Effect of Colistin, Fosfomycin and Meropenem/Vaborbactam on Carbapenem-Resistant Enterobacterales in Egypt: A Cross-Sectional Study.

Purpose: The increasing multi-drug carbapenem resistance among Enterobacterales are a severe health problem limiting therapeutic options and worsen the prognosis. This study characterizes carbapenemase genes and integrons among uropathogenic carbapenem resistant Enterobacterales (CRE) isolates recovered from Mansoura University Hospitals and evaluates the effect of colistin, fosfomycin and meropenem-vaborbactam on these isolates. Patients and Methods: A total of 200 Enterobacterales isolates were collected from patients with urinary tract infections. Antimicrobial susceptibility testing was performed by the disc diffusion method. Colistin susceptibility was tested using the broth microdilution method and fosfomycin and meropenem/vaborbactam susceptibility were tested by MIC Test Strips. Carbapenem resistant isolates were screened for carbapenemase activity phenotypically using the modified carbapenem inactivation method and EDTA-modified carbapenem inactivation method and genotypically by multiplex PCR. Integrons class 1 and 2 and fosA gene were assayed by PCR. Data were statistically analyzed using the Statistical Package for Social Sciences (SPSS) version 16. The Chi-square or Fisher's exact test was used to compare groups, as appropriate. Results: Ninety-two Enterobacterales isolates were resistant to meropenem (46%); 52 E. coli and 40 K. pneumoniae strains. All CRE isolates were multi-drug resistant (MDR). Sensitivity of CRE isolates to colistin, fosfomycin and meropenem/vaborbactam were 67.4%, 82.6% and 58.7%, respectively. Carbapenemase genes were detected by multiplex PCR in 69.6% of CRE isolates (Carbapenemase producing Enterobacterales (CPE) mainly blaNDM (37%). CPE isolates were significantly more resistant to meropenem/vaborbactam than non-CPE isolates; 51.6% vs 17.8%, respectively (P = 0.003) especially blaNDM carrying isolates (70.6%). Class 1 integrons and fosA gene were detected in 91.3% and 11.9% of CRE isolates, respectively. Conclusion: This study revealed that about half of the uropathogenic Enterobacterales isolates were MDR CRE. Carbapenemase gene blaNDM was the main gene among CRE isolates. Meropenem/vaborbactam sensitivity was significantly higher on non-CPE than CPE isolates and limited by the predominance of blaNDM .

Molecular Detection of Sapovirus in Children Under Five Years with Acute Gastroenteritis in Mansoura, Egypt between January 2019 and February 2020.

Background: Sapovirus has emerged as a viral cause of acute gastroenteritis. However, there are insufficient data about the presence of this virus among children with acute gastroenteritis. The present study aimed to evaluate the presence of sapovirus in children with acute gastroenteritis by reverse transcriptase-polymerase chain reaction (RT-PCR). Methods: A cross-sectional study enrolled 100 children patients with acute gastroenteritis from outpatient clinics with excluded bacterial pathogens and parasitic infestation. A stool sample was collected from each child for laboratory examination. Each stool sample was subjected to study by direct microscopic examination, study for rotavirus by enzyme-linked immunoassay (ELISA) and the remaining sample was subjected to RNA extraction and RT- PCR for sapovirus. Results: The most frequently detected virus was rotavirus by ELISA (25%). RT-PCR detected sapovirus in 7% of the stool samples. The children with sapovirus were all from rural regions and presented mainly during the winter season in Egypt (42.9%). The main presenting symptoms were fever (71.4%) and vomiting (57.1%). None of the children with sapovirus had dehydration. Rotavirus was significantly associated with sapovirus infections in 5 patients (71.4%, P=0.01). There was an insignificant difference between symptoms of gastroenteritis in children with sapovirus and children with gastroenteritis without sapovirus as regards vomiting (P=0.7), fever (P=0.46), and abdominal pain (P=0.69). Conclusion: The present study highlights the emergence of sapovirus as a frequent pathogen associated with acute gastroenteritis in children. There is a need for a national survey program for the study of sapovirus among other pathogens associated with acute gastroenteritis for better management of such infection.

Diagnostic Value of Multiplex Polymerase Chain Reaction in Detection of Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from Sepsis in Pediatrics.

