The incidence of sepsis is a growing problem worldwide with a high mortality rate.  K- capsule antigens are becoming more dangerous than before. The purpose of the study was to categorize the capsules virulence factors, as well as to find a safe and empirical antibiotic treatment for sepsis infection, and to determine if the existence of ESBL producing bacteria have increased exponentially in recent decades. A total of 101 isolates were collected for a period of 5 years, of which blood isolates (n=38) were collected at Uppsala university hospital and feces isolates from healthy mothers and their infants (n=63) at Cunghi hospital, China. Isolates were serotyped with agglutination test, using N. meningitides B / Ecoli K1 against K1 capsule antigen, and phage test, using K1, K5 and K13 bacteriophage to identify the corresponding E. coli K antigen. Results showed that K1- capsule antigen could be identified in 42% (16/38) of the blood-isolated bacterial strains, compared with 11% (7/63) in fecal-isolated bacterial strains, while K5- capsule antigen was serotyped in 5% (2/38) and 19% (12/63), respectively. In contrast, the K13 capsule antigen was not found in blood-isolated bacterial strains and was serotyped only in 4% (3/63) of the fecal-isolated bacterial strains. Overall, the investigated E.coli  K capsular antigens were identified in 47% (18/38, non -typeable n=20) of the blood-isolated cultures compared with 35% ( 22/36, non-typeable=41) in fecal-isolated cultures..

Immuncytokemi (ICC) har blivit en framgångsrik metod inom cancerdiagnostiken. ICC bygger på antikropps- och antigenreaktion då infärgning med specifika antikroppar kan lokalisera strukturer hos celler. Hos klinisk patologi i Kalmar utförs ICC med den traditionella prepareringsmetoden där materialet formalinfixeras, bäddas i paraffin och snittas enligt cell block teknik (CBT). Vid ICC med vätskebaserad cytologi (LBC, liquid-based cytology) kan infärgning utföras på en mindre mängd material samt ge ett resultat 1-2 dagar innan resultat fås med CBT. ICC med LBC saknar kontroller vilket medför att resultatet blir mindre tillförlitligt. Syftet med examensarbetet var att ta fram positivt kontrollmaterial för ICC av LBC-preparerat material. Kontrollmaterial selekterades från tidigare diagnostiserade och immunfärgade patientprover i form av exsudat och tonsillmaterial. En panel av 6 monoklonala antikroppar bestående av anti-CD20, anti-CD3, anti-CK5, anti-CK7, anti-Ki-67 och anti-WT1 användes. Kontrollmaterialet preparerades enligt ThinPrep™-metoden och sattes till objektglas med Cytospin™. När kontrollmaterialets positivitet bekräftats testades det i rutinarbete ihop med 10 patientprover. Patientproverna preparerades med ThinPrep™-metod och placerades på objektglasen med hjälp av en ThinPrep™5000 processor. Samtliga resultat jämfördes med infärgning gjord med CBT och majoriteten av resultaten var likvärdiga. Ur resultatet drogs slutsatsen att metoden verkar lovande men prepareringssteg och färgprotokoll behöver optimeras samt att större studier med fler patientprover behövs för att kunna beräkna tillförlitlig statistik. / Immunocytochemistry (ICC) has become a successful method of cancer diagnosis. ICC is based on antibody and antigen response, where staining with specific antibodies can localize structures in cells. Today at clinical pathology in Kalmar ICC is performed with the traditional method of preparation where the material is formalin fixed, embedded in paraffin and then cut into thin sections called cell block technique (CBT). ICC with liquid-based cytology (LBC) can be performed on a smaller amount of material and give results 1-2 days before results are obtained with CBT. ICC combined with LBC lacks controls so the result is not reliable. Purpose of this essay was to produce positive control material for the ICC of LBC-prepared material. Control materials were selected from previously diagnosed and immune-stained patient samples consisting of exudate and tonsillar material. A panel of 6 monoclonal antibodies, anti-CD20, anti-CD3, anti-CK5, anti-CK7, anti-Ki-67 and anti-WT1, was used. Control material was prepared according to the ThinPrep ™ method and was added to slides with Cytospin ™. After confirming the positivity of the control material, it was tested in routine work with 10 patient samples. Patient samples were prepared using the ThinPrep ™ method and added to the slides with the ThinPrep ™ 5000 processor. Results compared to staining with CBT showed correlation. Conclusion was that the method seems promising but stages of preparation and staining protocols need to be optimized and larger studies with more patient samples must be done in order to get reliable statistic data.

