PathoFinder BV

Maastricht, Netherlands

PathoFinder BV

Maastricht, Netherlands
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Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: NMP.2013.1.2-2 | Award Amount: 7.53M | Year: 2014

The main objective of PneumoNP is the development of a theragnostic system for the treatment of lung Gram-negative bacterial infections. As a proof of concept PneumoNP will focus the attention on Klebsiella pneumoniae caused infections. A diagnostic kit will be developed to enable a rapid and precise identification of the bacteria strain causing the infection and avoid the use of wide spectrum antibiotics. For the treatment a nanotherapeutic based inhalable antibiotic will be developed. The therapeutic nanosystem will be based on a nanocarrier combined with an antimicrobial peptide. 3 different types of NCs will be tested with 2 AMPs to be able to obtain a novel effective inhalable antimicrobial NS. Nanotherapeutics offers many advantages in pulmonary drug-delivery, due to the huge surface area available in the lungs and their potential to achieve uniform distribution of drug dose among the alveoli. To improve this delivery to the lungs an aerosol system will also be developed. Due to the characteristics related to pulmonary delivery of NCs, topical and systemic bioavailabilities are envisaged. A diagnostic kit will be generated to monitor the efficacy and efficiency of the therapy. Once this treatment is proved to be effective, it could then be applied to any Gram-negative lung bacterial infection. The number of antibiotic resistant bacteria strains is increasing rapidly, new types of therapy are urgently required to avoid the use of standard antibiotics. Gram-negative bacteria that cause pneumonia are one of the main sources of nosocomial infections, mainly in people with a weakened immune system. Apart from pneumonia they can cause bacteremia and other infections. Early detection of the infection source combined with the development of appropriate and effective NSs to treat multi-drug resistant (MDR) bacteria caused infections will definitely radically improve the healing process of patients and avoid complications for people in hospital.

Theelen W.,Maastricht University | Theelen W.,RWTH Aachen | Litjens R.J.N.T.M.,Maastricht University | Vinokurova S.,University of Heidelberg | And 9 more authors.
Human Pathology | Year: 2013

We evaluated the reliability of a novel multiplex ligation-dependent probe amplification (MLPA) assay in detecting integration of human papillomavirus (HPV) based on the viral E2/E6 copy number ratio in formalin-fixed and paraffin-embedded cervical lesions. The MLPA results were compared with those of amplification of papillomavirus oncogene transcripts for RNA, detection of integrated papillomavirus sequences for DNA, and HPV fluorescence in situ hybridization (FISH). DNA was isolated from 41 formalin-fixed and paraffin-embedded HPV-positive cervical lesions (cervical intraepithelial neoplasia grade 3 lesions, squamous cell carcinomas, and adenocarcinomas) for MLPA analysis. From 13 matching frozen samples, DNA and RNA were isolated for the detection of integrated papillomavirus sequences and/or the amplification of papillomavirus oncogene transcripts, respectively. Integrated HPV16, HPV18, or both were identified. The MLPA assay detected viral integration in 12 of these 13 cases, and episomal copies also were detected in 7 cases. In 20 of the 24 cases with exclusive viral integration or episomal viral copies as detected by FISH, MLPA confirmed the physical status of the virus. In the cases classified as mixed by FISH, the presence of excess episomal copies complicated the recognition of viral integration by MLPA. Furthermore, the feasibility of detecting gain of the telomerase genes with the HPV MLPA assay was evaluated. The MLPA confirmed the FISH data in 12 of 13 cases in which the status of copy number gain for telomerase RNA component was known. In conclusion, the HPV MLPA assay can be performed on routinely processed cervical lesions for the detection of viral load and HPV integration. © 2013 Elsevier Inc.

Theelen W.,Maastricht University | Speel E.-J.M.,Maastricht University | Herfs M.,University of Liège | Reijans M.,PathoFinder BV | And 7 more authors.
American Journal of Pathology | Year: 2010

