Applying Unsterile Microporous Tape onto Surgical Wounds: Tape Contamination and Clinical Rationale.

Fomites are surfaces that carry contaminants and may cause infection. We wanted to assess the bacterial load on rolls of nonsterile microporous tape in a hospital setting and explore the scientific rationale behind the existing practice of applying unsterile adhesives onto a surgical wound. METHODS: We analyzed the aerobic bacterial contamination in rolls of microporous tape collected from surgical theaters, outpatient clinics, and storage rooms at St. Olav's University Hospital, Trondheim, Norway between 2018 and 2020. We also reviewed the literature for relevant publications. RESULTS: A total of 58 rolls were collected; 55 were included for final analysis. Exposed tape surfaces were significantly more contaminated than unexposed surfaces. Tape rolls from outpatient clinics were significantly more contaminated and contained a significantly greater variety of microbes than rolls from operation theaters and storage rooms. Unexposed surfaces from both operation theaters and storage rooms demonstrated very little contamination. CONCLUSIONS: Rolls of tape may act as fomites, but widespread use of adhesives is inevitable in hospital settings. Removing the outer layer of a tape roll before use may significantly reduce bacterial contamination. Given sufficient vigilance to avoid cross-contamination, inner layers of tape may represent a close-to-sterile alternative as surgical dressing. However, the economic savings constitute a negligible fraction of the total costs of the surgery, and the risk of contamination seems apparent. Scientific support of dressing a fresh surgical wound with unsterile microporous tape is lacking, and we therefore do not recommend the practice except in situations with very limited resources.

Laminar airflow and mixing ventilation: Which is better for operating room airflow distribution near an orthopedic surgical patient?

BACKGROUND: There has been little research on the performance of laminar airflow (LAF) and mixing ventilation (MV) systems regarding clean airflow distribution near a surgical patient in operating rooms (ORs). The objective of this study was to examine the performance of LAF and MV systems in ORs at St Olavs Hospital in Norway. METHODS: Experimental measurements were conducted in 2 ORs equipped with LAF and MV systems. RESULTS: Under real operating conditions, airflow distribution from the LAF system was disrupted, and airflow velocity became significantly lower than that of MV above the lying patient. Airflow pattern was observed as distributed vertically downward and horizontally with LAF and MV, respectively. Turbulence intensity of supply airflow from LAF was much lower than that of MV. CONCLUSIONS: The airflow distribution by LAF system in close proximity to a patient is greatly affected by thermal plumes generated above incisions by both patients and surgical facilities. The effect of surgical facilities on airflow distribution by using MV is not significant compared to LAF ventilation. New guidelines are needed for the design of clean airflow distribution systems in the vicinity of surgical patients in ORs.

Investigation of an outbreak caused by antibiotic-susceptible Klebsiella oxytoca in a neonatal intensive care unit in Norway.

AIM: Klebsiella spp. have been stated to be the most frequent cause of neonatal intensive care unit (NICU) outbreaks. We report an outbreak of Klebsiella oxytoca in a NICU at a tertiary care hospital in Norway between April 2016 and April 2017. This study describes the outbreak, infection control measures undertaken and the molecular methods developed. METHODS: The outbreak prompted detailed epidemiological and microbial investigations, where whole-genome sequencing (WGS) was particularly useful for both genotyping and development of two new K. oxytoca-specific real-time PCR assays. Routine screening of patients, as well as sampling from numerous environmental sites, was performed during the outbreak. A bundle of infection control measures was instigated to control the outbreak, among them strict cohort isolation. RESULTS: Five neonates had symptomatic infection, and 17 were found to be asymptomatically colonised. Infections varied in severity from conjunctivitis to a fatal case of pneumonia. A source of the outbreak could not be determined. CONCLUSION: This report describes K. oxytoca as a significant pathogen in a NICU outbreak setting and highlights the importance of developing appropriate microbiological screening methods and implementing strict infection control measures to control the outbreak in a setting where the source could not be identified.

Molecular characteristics of Streptococcus agalactiae strains deficient in alpha-like protein encoding genes.

