Background: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit... Show moreBackground: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit components, buffers and even plastics has resulted in suboptimal testing procedures worldwide. Alternative workflows have been implemented to overcome these difficulties. Recently a liquid based sample prep has been launched as solution to overcome limitations in relation to nucleic acid extraction.Objective: Multicenter evaluation of the QlAprep& Viral RNA UM kit (QIA P&A) for rapid sample preparation and real-time PCR detection of SARS-CoV-2 in comparison to standardized laboratory testing methods.Study design: Selected samples of the routine diagnostic workflow at Clinical Microbiology Laboratories of four Dutch hospitals have been subjected to the rapid QIA P&A protocol and the results have been compared to routine diagnostic data.Results: Combining results of manual and automated procedures, a total of 377 clinical samples of which 202 had been tested positive with a wide range of C-T values, showed almost complete concordance in the QIA P&A assay for samples up to C-T values of 33 with one exception of C-T 31. Prospectively 60 samples were tested and also showed 100 % concordance with 5 positives. The method has been automated by two centres.Conclusions: Despite an input of only 8 mu L of clinical sample, the QIA P&A kit showed good performance for sample preparation and amplification of SARS-CoV-2 and can contribute as a rapid molecular testing strategy in managing the CoV-2 pandemic. Show less
To gain more insight into the evolutionary development of orchid flowers and fruits, the orchid species Erycina pusilla was studied. The evolutionary origin of the median petaloid sepal, the callus... Show moreTo gain more insight into the evolutionary development of orchid flowers and fruits, the orchid species Erycina pusilla was studied. The evolutionary origin of the median petaloid sepal, the callus on the labellum, and the stelidia was studied. These organs were found to be derived from a sepal, a stamen that gained petal identity, and stamens that became staminodes, respectively. The “Oncidiinae” model was proposed, explaining the duplications, diversifying selection and changes in spatial expression of different MADS-box genes that shaped the perianth, enabling the rewardless flowers of E. pusilla to mimic an unrelated rewarding flower for pollinator attraction.After pollination the inferior orchid ovary develops into a fruit. This process is described for E. pusilla up to fruit dehiscence. The fruit associated MADS-box genes and proteins together with other dehiscence-related genes were analyzed in order to propose a first “orchid fruit developmental protein and gene network” model. Fruit development was further studied by transcriptome analyses presenting data obtained from different developmental phases.By analyzing the anatomy of ripe fruits of different orchid species, possible correlations were found between fruit valve lignification patterns, life form, growth strategy, ecology, fruit orientation, dehiscence type, number of valves and slits, and phylogenetic relationships. Show less
Parasitic helminths are important organisms to study because their infections have both adverse and beneficial effects on the human host. Helminth infections are considered a burden, as these... Show moreParasitic helminths are important organisms to study because their infections have both adverse and beneficial effects on the human host. Helminth infections are considered a burden, as these infections cause significant morbidity in a large proportion worldwide. However, helminth infections, by means of their ability to modify host immune responses can also provide protection against inflammatory diseases (inflammatory bowel disease, diabetes, and asthma). It is important to better understand the underlying mechanisms of these Yin (positive) and Yang (negative) consequences of helminth infections. The general objective of this thesis is to track helminths at different levels. On the one hand to improve the detection of helminth infections, essential for the studying helminths and the interaction with their human host. Moreover, a more sensitive diagnostics is instrumental for monitoring the distribution of helminth infections and to evaluate the helminth infections elimination program. On the other hand, to understand the mechanistic insights of the interplay between helminths and the host immune system results in priming of Th2 and regulatory T cell responses. This could contribute to the identification of targeted pathways to manipulate immune responses, as part of developing therapeutics to treat inflammatory disorders characterized by deregulated Th2 and/or Treg responses. Show less
The detection of intestinal parasitic infections for routine diagnosis and for epidemiological research still depends mainly on microscopical examination of stool samples for the identification of... Show moreThe detection of intestinal parasitic infections for routine diagnosis and for epidemiological research still depends mainly on microscopical examination of stool samples for the identification of helminth eggs and protozoan trophozoites and cysts. Because microscopy has several limitations, additional diagnostic methods (e.g. culture, antigen detection) have been introduced to improve the detection of intestinal parasites. Although such additional methods increase sensitivity, the amount of hands-on time accumulates substantially. In chapter 1 various diagnostic methods are summarized and the diagnostic challenges are described for the most important intestinal parasitic infections. During the last years remarkable progress has been made in the development of molecular diagnostic methods