Oncogenic human papillomavirus (HPV) infection is the most important risk factor in cervical carcinogenesis cases; high viral loads, viral integration into the host genome, and gain of the telomerase-related genes, TERT and TERC, are all factors associated with progression to cancer. A recently developed multiparameter HPV 16/18 multiplex ligation-dependent probe amplification (MLPA) assay, which allows the simultaneous assessment of these factors, was applied to a series of 67 normal and (pre)malignant frozen uterine cervical samples, as well as to 91 cytological preparations, to test the ability of the MLPA assay to identify high-risk lesions on the basis of these factors. Validation was performed using quantitative PCR, the PapilloCheck and fluorescence in situ hybridization. Only 5 out of 37 normal tissue samples or low-grade cervical lesions (ie, CIN1 and condyloma) showed either an HPV16 viral load higher than 25 copies per cell, viral integration, and/or gain of one of the telomerase-related genes, whereas for the high-grade cervical lesions, one or more of these risk factors was found in 25 of 30 cases. The HPV MLPA assay showed a sensitivity of 83% and a specificity of 86% in frozen cervical specimens. Furthermore, the feasibility of the MLPA assay was shown for cytological samples, where in 57% of highgrade squamous intraepithelial lesion cases, the highrisk factors were detected using this assay. Copyright © American Society for Investigative Pathology.

Litjens R.J.N.T.M.,Maastricht University | Theelen W.,Maastricht University | van de Pas Y.,Maastricht University | Ossel J.,PathoFinder BV | And 7 more authors.
Journal of Medical Virology | Year: 2013

Current screening methods for uterine cervical cancer such as Papanicolaou smears and/or high risk human Papillomavirus (HR-HPV) detection have a high negative predictive value but a low positive predictive value for the presence of high grade cervical lesions. Therefore, new parameters are needed to reduce the rate of unnecessary referrals for colposcopy. The predictive value of the HPV multiplex ligation-dependent probe amplification (MLPA) assay, which can assess simultaneously HPV16/18 viral load and viral integration, was evaluated. The assay was applied to 170 cervical cytological samples, and the results were correlated with the matching histological follow-up. The GP5+/6+ assay and qPCR were used as a control for HR-HPV typing. The MLPA assay classified a higher percentage of cases as high-risk (high-viral load and/or viral integration) with higher grades of dysplasia. There was a high correlation between the HPV MLPA assay and qPCR for viral load and HPV genotyping, and between the MLPA assay and the GP5+/6+ assay for HPV genotyping. The sensitivity and specificity of the HPV MLPA assay for the detection of high-grade lesions were 44% and 93%, respectively. This study demonstrates that the HPV MLPA assay can reliably detect HPV 16/18, viral load, and viral integration in cytological samples. Also, high-risk classification correlated well with the presence of high-grade dysplasia. However, for the implementation of the MLPA assay into clinical practice, additional HR-HPV types need to be included to increase the sensitivity of the assay, and thereby increase its negative predictive value. © 2013 Wiley Periodicals, Inc.

Theelen W.,Maastricht University | Reijans M.,PathoFinder BV | Simons G.,PathoFinder BV | Ramaekers F.C.S.,Maastricht University | And 2 more authors.
International Journal of Cancer | Year: 2010

Oncogenic human papillomavirus (HPV) is the most important risk factor for cancer of the uterine cervix and a subgroup of head and neck cancers. Viral load has been associated with persistence of infection, whereas integration of HPV into the host cell genome is associated with transition to invasive disease. Viral integration is frequently correlated with loss of viral E2 and gain of the telomerase-related genes TERC and TERT. The objective of this study was to develop a rapid and sensitive multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous analysis of viral load, integration and copy number gain of TERC and TERT in HPV16/18-associated lesions. The performance of the assay was tested for HPV vs. human gene copy number ratios ranging from 0.1 to 100 and for percentages of integration ranging from 0 to 100%. The model systems used include plasmid mixtures and the HPV-positive cell lines SiHa, HeLa and CaSki described to contain a range of 2-600 viral copies per cell. In samples with low-viral load, viral integration can be reliably determined when more than 30% of the virus is integrated. Gain of the telomerase-related genes in the cell lines as determined by our MLPA assay was in accordance with data reported in the literature. Our study demonstrates that within a single MLPA-reaction viral type, load, integration and gain of TERC and TERT can be reliably determined, which will improve risk assessment for patients suspected for HPV infection. © 2009 UICC.

Muvunyi C.M.,Ghent University | Muvunyi C.M.,Center Hospitalier University Butare | Dhont N.,Ghent University | Verhelst R.,Ghent University | And 7 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2011

We evaluated a new multiplex polymerase chain reaction (mPCR), "STDFinder assay", a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing. © 2011 Elsevier Inc.

