BLT Stages

Helicobacter pylori (H. pylori) is a spiral-shaped bacterium. It colonizes the stomach and may be present in more than half of the world’s population. The clinical features of H. pylori range from asymptomatic gastritis to gastrointestinal malignancy. When the infection is left untreated, the patient can develop stomach cancer. Resistance to certain antibiotics has increased over the years and that is why there are various treatment options.

H. pylori can be detected using invasive and non-invasive methods of sampling. Invasive methods require a gastroscopy. Sometimes gastroscopy requires a procedure called an endoscopy. The gastroscopy is used to look inside the oesophagus (gullet), the stomach and the first part of the small intestine. During the endoscopy the doctor collects a sample of tissue from the lining of the stomach or upper intestine. These biopsies are used for the detection of the bacteria. Due to the fact that the doctor inserts a flexible tube into the stomach during the surgery this procedure is invasive and very unpleasant for the patient.

Since this method is very invasive for the patient and not without any risk, research is being conducted into non-invasive methods for obtaining representative samples. The aim of this study is therefore to evaluate if faeces can be used to detect H. pylori using real-time PCR. Faeces is a non-invasive matrix. PCR is a DNA-based method which requires a DNA-extraction. Due to the COVID19 virus only a part of the study could be conducted. Therefore this work will focus mainly on the optimization and comparison of DNA-extractions of faeces. These extractions can be used when the study will be continued. To obtain the DNA-extractions two different kits were used, namely the Qiaamp fast DNA stool mini kit and the Dneasy mericon food kit. During optimization different conditions were modified (such as more sample, longer incubation time, etc) to achieve higher DNA-concentrations. These conditions as well as the different kits will be compared with each other to conclude which condition and kit has the highest DNA-concentration.

Based on the yield of DNA, different conditions and kits were evaluated. After comparison of the two kits, it was seen that the yield of DNA was highter using the Qiaamp kit compared to the food kit (p-value 0.0034). After comparison of the different conditions, it can be concluded that when more sample (p-value 0.0002 for the qiaamp kit and 0.031927 for the food kit), a longer incubation time (p-value 0.0102 for the qiaamp kit and 0.044419 for the food kit) and a higher incubation temperature (p-value 0.00103 for the food kit)  are used a significant difference can be seen. Only when the elution step was executed twice there was no significant difference to be seen.

It can be concluded from these results that the highest DNA-concentrations are obtained when the Qiaamp kit with longer incubation times is used.

Helicobacter pylori is a pathogenic bacterium that colonizes the stomach of more than 40% of the world’s population. Colonization with H. pylori can lead to infection and causes diseases like gastritis and peptic ulcers. When left untreated, it is a high-risk factor for gastric cancer development. Due to increasing resistance rates of the Helicobacter pylori strains to antibiotics, therapy failure is an rising problem.

Histology is used for routine detection of H. pylori in combination with urea breath testing and culture if therapy failed. Histology is faster and detects H. pylori along with the inflammatory reaction in the tissue, however, this method cannot be applied for antibiotic susceptibility testing. Culture allows testing to antibiotic sensitivity, unfortunately H. pylori is a highly fastidious organism, requiring stringent culture conditions for at least three days. The aim of this study is to evaluate the use of PCR for the detection of both H. pylori and antibiotic resistance to clarithromycin.

In total, 143 gastric biopsies from patients with clinical suspicion of H. pylori infection and indication for gastrointestinal endoscopy were included and were tested for histology, culture and PCR. Phenotypic susceptibility to clarithromycin was evaluated using gradient strip (E-test, bioMérieux). For molecular detection the commercially available Allplex H. pylori & ClariR real-time PCR assay (Seegene) is used.

The prevalence of H. pylori in this study is 27% for culture, 28% for histological methods and 40% for molecular detection. H. pylori (n=31) showed a high rate of clarithromycin resistance (25.8%) of which three phenotypic resistance results could not be confirmed by PCR. The sensitivity and specificity of the molecular assay are 95% and 83% respectively according to the golden standard (histology). Relative to culture, the sensitivity and specificity are respectively 100% and 83%. By implementing a cutoff threshold, i.e. all PCR results with Ct values higher than 36.0 are considered negative, the specificity and positive predictive values for PCR increased strikingly.

Further investigation of the weak positive results is necessary before implementing this molecular assay for routine diagnosis. The results of this study suggest that a clinical threshold is necessary. The study proved that molecular methods are suitable for a more rapid determination of both H. pylori and clarithromycin resistance, in comparison to histological staining and microbiological culture. Since molecular methods are sensitive, it should be examined if non-invasive faecal samples are also useful for the accurate detection of H. pylori.

The aim of this research is to evaluate the performance of the new body fluid module on the Sysmex-XN hematology analyzer (XN-BF) for blood cell count and differential in body fluids in the laboratory of AZ Sint-Lucas Ghent. Along with other validation parameters, a method comparison with manual microscopy was performed to evaluate the accuracy of the body fluid module.

Automated blood cell count and differential in body fluids add a great value to medical laboratories in comparison to the labor-intensive and time-consuming manual microscope method. The hematology analyzer needs to be validated according to the laboratories standards; therefore the XN-BF was evaluated according to these standards.

During a period of nine weeks, 110 samples (42 pleural fluids, 17 synovial fluids, 22 ascites, 2 continuous ambulatory peritoneal dialysis (CAPD) and 27 cerebrospinal fluids (CSF)) were used for method comparison between the XN-BF and manual microscopy for blood cell counting (Fuchs-Rosenthal counting chamber) and differential (cytospins). Further evaluation included inter-observer variation (manual method), precision, carry-over, linearity and Lower Limit of Quantitation (LLoQ).

Inter-observer variation for the manual counting method showed a coefficient of variation of 8,17% for total nucleated cell count (TC) and 4,66% for red blood cell count (RBC). For the method comparison in pleural fluids, good correlations (R²=0,972; R²=0,994) were found for TC and RBC >1000/µl counts, despite the bias found with the Passing and Bablok regression analysis in TC (y=80,7980+1,1919x). Excellent correlation for TC and RBC (R²>0,980) was found in synovial fluids, yet a constant bias was found again for TC (y=154,4702+0,9890x). In ascites and CAPD, good correlations are found as well (TC: R²=0,964; RBC: R²=0,989). However, the XN-BF systematically counted more TC compared to the manual microscopy (y=-0,03084+1,2419x). In CSF <10 TC/µl (n=21) no significant or constant bias was found, but correlations were worse compared to the other fluids (R²=0,817). Regardless, good correlations were found for RBC (n=25) and TC >5/µl (n=7). For the differential count, the XN-BF systematically counted less monocytes compared to the manual method in all fluids. Results of comparing the manual reporting for malignant cells with the High-Fluorescent fraction on the XN-BF showed that these cells are located in this region, but that the XN-BF is not specific enough for quantitation of these cells. Furthermore carry-over was negligible, precision was excellent (CV%: TC=2,3%; RBC=0,0%; neutrophils=6,4%; lymphocytes=4,2%; monocytes=5,1%), linearity was good for both RBC and TC (R²=0,99) and the LLoQ for TC was defined at 4/µl.

The XN-BF can be used in the laboratory for fast counting of TC and RBC, if nucleated cell counts <10/µl are counted again manually (CSF). Differentiation through microscopy is still needed due to the low correlations in monocyte differentiation and the lack of precision for reporting of malignant cells.