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Medisch Labo Bruyland

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Abstract Bachelor Project MLT 2019-2020Diagnosis of a bacterial gastroenteritis in an outpatient setting: Molecular assay versus culture

Gastroenteritis is a frequently diagnosed infectious disease in both the developing and developed countries, and often results in some form of diarrhea, along with other symptoms. The different causes of an infectious gastroenteritis are: bacteria, parasites, viruses, or fungi. This study will only focus on the bacterial cause. Most of the time bacterial gastroenteritis is self-limiting, however, identification of a pathogenic agent by bacterial stool culture is required for the management of patients with severe or prolonged diarrhea, symptoms consistent with invasive disease, or a patient history predicting a complicated course of disease. These days, determination of the cause of infection is prone to delay partly due to the long turn-around-time (TAT) of conventional diagnostic tests (e.g. bacterial culture). Apart from diarrhea related to infection, symptoms could also result (especially in developed countries) from an underlying disease or the use of medication. It is therefore important that diagnostic tests could be performed in order to either exclude or confirm a possible bacterial causative agent.

The aim of this study is to examine fecal samples from 100 patients for the presence of bacterial pathogens. Therefore, both bacterial stool culture and multiplex PCR are performed on all fecal samples. Both methods will be compared and discussed.

100 fecal samples were collected (both from human and veterinary origin) and inoculated on different culture media. These culture media were used for the detection of Campylobacter sp., Yersinia sp., Salmonella sp., Aeromonas sp. and Shigella sp.. Colonies that presumably indicated pathogenic species, were identified using MALDI-TOF MS. In addition, all 100 fecal samples were investigated with multiplex real-time PCR. Both the FLOW Solution and MagNA Pure LC 2.0 instruments were used for the processing and purification of the samples.

A total of seven cases out of 100 were reported positive when investigated by bacterial culture, while multiplex PCR reported a total of 30 positive cases. Within the group of samples positive by culture, three were positive for Aeromonas sp. and four were positive for Campylobacter sp.. Discordant results were shown in three cases where culture revealed growth of Aeromonas sp. while negative results were obtained when tested by PCR. Multiplex PCR showed superior sensitivity for detection of gastro-intestinal bacterial pathogens compared to culture (1 positive case for Shigella sp., five for Aeromonas sp., six for Yersinia sp. and up to eighteen for Campylobacter sp.). As for Salmonella sp., not a single case has been reported by both methods. Finally, Cohen’s kappa statistic was used to measure the interrater reliability between both methods which showed a moderate agreement.

Although it is clear that multiplex PCR exhibits a higher sensitivity than conventional stool culture, a significant number of these pathogens may not be clinically relevant. For example, there is the possibility that a pathogen is present in the intestinal flora to a small extent, but is not responsible for the occurrence of the symptoms. This could lead to unnecessary antibiotic prescriptions. Culture media, on the other hand, are designed to be more specific for the identification of clinically relevant pathogen levels because of the better adapted cut-off values for detection. Culture and multiplex PCR both have their advantages and disadvantages. Because of its higher sensitivity and short TAT, multiplex PCR is often preferred. However, a clinical interpretation of the data obtained by PCR is essential.

Abstract bachelorproef 2016-2017The detection of Extractable Nuclear Antibodies with two methods 

The aim of this study is to compare two devices (EuroBlotMaster and Phadia 250) for analysing antinuclear antibodies.

Antinuclear antibodies are autoantibodies that are used in the diagnosis of systemic autoimmune diseases such as Lupus Erythematosus, Sjögren’s syndrome, Systemic sclerosis, Mixed connective tissue disease, Polymyositis and Dermatomyositis. Examples of autoantibodies are anti-dsDNA, ant-Ro, anti-La, anti-Sm, anti-RNP, anti-Scl etc. This autoantibodies  are also called antinuclear antibodies.

The first step is a screening with Hep-2000® cells. The result is a fluorescent pattern and a titer. If the titer is at least 1/80, additional tests are done. This can be done on the EuroBlotMaster or/an Phadia 250.

The Phadia 250 uses the Fluorescence Enzyme immunoassay (FEIA) technique. First there is a screening with the Symphony. If this is positive, all the autoantibodies are separately determined. Autoantibodies that are tested; anti-dsDNA, anti-CENP, anti-Jo1, anti-Scl-70, anti-Sm, anti-RNP, anti-Ro52/60, anti-La and anti-RNP-70.

The EuroBlotMaster with the immunoblot technique, uses strips which are stained by indirect immunostaining. From this, a band pattern is observed by using the EuroLineScan. This scan converts an intensity to a qualitative observation. This strip can detect sixteen autoantibodies.

There is a good agreement between the two methods for most of the tested antibodies. Another advantage of the EuroBlotMaster is the detection of six other antibodies, that can help in the further diagnostic of auto-immune diseases. The workflow for ENA detection will be changed. After a screening with Symphony on Phadia 250, positive results are further analysed with the immunoblot technique with the EuroBlotMaster.

Generally it can be decided that the results on the EuroBlotMaster and the results on the Phadia 250 are comparable. 

Samenvatting eindwerk 2014-2015: Verification (implementation validation) of a HPLC analyser for the measurement of HbA1c
Haemoglobin is a protein in the erythrocytes that caries oxygen to the tissues. When glucose binds to haemoglobin, haemoglobin A1c is formed.
The detection of HbA1c is useful in the diagnosis and evaluation of treatment in diabetics.
In this study the Menarini HA 8180V HPLC analyser for HbA1c is validated and compared with Tosoh G8. The principle of both analysers is reversed phase cation-exchange chromatography.
The validation/verification protocol includes verification of within-run, between-run, correlation and trueness. 1000 patient samples were tested and samples from extern quality controle 2014 were requested in the WIV (institute of health).
Within-run variation for low (42 mmol/mol), middle (61,1 mmol/mol) and high (88,7 mmol/mol) concentrations, are respectively 0%, 0,36% and 0,54%.
Between-run variation for level 3 (25,0 mmol/mol), 4 (33,0mmol/mol) and 5 (73,6 mmol/mol) concentrations, are respectively 0%, 0% an 1%.
Correlation between Tosoh G8 and Menarini HA 8180V is good, the correlation coefficient is 0,996.
The results of trueness, both analysers had for low IFCC levels and middle IFCC levels an aberration of 1, for high IFCC levels the Tosoh G8 had an aberration of 1 and the Menarini HA 8180V had an aberration of 0. The results for trueness are excellent according to the criteria of the WIV for both analysers.


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