Streptococcus agalactiae (group B streptococci, GBS) are important human and animal pathogens, which can be subdivided based on different capsular polysaccharides and surface-anchored alpha-like proteins (Alps), as well as other proteins. Nearly all GBS strains possess an Alp (Alp GBS), although Alp-negative GBS (non-Alp GBS) do occur. In this study, 10 (1.1 %) of 932 clinical human GBS tested lacked an Alp encoding gene. All 10 strains were from patients with bloodstream infection, confirming that non-Alp GBS can be highly virulent. All non-Alp GBS expressed one or more of the surface-anchored proteins R3, Z1 and Z2, while less than 10 % of unselected clinical strains express any of these proteins. In contrast to Alp GBS, all non-Alp strains tested were PCR negative for the upstream sequence of the insertion site of the Alp encoding gene of Alp GBS. Genome sequencing showed that all but one of the 10 clinical non-Alp strains and the non-Alp reference strain CNCTC 10/84 lacked a region surrounding the Alp gene commonly present in Alp GBS strains. These strains instead harboured an 849 bp region not present in the Cα prototype strain A909. We have shown that non-Alp GBS differ from Alp GBS in the region surrounding the insertion site of Alp genes of Alp GBS as well as in their content of other surface proteins and that PCR for the upstream flanking region of the Alp gene may be useful for differentiation between Alp and non-Alp GBS.

A Silenced vanA Gene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci.

We report an outbreak of vancomycin-variable vanA(+) enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA(+) Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 µg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised.

Comparison of PCR and serotyping of Group B Streptococcus in pregnant women: the Oslo GBS-study.

Streptococcus agalactiae (GBS) is a leading cause of invasive neonatal infection. Serotyping of GBS is important in following epidemiological trends and vaccine development. Capsular serotyping of GBS by latex agglutination has been the predominant typing method, but more recently capsular genotyping has been introduced as an alternative method. The purpose of this study was to compare the relative performance of these methods in a contemporary population of pregnant women. We typed isolates from an unselected population of 426 colonized women at delivery using latex agglutination and a combination of four PCR methods. Antibiotic resistance was tested in 449 isolates. Capsular genotyping gave a result in all except three of 426 isolates. Fifty-nine of 426 isolates could not be typed by latex agglutination. Agreement between serotyping and genotyping was shown in 303 (71.1%) of the isolates. 10.2% of the isolates were resistant to erythromycin, 9.6% to clindamycin, 76.6% to tetracycline and none to penicillin. In conclusion, a substantial proportion of the colonizing strains were non-typeable by serotyping, but typeable by genotyping. This suggests that a diagnostic genotyping strategy is preferable to serotyping of the GBS polysaccharide capsule in colonized, pregnant women.

Survey of immunological features of the alpha-like proteins of Streptococcus agalactiae.

Nearly all Streptococcus agalactiae (group B streptococcus [GBS]) strains express a protein which belongs to the so-called alpha-like proteins (Alps), of which Cα, Alp1, Alp2, Alp3, Rib, and Alp4 are known to occur in GBS. The Alps are chimeras which form mosaic structures on the GBS surface. Both N- and C-terminal stretches of the Alps possess immunogenic sites of dissimilar immunological specificity. In this review, we have compiled data dealing with the specificity of the N- and C-terminal immunogenic sites of the Alps. The majority of N-terminal sites show protein specificity while the C-terminal sites show broader cross-reactivity. Molecular serotyping has revealed that antibody-based serotyping has often resulted in erroneous Alp identification, due to persistence of cross-reacting antibodies in antisera for serotyping. Retrospectively, this could be expected on the basis of sequence analysis results. Some of the historical R proteins are in fact Alps. The data included in the review may provide a basis for decisions regarding techniques for the preparation of specific antisera for serotyping of GBS, for use in other approaches in GBS research, and for decision making in the context of GBS vaccine developments.

Comparison of Z and R3 antigen expression and of genes encoding other antigenic markers in invasive human and bovine Streptococcus agalactiae strains from Norway.

Streptococcus agalactiae (GBS) may cause a variety of infectious diseases in humans caused by human GBS and mastitis in cattle caused by bovine GBS. Over the last few years molecular testing has provided evidence that human and bovine GBS have evolved along diverse phylogenetic lines. In the present study 173 invasive human GBS strains and 52 invasive bovine strains were tested for altogether 18 strain-variable and surface-localized antigenic markers including all 10 capsular polysaccharides (CPS) and proteins including Cß, the alpha-like proteins, R3 and the recently described Z1 and Z2 antigens. PCR was used to detect encoding genes and antibody-based methods to detect expression of antigens. Thirteen of the 18 markers were detected in isolates of both strain categories. Seven of the ten CPS antigens were detected in both groups with types III and V predominating in the human GBS strains, types IV and V in the bovine isolates. Z1, Z2 and/or R3 expression and the genes encoding Cß, Cα, Alp1, Alp2/3 or R4 (Rib) were detected in both groups. Protein antigen-CPS associations well known for human strains were essentially the same in the bovine isolates. The results show that in spite of evolution along different lines, human and bovine GBS share a variety of surface-exposed antigenic markers, substantiating close relationship between the two GBS subpopulations.

