection of genes encoding carbapenemases, KPC, NDM, VIM, OXA-48, OXA-181 and extended-spectrum beta-lactamases from the CTX-M-1 and CTX-M-9 groups. A total of 120 clinical specimens were included in the study. The test gave valid results for 58 (96.7%) urine samples and 57 (95.0%) positive blood cultures. ESBL and/or carbapenemase enzymes genes were detected in 56 (93.3%) urine and 55 (91.7%) blood samples, respectively. The Eazyplex® SuperBug CRE assay can be used for a rapid detection of the genes encoding the most important resistance mechanisms to beta-lactams in Gram-negative rods also without the necessity of bacterial culture

Reliable Diagnostics of SARS-CoV-2 Infections Using One- and Two-Gene Molecular Tests for a Viral RNA Detection—Results Questioning Previous Observations

SARS-CoV-2 is a new virus from the Coronaviridae family and its rapid spread is now the most important medical problem worldwide. Currently used tests vary in the number and selection of SARS-CoV-2 target genes. Meanwhile, the choice of the appropriate target gene may be important in terms of a reliable detection of a viral RNA. As some researchers questioned the sensitivity of the monogenic VIASURE SARS-CoV-2 S gene Real Time PCR Detection Kit (CerTest Biotec, Zaragoza, Spain) in mid-2020, the aim of the study was to evaluate the usefulness of this kit, used along with the BD MAX™ System (Becton Dickinson, East Rutherford, NJ, USA), and compare the results with two-gene Bosphore Novel Coronavirus (2019-nCoV) Detection Kit v1 (Anatolia Diagnostics and Biotechnology Products Inc., Istanbul, Turkey). Both tests were carried out on 306 nasopharyngeal/oropharyngeal swabs. The consistent results (72 positive and 225 negative results found simultaneously in both kits) were obtained for 297 (97.1%) samples altogether, while discrepancies between the results of the evaluated tests were observed for nine (2.9%) specimens. There were no statistically significant differences between the method used and the frequency of positive results. Both tests, targeted at detecting one and two genes, are effective in SARS-CoV-2 RNA detection

Agarose Gel Electrophoresis-Based RAPD-PCR—An Optimization of the Conditions to Rapidly Detect Similarity of the Alert Pathogens for the Purpose of Epidemiological Studies

Agarose gel electrophoresis is a well-known tool to detect DNA fragments amplified in polymerase chain reaction (PCR). Its usefulness has also been confirmed for epidemiological studies based on restriction fragments length polymorphism (RFLP), usually performed using pulsed-field gel electrophoresis (PFGE). Little is known on the effectiveness for alert-pathogen epidemiological studies of another less time-consuming and costly technique called randomly amplified polymorphic DNA-PCR (RAPD-PCR). Meanwhile, its usefulness is believed to be comparable to RFLP-PFGE. Therefore, the aim of the study was to establish and optimize the conditions of agarose gel electrophoresis following RAPD-PCR for 19 Enterococcus faecium strains derived from epidemic outbreaks at intensive care units. An application of different PCR primers, primer combinations, and, in particular, agarose gel concentrations and electrophoresis conditions revealed the usefulness of this relatively fast and inexpensive method based on RAPD-PCR for epidemiological studies without a compulsion to use the specialized equipment necessary for RFLP-PFGE

Association of fungi and archaea of the gut microbiota with Crohn's disease in pediatric patients : pilot study

The composition of bacteria is often altered in Crohn’s disease (CD), but its connection to the disease is not fully understood. Gut archaea and fungi have recently been suggested to play a role as well. In our study, the presence and number of selected species of fungi and archaea in pediatric patients with CD and healthy controls were evaluated. Stool samples were collected from children with active CD (n = 54), non-active CD (n = 37) and control subjects (n = 33). The prevalence and the number of selected microorganisms were assessed by real-time PCR. The prevalence of Candida tropicalis was significantly increased in active CD compared to non-active CD and the control group (p = 0.011 and p = 0.036, respectively). The number of Malassezia spp. cells was significantly lower in patients with active CD compared to the control group, but in non-active CD, a significant increase was observed (p = 0.005 and p = 0.020, respectively). There were no statistically significant differences in the colonization by archaea. The obtained results indicate possible correlations with the course of the CD; however, further studies of the entire archeobiome and the mycobiome are necessary in order to receive a complete picture

An Application of Real-Time PCR and CDC Protocol May Significantly Reduce the Incidence of <i>Streptococcus agalactiae</i> Infections among Neonates

Streptococcus agalactiae is an important human opportunistic pathogen, especially infectious for pregnant women and neonates. This pathogen belongs to beta hemolytic Streptococcus spp. representatives and accounts for a significant part of early infections in newborns, including serious life-threatening infections. This research investigated the usefulness of Centers for Disease Control and Prevention (CDC) protocol for S. agalactiae DNA detection in 250 samples of recto-vaginal swabs collected from pregnant women (at 35-37 weeks of gestation) and pre-cultured overnight in liquid medium. With an application of the CDC protocol-based real-time PCR, the cfb gene was detected in 68 (27.2%) samples compared to 41 (16.4%) for the standard culture-based methodology. The applied molecular method presented high sensitivity (100.0%) and specificity (87.1%). Therefore, it allowed for more precise detection of S. agalactiae bacteria, compared to the reference diagnostic method, culture on solid media with the following strain identification. The increased sensitivity of GBS detection may result in a reduced number of infections in newborns and leads to more targeted antimicrobial prophylaxis therapy of GBS infections in pregnant women. In addition, the use of the molecular method allows for a significant reduction in the time needed to obtain a result for GBS detection, and interpretation of the results is relatively simple. Therefore, it enables a faster intervention in case of a necessity of an antibiotic therapy introduction in pregnant women whose GBS status is unknown at the time of delivery