based on the Polymerase Chain Reaction (PCR) technique. For the detection of intestinal parasites, DNA isolation from stool can be processed in a semi- or fully-automated system where after specific DNA of multiple targets can be amplified simultaneously, visualized on screen and semi-quantified in a closed tube system with a multiplex real-time PCR. In this thesis, a diagnostic approach using multiplex real-time PCR is assessed for the routine clinical diagnosis and epidemiology of intestinal parasites. In chapter 2 the diagnostic results that were obtained with multiplex-real-time PCR for the detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum / C._hominis (HGC PCR) were compared with the results obtained by routine microscopy on faecal samples from patients visiting their general practitioner because of gastrointestinal symptoms. The study revealed that significant numbers of G._lamblia and Cryptosporidium infections remained undetected with microscopy, even with the use of a multiple sampling procedure of faecal specimens in combination with fixatives (also referred to as the triple faeces test (TFT) procedure). Parasites that had been detected with microscopy but not with real-time PCR consisted mainly of non-pathogenic parasites and Dientamoeba fragilis, of which its pathogenicity is still disputed upon. Hence, compared to the molecular approach, microscopy provided limited sensitivity in a routine diagnostic setting of general practice patients with gastro-intestinal complaints. For the same patient group as described in chapter 2, the results of Cryptosporidium detection with the HGC-PCR were studied in more detail in chapter 3. Microscopic examination for Cryptosporidium, which requires an additional staining procedure of the faecal smear, was specifically requested by general practitioners twenty-one times over a period of approximately seven months and was found positive in 13 cases. In contrast to the conventional diagnostic approach, with HGC PCR 80 cases were detected. The prevalence of Cryptosporidium in Dutch patients with gastro-intestinal complaints attending their general practitioner has so far exceeded the figures in previous studies. The highest infection rate has been detected among children aged under five years with a peak in the month of September: almost one-third of them had been infected with Cryptosporidium, mainly with the species Cryptosporidium hominis. The lack of request for an additional diagnostic procedure to detect Cryptosporidium is not the only reason for missing infections. Basic microscopic stool examination was often not requested, leaving a substantial number of gastro-intestinal parasitic infections undiagnosed. Besides the Dutch general practice patients group, the diagnostic approach with real-time PCR was also assessed for the routine diagnosis of intestinal parasites in returning travellers. Chapter 4 describes a prospective study where the results of 2591 microscopic examinations and antigen detections (i.e. care as usual) were compared with the results of those from the HGC-PCR and an additional Strongyloides stercoralis real-time PCR. The detection rates of all four targeted parasite species were increased with real-time PCR whereas the prevalence of ten additional pathogenic parasite species found with microscopy was 0.5% at most. A fully automated DNA isolation process and extension of the molecular diagnostic targets could further increase the detection rates of parasites while having considerable impact on cost-efficiency of the diagnostic procedures in the laboratory. Chapter 5 describes the molecular epidemiology of G. lamblia infections in the group of returned travellers. Symptoms of G._lamblia infections in this group, consisting mainly of adults, were highly variable and ranged from asymptomatic to the presence of severe gastro-intestinal complaints. Data in this study showed that although the G._lamblia Cycle-threshold (Ct)-values, which reflect the intensity of infection, correlated with the number of specified gastro-intestinal complaints, the parasite was also detected in 4.7 % of the travellers who did not have any intestinal symptoms. The reasons for the clinical heterogeneity of G._lamblia infections are still not fully understood. In previous studies the epidemiological role of G._lamblia assemblages (i.e. group of genotypes) have been suggested as an important factor associated with gastro-intestinal complaints, however, this could not be proven in this group of travellers. In chapter 6 a real-time PCR for the detection of Isospora belli was evaluated and in chapter 7 a multiplex real-time PCR was evaluated for the detection of Encephalitozoon intestinalis and Enterocytozoon bieneusi. These real-time PCRs have been developed in addition to the panel of molecular diagnostic assays to be used in a clinical setting and in epidemiological studies. As such, a phylogenetic study of E._bieneusi infections in persons with different clinical backgrounds is described in chapter 8. The results indicate a dynamic evolutionary process between genotypes of E._bieneusi. One specific genotype was restricted to transplantation patients receiving immuno-supressives and another genotype showed its preferential habitat in patients living with HIV/AIDS, which further emphasizes the predisposition for specific hosts by different E._bieneusi genotypes. Although Schistosoma mansoni and Schistosoma haematobium are living in the blood vessels and not in the intestinal lumen, this thesis also elaborates on real-time PCR for the detection of Schistosoma in humans. In chapter 9 a multiplex real-time PCR was evaluated for the detection of both S. mansoni and S._haematobium in stool samples collected in an area endemic for both Schistosoma species. The detected Ct-values