BACKGROUND: Proper identification of the causative organism in pediatric sepsis is crucial for early diagnosis and prevention of septic shock and organ failure. OBJECTIVES: The aim of the present study was to evaluate the multiplex polymerase chain reaction (PCR) for detection of Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from positive blood cultures for these pathogens isolated from children with hospital- acquired sepsis compared to the conventional biochemical reactions for identification of these organisms. METHODS: This study was a cross-sectional study performed on 100 isolates from pediatric blood cultures, including Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The study also included 100 isolates of Escherichia coli as a negative control. All isolates were identified by API 20NE and the multiplex PCR with primers specific to the 3 tested bacteria. RESULTS: Multiplex PCR was positive in 96% of isolates and 4 isolates had negative results. Falsepositive results were reported with three E. coli strains. Multiplex PCR identified all the isolates of Acinetobacter baumannii, 29 isolates of Pseudomonas aeruginosa and 27 isolates of Stenotrophomonas maltophilia. The diagnostic value of the multiplex PCR compared to the biochemical identification revealed sensitivity 96.04%, specificity 96.9%, positive predictive value 97.00%, negative predictive value 96.00% and accuracy 96.50%. CONCLUSION: The present study highlights the diagnostic value of multiplex PCR to identify Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from positive blood cultures. Multiplex PCR was sensitive, specific and accurate. The accuracy differs according to the organisms with 100% accuracy for Acinetobacter baumannii.

Characterization of E. coli Phylogroups Causing Catheter-Associated Urinary Tract Infection.

PURPOSE: Characterization of different uropathogenic E. coli (UPEC) phylogroups is crucial to understand pathogenesis of urinary tract infection (UTI). The objective of our study was to evaluate the antibiotic resistance pattern, biofilm formation and pathogenicity islands (PAIs) of UPEC phylogroups isolated from catheter-associated UTI (CAUTI) compared to community UTI (Com-UTI). PATIENTS AND METHODS: This study included 90 UPEC strains recovered from CAUTI and Com-UTI. Antimicrobial susceptibility was tested by the Kirby-Bauer method and extended spectrum beta-lactamase (ESBL) production was confirmed using the combined disk. The biofilm formation was tested using the microtiter plate assay. Main E. coli phylogroups (A, B1, B2 and D) were detected by multiplex PCR and 2 multiplex PCR detected the 8 PAIs. RESULTS: Antibiotic resistance of UPEC strains showed a similar high resistance in CAUTI and Com-UTI. Isolates from CAUTI significantly produced biofilm higher than Com-UTI strains (68.9% vs 44.4%). In CAUTI and Com-UTI isolates, phylogroup A was the commonest (53.3% vs 48.9%, respectively). PAI IV536 was the most common in the strains from CAUTI (71.1%) and Com-UTI (73.3%). No significant relationship was detected between the studied characters and different phylogroups except the significant resistance to cefotaxime, ceftazidime and aztreonam among phylogroups from CAUTI isolates. CONCLUSION: Increased antibiotic resistance and ESBLs were detected in UPEC strains from CAUTI and Com-UTI. The strains from CAUTI significantly produced biofilm higher than Com-UTI strains. Phylogroup A was the predominate phylogroup and PAI IV536 was the most prevalent marker in all phylogroups from both types of UTI.

Study of the diagnostic value of interleukin-6 and interleukin-8 in children with acute gastroenteritis.

INTRODUCTION: Gastroenteritis in children is responsible for high morbidity and mortality. Our aim was to determine the serum and fecal levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) in children with acute gastroenteritis of viral and bacterial etiology to assess their utility as diagnostic biomarkers for these infections. METHODS: In this case-control study, the children were classified according to the pathogen recovered from the stool by bacterial culture or by direct viral antigen detection by enzyme immunoassay (EIA) into 50 children with acute bacterial gastroenteritis and 50 children with acute viral gastroenteritis. In addition, 50 apparently healthy children were included as a control group. Blood and stool samples were subjected to detection of IL-6 and IL-8. RESULTS: There were statistically significant elevations of total leucocytes counts, absolute neutrophils count, C-reactive protein, serum IL-6 and serum IL-8 in children with gastroenteritis compared to healthy children (p<0.001). CRP, serum IL-6 and IL-8 had significantly elevated levels in children with bacterial gastroenteritis compared to viral gastroenteritis. Fecal IL-6 and IL-8 had significantly elevated levels in children with acute gastroenteritis than in healthy control (p<0.001). The area under the curve (AUC) showed that CRP and serum IL-6 could be used as discriminative markers for acute bacterial gastroenteritis in children, in comparison to serum IL-8. CONCLUSIONS: Elevated serum IL-6 and CRP can aid in differentiation between viral and bacterial gastroenteritis. Serum IL-8 had limited discrimination ability between viral and bacterial gastroenteritis. Stool levels of IL-6 and IL-8 were elevated in children with viral and bacterial gastroenteritis, however, their assessment by enzyme linked immunosorbent assay had technical limitations to be used as differentiation biomarkers.

Candida glabrata complex from patients with healthcare-associated infections in Mansoura University Hospitals, Egypt: distribution, antifungal susceptibility and effect of fluconazole and polymyxin B combination.