Usefulness of Molecular Methods for Helicobacter pylori Detection in Pediatric Patients and Their Correlation with Histopathological Sydney Classification

Helicobacter pylori infections, as one of the most prevalent among humans, are generally acquired during childhood, and are one of the main causes of chronic gastritis and peptic ulcer disease. A bacterial culture from a gastric biopsy is the gold standard and is the only method that has 100% specificity. However, its sensitivity varies, depending on experience of the laboratory staff, applied culture media, specimen transport conditions, biopsy site, and quality of the sample. The same factors compromise all invasive methods and a culture-based H. pylori infection diagnostic, as well as a recent intake of antibiotics, bismuth-containing compounds, and proton pump inhibitors. Molecular methods have been used for clinical microbiology investigation since the beginning of the 21st century. However, their usefulness for H. pylori infections diagnosis remains unclear, especially in pediatric patients. The aim of the study was to assess the incidence of H. pylori infections in a group of 104 pediatric patients and to compare the results of the PCR test with the corresponding histopathological investigation effects. Among the biopsy samples collected from 104 children, 44 (42.3%) were positive in PCR, while 43 (41.3%) and 39 (37.5%) presented histologically-confirmed signs of inflammation and H. pylori colonization, respectively. Moreover, the mean grades of the parameters of the histopathological examination were higher in the group of PCR-positive samples. The compatibility of both research methods was confirmed, emphasizing the usefulness of molecular methods for detecting H. pylori infections in pediatric patients. Considering that the PCR-based method gives reliable results and is less time-consuming and costly, it is worth discussing this method as a new standard in the diagnosis of H. pylori infections, at least among pediatric patients, for which culture-based diagnostics is not sufficient or histopathological examination is negative, while inflammation signs are observed macroscopically

Diagnostic Value of Whole-Blood and Plasma Samples in Epstein–Barr Virus Infections

Epstein–Barr virus (EBV) is an oncogenic virus classified by the World Health Organization as a class 1 carcinogen. Post-transplant lymphoproliferative disorders are believed to be strongly related to an EBV infection. Monitoring of EBV DNAemia is recommended to assess the risk of reactivation of latent infection and to assess the effectiveness of therapy. Currently, various types of clinical specimens are used for this purpose. The aim of the study was to assess a reliable method of EBV viral load investigation depending on the clinical material used: whole blood or plasma samples. We found that of 134 EBV-DNA-positive whole-blood samples derived from 51 patients (mostly hemato-oncology or post-transplantation), only 43 (32.1%) were plasma-positive. Of these, 37 (86.0%) had lower plasma DNAemia compared to the corresponding whole-blood samples. We conclude that whole-blood samples have a higher sensitivity than plasma samples in EBV DNA detection. The clinical utility of the tests is unclear, but our results suggest that either whole blood or plasma should be used consistently for EBV viral load monitoring

Usefulness of Molecular Methods for <i>Helicobacter pylori</i> Detection in Pediatric Patients and Their Correlation with Histopathological Sydney Classification

Helicobacter pylori infections, as one of the most prevalent among humans, are generally acquired during childhood, and are one of the main causes of chronic gastritis and peptic ulcer disease. A bacterial culture from a gastric biopsy is the gold standard and is the only method that has 100% specificity. However, its sensitivity varies, depending on experience of the laboratory staff, applied culture media, specimen transport conditions, biopsy site, and quality of the sample. The same factors compromise all invasive methods and a culture-based H. pylori infection diagnostic, as well as a recent intake of antibiotics, bismuth-containing compounds, and proton pump inhibitors. Molecular methods have been used for clinical microbiology investigation since the beginning of the 21st century. However, their usefulness for H. pylori infections diagnosis remains unclear, especially in pediatric patients. The aim of the study was to assess the incidence of H. pylori infections in a group of 104 pediatric patients and to compare the results of the PCR test with the corresponding histopathological investigation effects. Among the biopsy samples collected from 104 children, 44 (42.3%) were positive in PCR, while 43 (41.3%) and 39 (37.5%) presented histologically-confirmed signs of inflammation and H. pylori colonization, respectively. Moreover, the mean grades of the parameters of the histopathological examination were higher in the group of PCR-positive samples. The compatibility of both research methods was confirmed, emphasizing the usefulness of molecular methods for detecting H. pylori infections in pediatric patients. Considering that the PCR-based method gives reliable results and is less time-consuming and costly, it is worth discussing this method as a new standard in the diagnosis of H. pylori infections, at least among pediatric patients, for which culture-based diagnostics is not sufficient or histopathological examination is negative, while inflammation signs are observed macroscopically