The mesenchymal stromal cell (MSC) is of great interest for its immunomodulatory and regenerative properties. However, to research and use these MSCs it is essential to identify and characterize them as such. They need to fulfill the MSCs' minimal criteria which assess the differentiation potential, cell surface markers, and adherence. In this study, cells donated from human bone marrow were identified as MSC according to the minimal criteria. Methods used were flow cytometry, immunofluorescent staining, and ELISA. Furthermore, the population was cultured in three different media (DMEM-LG with either 10% FBS, 2% FBS, or 10% FBS supplemented with 10% conditioned media from human urinary bladder carcinoma cells (T24)) for 21 days whereupon tested for the mesenchymal characteristics, cells were counted and size measured at every passage. All cultures maintained their mesenchymal character, however, cells grown in 2% FBS became a considerably more heterogenous population regarding cell size and granularity, perhaps because of senescence. Additionally, these cells somewhat decreased in proliferation and resulted in 1 x 106 cells after 21 days, however, this was not a significant decrease when compared to the 10% FBS culture which had 2.16 x 106 cells after 21 days (p=0.061). On the contrary, the culture supplemented with T24 conditioned media resulted in a significantly higher cell count with 4.75 x 106 cells (p=0.008). Further studies could investigate which components in the conditioned media contributed to the proliferation. Moreover, the cell population in this study could not be characterized as MSC with certainty as additional cell surface markers should be tested.

Immunocytokemi på utstryk är en metod för sortering av celltyper och identifiering av maligna celler. Metoden anses som ett aktuellt alternativ till den redan etablerade immunocytokemi på cellblock-metoden vid diagnostik av maligniteter. Syftet med examensarbetet är metodutvärdering och etablering av immunocytokemi på utstryk för sortering och identifiering av olika celltyper och maligna celler, samt metodjämförelse mellan utstryk och cellblock. Fem prover från pleuravätska framställdes till utstryk och cellblock. Immunocytokemi med antikropparna anti-Epithelial Specific Antigen, anti-Cytokeratin 7 och anti-Carcinoembryonic Antigen tillämpades på dessa. Resultatbedömning omfattade bevarandet av morfologin, färgupptag och ospecifik färgning hos celler och bakgrund, samt möjligheten att ställa säker diagnos för utstryk. Resultat visar färgupptag och viss bevarad morfologi hos utstryk. Samtliga utstryk har ospecifik färgning och kontamination som medför svårighet att urskilja celltyper och identifiera maligna celler. Möjlighet att ställa diagnos med utstryk varierar med preparaten. Färgningen hos samtliga cellblock är robust och mer specifik för celler och bakgrund. Examensarbetet når slutsatsen att etablering av immunocytokemi- metoden på utstryk är möjligt och bör tillämpas i vissa situationer. Etablering som rutinmetod är olämpligt. Framställning av preparat med hög kvalitet och specifika markörer är utvecklingsområden för laboratoriet och vidare forskning. / The method of immunocytochemistry on smears is utilized for categorizing cell types and identifying malignant cells. Immunocytochemistry on smears is a prospective alternative to the already implemented cell block method when diagnosing malignancies. The thesis aims to evaluate and implement immunocytochemistry on smears for categorizing and identifying cell types and malignant cells, along with comparing smears and cell blocks. Five pleural samples were processed into smears and cell blocks. Staining of these occurred with antibodies anti- Epithelial Specific Antigen, anti-Cytokeratin 7 and anti-Carcinoembryonic Antigen. Evaluated factors included preservation of morphology, stain uptake and non-specific staining for cells and background and finally the ability to generate a definitive diagnosis for smears. Results demonstrate stain uptake and preservation of morphology in some smears. All smears demonstrate non-specific staining and contamination, causing difficult categorization and identification. Generating a definitive diagnosis varies among samples. The thesis concludes that immunocytochemistry is a suitable platform for smears and a prospective implementation at the laboratory in certain situations. Immunocytochemistry on smears is not eligible as a routine method. Preparation of high-quality smears and specific antibody markers are deemed points of interest for the laboratory and future studies.