Novel aspects of the Z and R3 antigens of Streptococcus agalactiae revealed by immunological testing.

Group B streptococci (GBS) are important human and bovine pathogens which can be classified by a variety of phenotype- and gene-based techniques. The capsular polysaccharide and strain-variable, surface-anchored proteins are particularly important phenotypic markers. In an earlier study, a previously unrecognized protein antigen called Z was described. It was expressed by 27.2% of GBS strains from Zimbabwe, usually in combination with R3 protein expression. In this study, a putative Z-specific antiserum actually contained antibodies against two different antigens named Z1 and Z2; Z1 was >250 kDa in molecular mass. Z1, Z2, and R3 generated multiple stained bands on Western blots and showed similar chromatographic characteristics with respect to molecular mass, aggregate formation, and charge. Of 28 reference and prototype GBS strains examined, 8/28 (28.5%) isolates expressed one, two, or all three of the Z1, Z2, and R3 antigens; 4/28 expressed all three antigens; 2/28 expressed Z2 and R3; 1/28 expressed Z1 only; and 1/28 expressed R3 only. Twenty (71.5%) of the 28 isolates expressed none of the three antigens. Expression of one or more of these antigens was shown by isolates of the capsular polysaccharide types Ia, Ib, V, and IX and NT strains and occurred in combination with expression of various other strain-variable and surface-localized protein antigens. When used as serosubtype markers, Z1, Z2, and R3 affected existing GBS serotype designations for some of the isolates. For instance, the R3 reference strain Prague 10/84 (ATCC 49447) changed serotype markers from V/R3 to V/R3, Z1, and Z2. Other isolates may change correspondingly, implying consequences for GBS serotyping and research.

Multiple-locus variant-repeat assay (MLVA) is a useful tool for molecular epidemiologic analysis of Streptococcus agalactiae strains causing bovine mastitis.

Group B streptococci (GBS) were considered a major cause of mastitis in cattle until preventive measures succeeded in controlling the disease in the 1970s and 1980s. During the last 5-6 years an increasing number of cases have been observed in some Scandinavian countries. A total of 187 GBS isolates from mastitis cases were collected from 119 animals in 34 Norwegian farms in the period from April 2007 to November 2010. 133 (71%) of the isolates were from farms with automated milking systems. The strains underwent typing of capsular polysaccharides (CPS) and surface proteins, and were analyzed by multi-locus variable repeat assay (MLVA) to investigate the epidemiological relationship of strains within and between farms. The GBS strains were differentiated into 12 types by CPS and surface protein analysis, with CPS types V (54%) and IV (34%) predominating. MLVA was superior to CPS and protein typing for strain differentiation, resolving the 187 strains into 37 types. In 29 of 34 farms all GBS strains had identical MLVA profiles specific for each farm. However, in one farm represented with 48 isolates, four MLVA variants with differences in one repeat locus were observed during the almost 3-year long collection period. Similar variations were observed at four other farms. This might reflect the stability of repeat loci under in vivo conditions. Farms with automated milking systems were overrepresented in this material. In conclusion, the five-loci MLVA allowed rapid high-resolution genotyping of the bovine GBS strains within and between farms.

Streptococcus agalactiae alpha-like protein 1 possesses both cross-reacting and Alp1-specific epitopes.