correlated with the results from quantitative microscopy on stool and on urine samples. Furthermore, the use of ethanol-suspended stool samples for storage and transport at ambient temperatures, as was done in this study, makes collection of samples at remote areas more attractive. In chapter 10, the performance of the Schistosoma real-time PCR was evaluated for the diagnosis of female genital schistosomiasis on DNA isolated from vaginal lavages. The preliminary results in this study showed that the semi-quantitative outcome of the PCR can be used in the differential diagnosis of vaginal lesions and as a predictor of disease pathology. In conclusion, the comparative studies in this thesis revealed that the introduction of real-time PCR for routine detection of diarrhoea causing protozoa and helminths will improve the diagnostic efficiency of laboratories dealing with faecal samples. Standard diagnostic procedures can further be improved by the design of real-time PCR panels for specific patient groups containing parasitic, bacterial, fungal and / or viral targets. Furthermore, with the simple sample collection procedure and the high throughput potential, the multiplex real-time PCR showed to be a powerful tool for epidemiological studies, even in remote areas. The real-time PCR method has little value for clinical diagnostics in low-income countries but can certainly play a prominent role as a gold standard in quality control for the evaluation of new, inexpensive rapid tests for field diagnostics. Show less
To date, 128 mycobacterial species have been characterised, ranging from non-pathogenic to pathogenic for humans. Molecular methods contributed significantly to the identification of the species,... Show moreTo date, 128 mycobacterial species have been characterised, ranging from non-pathogenic to pathogenic for humans. Molecular methods contributed significantly to the identification of the species, replacing conventional laborious methods. In this thesis, the design and application of a genus-specific real-time PCR, for the rapid detection of non-tuberculous mycobacteria in clinical materials, was described. The technique was extremely useful for the rapid detection of the slowgrowing Mycobacterium species. Addition of species-specific probes to the ITS assay, identified M. haemophilum to be present in previously undiagnosed skin inflammation and resulted in the recognition of M. haemophilum as the second most common mycobacterial species causing lymphadenitis. Subsequent Amplified Fragment Length Polymorphism analysis of M. haemophilum isolates showed this species to posses an extremely low mutation rate. Also, M. haemophilum lymphadenitis cases are suspected to have a common source, most likely piped water, in contrast to M. avium infections, which appear to originate from variable environmental sources as was underscribed by Restriction Fragment Length Polymorphism analysis. Contamination with saprophytic mycobacterial DNA is problematic for the current NTM detection in clinical materials. This and other bottlenecks in the molecular diagnostics of NTM were addressed in this thesis as well. Show less
Clostridium difficile was first discovered in 1935, but it was not until 1977 that this bacterium was found to be associated with pseudomembranous colitis. The disease was considered to be caused... Show moreClostridium difficile was first discovered in 1935, but it was not until 1977 that this bacterium was found to be associated with pseudomembranous colitis. The disease was considered to be caused by the production of two C. difficile toxins, toxins A and B (TcdA and TcdB). TcdA was shown to exhibit enterotoxic effects on the intestine, whereas TcdB was shown to be more cytotoxic. Strains of C. difficile that do not produce these toxins are known to be non-toxinogenic and therefore non-pathogenic. However, outbreaks and more severe infections due to strains producing only TcdB have been described. To diagnose C. difficile -associated disease, a method with a high sensitivity and specificity is necessary. This thesis describes the application of methods based on the molecular detection of the pathogen in comparison with conventional microbiological methods. After isolation of the pathogen, the epidemiology of C. difficile -associated disease can be studied using molecular typing methods on the isolates. These typing methods need to be highly discriminatory, depending on the epidemiological data to be studied, and methods need to be stable en reproducible, with the possibility to exchange data between different laboratories all over the world. Show less
Real-time monitoring of PCR has strongly supported the increased diagnostic use of nucleic acid detection assays in clinical virology. Particularly the improvements in the ability to quantify... Show moreReal-time monitoring of PCR has strongly supported the increased diagnostic use of nucleic acid detection assays in clinical virology. Particularly the improvements in the ability to quantify target nucleic acid sequences offer new opportunities in the management of viral infections. Real-time PCR is rapidly replacing traditional PCR, and new diagnostic uses will likely emerge. This thesis explores the wide range of potential applications of real-time quantitative PCR technology in clinical virology. This exploration is directed to the design of methods, the application to relevant patient categories, the comparison with established methods where available, and the definition of the clinical relevance of the approach. The focus comprises viral targets where an elaborate balance between viral replication and the host immune system has been established, which brings about viral maintenance without affecting the host, until this balance is disturbed. Show less