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Background: Vancomycin-resistant enterococci (VRE) is a common nosocomial infection. It classifies as Enterococcus faecalis or faecium carrying vanA or vanB gene that alters the bacterial cell wall hence lowering affinity for Vancomycin. Screening for VRE in Swedish hospitals are performed with stool sample pre-grown in selective broth followed by PCR and culture on selective media. Viasures Vancomycin resistance, Real Time PCR Detection Kit indicates that pre-growth in broth is not needed for the analyze. Aim: Comparison between the PCR kit and the subsequent culture on chromogenic agar with or without pre-growth in selective broth. Method: E. faecium with vanA gen (CCUG 36804) and E. faecalis with vanB gen (ATCC 51299) were suspended in different concentrations and added to ESwab transport medium. Thereafter small samples from the ESwab tube were enriched in selective broth. Samples from both selective broth and ESwab medium were analyzed with Viasures PCR kit on BD-MAX system and cultivated on chromogenic agar. Results: With pre-growth in selective broth the genes were found in every sample regardless of pre-concentration in the ESwab medium. Without enrichment the PCR kit always amplified the genes when the concentration was 40 000 cfu/ml for E. faecium (vanA) and ≥ 10 000 cfu/ml for E. faecalis (vanB). Colonies grew on chromogenic agar in every concentration from both ESwab and selective broth. Conclusion: Culture on chromogenic agar is comparable with or without pre-growth in selective broth but Viasure’s PCR kit is not equal for both methods in lower concentrations of the bacteria.

Practicing Motor Imagery (MI) with a Brain-Computer Interface (BCI) has shown promise in promoting motor recovery in stroke patients. A BCI records a person’s brain activity and provides feedback to the person in real time, which allows the person to practice his or her brain activity. By imagining a movement (performing MI) such as gripping with their hand, cortical areas in the brain are activated that largely overlaps with those activated during the actual hand movement. A BCI can provide positive feedback when the hand-related cortical areas are activated during MI, which helps a person to learn how to perform MI. Despite evidence that stroke patients may recover some motor function from practicing MI with BCI feedback thanks to the feedback provided from a BCI, the effectiveness and reliability of BCI-based rehabilitation are still poor.  A BCI can detect MI by analyzing patterns of features from the brain activity. The most common features are extracted from the oscillatory activity in the brain.  In BCI research, MI is often treated as a static pattern of features, which is detected by using machine learning algorithms to assign activity into a binary state. However, this model of MI may be inaccurate. Analyzing brain activity as dynamically varying over time and with a continuous measure of strength could better represent the cortical activity related to MI.  In this Licentiate thesis, I explore a method for analyzing the temporal dynamic of MI-activity with a continuous measure of strength. Brain activity was recorded with electroencephalography (EEG) and subject-specific feature patterns were extracted from a group of healthy subjects while they performed MI of two opposing hand movements: opening and closing the hand. Although MI of the two same-hand movements could not be discriminated, the continuous output from a machine learning algorithm was shown to correlate well with MI-related feature patterns. The temporal analysis also revealed that MI is dynamically encoded early, but later stabilizes into a more static pattern of brain activity. Last, to accommodate for higher temporal resolution of MI, I designed and evaluated a BCI framework by its feedback delay and uncertainty as a function of the stress on the system and found a non-linear correlation. These results could be essential for developing a BCI with time-critical feedback. To summarize, in this Licentiate thesis I propose a promising method for analyzing and extracting a temporal representation of MI, enabling relevant and continuous neurofeedback which may contribute to clinical advances in BCI-based stroke rehabilitation.

Methicillin-resistant Staphylococcus aureus (MRSA) is a variant of the more common Methicillin-Susceptible Staphylococcus aureus (MSSA), an opportunistic pathogen a portion of the human population carries as normal bacterial flora. When an outbreak of MRSA occurs, it is often important to determine if and how these strains are related to each other. In this report two different types of epidemiological methods were combined (namely Multi-Locus Sequence Typing and staphylococcal protein A-typing), in order to reduce workload, costs and time. A panel of resistance and virulence markers was also added to gather as much information about the culture as possible in a single analysis. To test the viability of the method extracted DNA and heat-treated bacterial cultures of both MRSA and MSSA were amplified with a curated panel of primers. These products were later sequenced with Nanopore’s MinION using the Flongle flow-cell. The method showed promise and worked as intended regarding the staphylococcal protein A-typing and the panel of resistance and virulence markers. However, the Multi-Locus Sequence Typing did still require optimization in order to be used clinically. In summary the project can be viewed as a success since it succeeded in being more time, cost and work efficient than many of its predecessors, when the problems with the Multi-Locus Sequence Typing are solved.

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