Most isolates of group B streptococci (GBS) express an alpha-like protein (Alp), Cα (encoded by bca), Alp1 (also called epsilon; alp1), Alp2 (alp2), Alp3 (alp3), Alp4 (alp4), or R4/Rib (rib). These proteins are chimeras with a mosaic structure and with antigenic determinants with variable immunological cross-reactivities between the Alps, including Alp1 and Cα cross-reactivity. This study focused on antigenic domains of Alp1, studied by using rabbit antisera in immunofluorescence, Western blotting, and enzyme-linked immunosorbent assay (ELISA)-based tests and whole cells of GBS or trypsin-extracted and partially purified antigens from the strains A909 (serotype Ia/Cα, Cß) and 335 (Ia/Alp1). Alp1 and Cα shared an antigenic determinant, Alp1/Cα common, not harbored by other Alps, probably located in the Alp1 and Cα repeat units, as these units are nearly identical in genomic sequence. An antigenic Alp1 determinant was Alp1 specific and was most likely located in the N-terminal unit of Alp1 in which an Alp1-specific primer site for PCR is also located. In addition, Alp1 possessed a domain with low immunogenicity which cross-reacted immunologically with Alp2 and Alp3, with unknown location in Alp1. Alp1 was partially degraded by trypsin during antigen extraction but with the antigenic domains preserved. The results indicate that Cα and Alp1 are immunologically related in the same manner that R4 (Rib) and Alp3 are related. The domain called Alp1 specific should be important in GBS serotyping as a surface-anchored serosubtype marker. The Alp1/Cα common determinant may be of prime interest as an immunogenic domain in a GBS vaccine.

International external quality assurance for laboratory identification and typing of Streptococcus agalactiae (Group B streptococci).

We report the results from the first international multicenter external quality assessment (EQA) studies for molecular and serological typing of group B streptococcus (GBS) strains as part of DEVANI (Design of a Vaccine against Neonatal Infections), a pan-European program. A questionnaire-based surveillance was undertaken among eight laboratories participating in DEVANI and six laboratories not participating in DEVANI from 13 countries in order to assess their current microbiological procedures for GBS screening, diagnosis, and typing. GBS strains from three EQA distributions were characterized using molecular and serological methods based on GBS capsular polysaccharide typing. Participants were asked to test the first distribution using their current serotyping and genotyping methods. The Strep-B-Latex agglutination method was the most widely used method, with a typeability value of >90%. A multiplex PCR assay for GBS capsular gene typing was also used by 2 of 14 centers, which achieved a typeability value of 93%; this assay detected only 9 of 10 GBS capsular polysaccharide genes. From the second and third EQA studies, standardized protocols were prepared for serological and molecular typing of GBS strains based on the Strep-B-Latex agglutination method and a novel multiplex PCR assay that detected all 10 GBS capsular types (Ia to IX). These standardized protocols are being used by many European laboratories, and as the use of these methods increases, it is imperative to continuously improve and assess laboratory performance and offer training to any laboratories that have technical difficulties.

Rapid multiple-locus variant-repeat assay (MLVA) for genotyping of Streptococcus agalactiae.

Several methods have been used for typing of Streptococcus agalactiae (group B streptococci [GBS]). Methods currently in use may provide inadequate resolution (e.g., typing of capsular polysaccharides and surface protein) or are labor-intensive and expensive (e.g., multilocus sequence typing [MLST] or pulsed-field gel electrophoresis). This work describes the construction and use of a multiple-locus variant-repeat assay (MLVA) on 126 well-characterized human GBS strains, consisting mostly of invasive Norwegian strains and international reference strains. Based on in silico whole-genomic analysis of the genomes of strains A909, NEM316, and 2603V/R, 18 candidate loci were selected and investigated by PCR. Eleven loci showed diversity, and the five most diverse loci were used for the construction of an MLVA, consisting of a multiplex PCR followed by fragment analysis with capillary electrophoresis. The assay generated clusters which corresponded well with those observed by other methods. However, it provided a considerably higher degree of diversity, with 70 different MLVA types compared to 36 types generated by MLST. Simpson's index of diversity for the 5-locus MLVA was 0.963, compared to 0.899 for the MLST in this strain collection. MLVA results will generally be available within 2 days, which is usually faster than MLST. In our hands, MLVA of GBS represents a rapid, easy, and comparably inexpensive method for high-resolution genotyping of GBS.

Identification of surface proteins of group B streptococci: serotyping versus genotyping.

We compared serotyping to genotyping of group B streptococcal (GBS) surface proteins in 147 Australasian isolates. Results were concordant for the two methods in 73.8% of 122 isolates, discordant for three and partially discordant for 29 isolates. For the purpose of epidemiological typing of GBS, genotyping is superior to serotyping.

[Human metapneumovirus--occurrence and clinical significance]. / Humant metapneumovirus--forekomst og klinisk betydning.