INTRODUCTION: Candida glabrata complex is composed of three cryptic species, Candida glabrata sensu stricto, C. bracarensis and C. nivariensis. Its reduced susceptibility to fluconazole is responsible for treatment failure of its infections. Combination therapy is recommended to treat these resistant strains. This study assessed the distribution of C. glabrata complex collected from patients in Mansoura University Hospitals and their antifungal susceptibility, with evaluation of fluconazole and polymyxin B combination against them. METHODS: C. glabrata complex was collected from patients with healthcare-associated infections. The isolates were identified biochemically then the species were detected using multiplex PCR. The susceptibility of isolates to antifungals and polymyxin B was tested by the microdilution assay. The effect of fluconazole and polymyxin B combination was assessed by checkerboard microdilution and time kill assays. RESULTS: This study included 45 isolates of Candida glabrata complex. The common isolate was Candida glabrata sensu stricto (38 isolates). There were 4 isolates of C. bracarensis and three isolates of C. nivariensis. All isolates were susceptible to amphotericin B, and 17.8% were susceptible to itraconazole. Regarding fluconazole, 48.9% of isolates were susceptible dose-dependent and 51.1% were resistant. Synergistic effect of the combination was observed in 68.9% of isolates by the checkerboard method and in 66.7% of isolates using the time kill assay. CONCLUSIONS: Candida glabrata sensu stricto is the main member of the complex. Combination of fluconazole and polymyxin B can be considered a treatment option for fluconazole resistant C. glabrata complex infections. In vivo studies are needed to validate these results.

Antifungal treatment affects the laboratory diagnosis of invasive aspergillosis.

AIMS: The purpose of this study was to investigate the performance of non-invasive diagnostic tests such as galactomannan enzyme immunoassay and quantitative PCR in the early diagnosis of invasive aspergillosis (IA), and how these tests are impacted upon by the use of different classes of antifungal agents in an in-vivo model of IA. METHODS: A standardised rat inhalation model of IA was used to examine the effects of an azole, posaconazole, a polyene, amphotericin B and an echinocandin caspofungin. Daily blood samples were collected for subsequent analysis using a commercially available galactomannan assay and an inhouse qPCR assay. RESULTS: No significant differences were observed in the CE/g of Aspergillus fumigatus in the lungs of each group. qPCR was statistically more sensitive than galactomannan for both the early detection of infected controls (p=0.045) and for overall detection (p=0.018). However, antifungal treatment significantly reduced the overall sensitivity of qPCR (p=0.020); these effects were due to posaconazole and caspofungin. In the latter stages of infection (days 4 and 5) there were no significant differences in the numbers of infections detected by galactomannan and qPCR; however, the antifungal class used caused significant qualitative differences (p=0.041). Galactomannan showed improved detection in posaconazole-treated animals. CONCLUSIONS: Previous exposure to antifungal therapy must be considered when interpreting either qPCR or galactomannan-based IA diagnostics as this study has shown that individual classes of antifungal agents impact upon the dynamics of antigen and DNA release into the circulation.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Detection of Aspergillus fumigatus in a rat model of invasive pulmonary aspergillosis by real-time nucleic acid sequence-based amplification.

Rapid and sensitive detection of Aspergillus from clinical samples may facilitate the early diagnosis of invasive pulmonary aspergillosis (IPA). A real-time nucleic acid sequence-based amplification (NASBA) method was investigated by use of an inhalational rat model of IPA. Immunosuppressed male Sprague-Dawley rats were exposed to Aspergillus fumigatus spores for an hour in an aerosol chamber. Bronchoalveolar lavage (BAL) fluid, lung tissues, and whole blood were collected from five infected rats at 1, 24, 48, 72, and 96 h postinfection and five uninfected rats at the end of the experiment. Total nucleic acid (TNA) was extracted on an easyMAG instrument. A primer-molecular beacon set targeting 28S rRNA was designed to detect Aspergillus spp. The results were compared to those of quantitative PCR (qPCR) (18S rDNA) and quantitative culture. The analytical sensitivity of the real-time NASBA assay was <1 CFU/assay. A linear range of detection was demonstrated over 5 log units of conidia (10 to 10(5) spores). Both NASBA and qPCR showed a progressive increase in lung tissue burdens, while the CFU counts were stable over time. The fungal burdens in BAL fluid were more variable and not indicative of a progressive infection. The results of both real-time assays correlated well for both sample types (r = 0.869 and P < 0.0001 for lung tissue, r = 0.887 and P < 0.0001 for BAL fluid). For all whole-blood specimens, NASBA identified Aspergillus-positive samples in the group from which samples were collected at 72 h postinfection (three of five samples) and the group from which samples were collected at 96 h postinfection (five of five samples), but no positive results were obtained by culture or PCR. Real-time NASBA is highly sensitive and useful for the detection of Aspergillus in an experimental model of IPA.