PubMed | Maastricht University and PathoFinder BV
Type: Evaluation Studies | Journal: Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology | Year: 2014

High-risk (hr) human papillomavirus (HPV) infections play a causal role in the development of cervical cancer. The detection of hrHPV is, therefore, advocated in cervical cancer screening programs.The aim of this study was to determine the performance of a novel HPV typing assay, PapillomaFinder SMART 20. This is a one-tube-per-sample method, to be performed on standard real-time PCR platforms, using melting curve analysis to distinguish targets. The assay detects all 14 hrHPV types, of which 16, 18, 31, 33, 35, 39, 45, 52, 56 and 58 individually. HrHPV types 51, 59, 66 and 68 are detected in an hrHPV pool, and low-risk (lr) HPV types 6, 11, 40, 42, 43 and 44 in an lrHPV pool.The method was tested on HPV plasmid models, WHO and QCMD proficiency panels and a series of clinical cytological samples (n=45), the latter in comparison with a clinically validated real-time quantitative PCR.Type-specificity of the test was 100% using plasmids, the WHO and QCMD panels. Sensitivity for hrHPV in single infections was 100% using the WHO and QCMD panels and cytological samples, with an analytical sensitivity of 10-25 copies per reaction for all HPV types tested. Of the 34 HPV types present in the 8 multiple infections in the WHO panel, 30 were detected. In all cytological samples at least one hrHPV type was found, in concordance with the clinically validated method. Only when the viral load of the dominant HPV types in multiple infections greatly exceeded that of the other types in the infection, those other types were not always detected.PapillomaFinder SMART 20 is a rapid, easy to perform, single tube HPV typing assay. The assay detects the 14 hrHPV types, and the 6 most important lrHPV types with a high sensitivity and type-specificity.

Overmeire Y.,Laboratory of Clinical Microbiology | Vanlaere E.,Laboratory of Clinical Microbiology | Hombrouck A.,Scientific Institute of Public Health | De Beenhouwer H.,Laboratory of Clinical Microbiology | And 5 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2016

The 2014-2015 influenza season in Belgium was dominated by the circulation of 2 influenza A(H3N2) subgroups: 3C.2a and 3C.3b. Analysis of 166 nasopharyngeal aspirates, collected in patients with respiratory illness at the start of the epidemic season, showed a decreased sensitivity for the detection of influenza A(H3N2)/3C.2a using a commercially available multiplex assay. Gene sequencing of the matrix protein showed a point mutation (C163T) leading to a mismatch with the assay probes. © 2016 Elsevier Inc.

Simons G.,PathoFinder BV
Expert Review of Molecular Diagnostics | Year: 2010

The conference Applications of Nucleic Acids Technologies in Molecular Diagnostics (part of TIDES) consisted of four sessions: Regulatory Pathways and Quality Strategies, Manufacturing and Business Considerations for Successful Launch in the Clinical Market, Analytical Methods and Validation, and New Technologies. The conference brought together approximately 100 representatives from academia, clinical laboratories and industry and comprised 26 presentations. This article only focuses on new developments discussed in the session regarding New Technologies, with special emphasis on presentations highlighting real-time amplification and detection. © 2010 Expert Reviews Ltd.

Agency: European Commission | Branch: H2020 | Program: SME-1 | Phase: PHC-12-2014-1 | Award Amount: 71.43K | Year: 2015

Infection with certain Human Papilloma Virus (HPV) types has been demonstrated as the most important risk factor in the development of cervical dysplasia, found to be present in nearly 100% of women with cervical cancer. Currently implemented mass-screening approaches based on imaging, Pap testing and DNA testing display some relevant drawbacks in terms of specificity and sensitivity, and even when one is diagnosed with HPV, there is practically no way to determine whether this infection will regress (90% of the cases), or will turn into cancer, with obvious economic and societal consequences. Through the SixthSense Project, PathoFinder will validate a diagnostic algorithm for HPV screening able to detect the presence of oncogenic/high-risk types HPV DNA, and to measure their viral load and expression of oncogenic viral proteins E6/E7, which will be used as risk indicators for the development of cervical cancer, thus allowing to timely diagnose who will develop cancer, and initiate the treatment accordingly. The objective is to manufacture and worldwide distribute this new assay and the associated analytical device, leveraging the proprietary new generation multiplex and Real Time PCR technology for rapid detection and identification of human pathogens in clinical specimens. This result will allow women to accurately know their risk to develop cancer undergoing only one gynaecological sampling/visit for both first line and second line testing, avoiding unnecessary and excessive follow up procedures. At the same time National Healthcare Systems would reduce the costs of unnecessary follow up visits and of more invasive investigations, while gynaecologist would also be favoured in the management of patients with low grade dysplasia.

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