BACKGROUND: Human metapneumovirus (hMPV) was isolated for the first time in 2001 from young children with acute respiratory tract infection. MATERIALS AND METHODS: Review of published data on the clinical impact of hMPV and our own experience with hMPV during three winter seasons. We present four cases of severe hMPV childhood infections. RESULTS: Human metapneumovirus accounts for approximately 10% of respiratory tract infections that are not related to previously known etiologic agents. The virus seems to be distributed worldwide and to have a seasonal distribution. During a short epidemic, 60% of our isolates from children with respiratory infection tested positive for hMPV. Serologic studies have shown that by the age of five, virtually all children have been exposed to the virus and reinfections appear to be common. Human metapneumovirus may cause mild respiratory tract infection. Small children, elderly and immunocompromised individuals are, however, at risk of severe disease and hospitalization. Although the clinical manifestations of hMPV resemble those of respiratory syncytial virus, we have observed that hMPV more often causes severe pneumonia in hospitalized children. INTERPRETATION: Human metapneumovirus is an important cause of acute respiratory tract infection in children. The virus may cause severe disease in patients at risk. We recommend identification and isolation of hospitalized hMPV-infected patients.

[Human metapneumovirus--virologic and diagnostic aspects]. / Humant metapneumovirus--virologiske og diagnostiske aspekter.

Human metapneumovirus (hMPV) is a pathogenic respiratory virus, discovered in 2001. The virus is part of the family paramyxoviridae and is comparatively closely related to respiratory syncytial virus (RSV). The clinical manifestations of a hMPV infection are rather similar to infections caused by RSV, ranging from mild upper airway disease to severe pneumonia. Detection of hMPV RNA by polymerase chain reaction (PCR) is the only established routine diagnostic method. The virus is easily cultivated in several types of cell culture lines, however, cell pathology (cytopathic effect) is irregularly observed. Our department has developed an indirect immunofluorescence test based on polyvalent rabbit antibodies. This method appears to give reliable results, detecting hMPV both in cell culture and directly on patient samples. Nasopharyngeal aspirate is the preferred material for diagnostic purposes in small children. A nasopharyngeal swab, or alternatively a throat swab, can be used for older children and adults. Samples from lower airways are appropriate under special circumstances. Serology is not available for routine diagnostic use.

Outbreak of human metapneumovirus infection in norwegian children.

INTRODUCTION: Human metapneumovirus (hMPV) was recently discovered in children with acute respiratory tract infection. We have studied the occurrence of hMPV and report clinical findings of 50 hMPV-infected children who were hospitalized during an outbreak in Norway. METHODS AND POPULATION: During 5 months from November 15, 2002 to April 14, 2003 we collected nasopharyngeal aspirate specimens from 236 children admitted because of respiratory tract infection (RTI). Samples were analyzed for influenza virus A/B, parainfluenza viruses 1, 2 and 3 and respiratory syncytial virus by direct immunofluorescence assays and cell culture. Rhinovirus, adenovirus and hMPV were identified by polymerase chain reaction. RESULTS: Human metapneumovirus was identified in 50 of 236 children (21%). Most (41 of 50) hMPV-infected children were hospitalized between November 15 and January 15, and during these 2 months hMPV was the most common isolate (41 of 72 isolates; 57%). Respiratory syncytial virus was identified in 36 children (15%), among whom 34 were admitted after the hMPV outbreak. The median age of hMPV-infected children was 12 months (range, 1 to 115 months), and one-half of the children had an underlying chronic disease. The most common symptoms were fever (86%), cough (90%), dyspnea (80%), wheezing (56%), rhinorrhea (44%), anorexia (48%) and vomiting (36%). Eight (16%) had an upper respiratory tract infection (rhinopharyngitis, n = 6; laryngitis, n = 2), 24 (48%) had bronchiolitis and 17 (34%) had pneumonia. Two-thirds with a lower RTI also had signs of upper RTI. Fourteen (28%) children needed supplemental oxygen, 1 was treated with continuous positive airway pressure and 2 were ventilated mechanically. CONCLUSION: Human metapneumovirus was the most common virus isolate during the winter season 2002 to 2003 in children hospitalized for respiratory tract infection. Upper respiratory tract infections and mild to severe bronchiolitis were most common, but a relatively high proportion of hospitalized children developed severe pneumonia.