25 results on '"Molecular Diagnostic Techniques/methods"'
Search Results
2. Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: comparison with automated systems
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Hanne Larsen, Jesper B. Moeller, Henrik Dimke, Sanne Løkkegaard Larsen, Gitte Nyvang Hartmeyer, and Marianne Nielsine Skov
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RNA viruses ,0301 basic medicine ,Viral Diseases ,COVID-19/diagnosis ,Chloroform/chemistry ,Coronaviruses ,Molecular biology ,Computer science ,viruses ,Molecular biology assays and analysis techniques ,Purification techniques ,Medical Conditions ,COVID-19 Testing ,0302 clinical medicine ,RNA analysis ,COVID-19 Testing/methods ,030212 general & internal medicine ,Phenol–chloroform extraction ,skin and connective tissue diseases ,Pathology and laboratory medicine ,Virus Testing ,Multidisciplinary ,Nucleic acid analysis ,Newcastle Disease Virus ,virus diseases ,Medical microbiology ,RNA, Viral/genetics ,RNA isolation ,Infectious Diseases ,Molecular Diagnostic Techniques ,RNA purification ,Viruses ,RNA, Viral ,Medicine ,Chloroform ,RNA extraction ,SARS CoV 2 ,Pathogens ,Research Article ,2019-20 coronavirus outbreak ,SARS coronavirus ,Coronavirus disease 2019 (COVID-19) ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Science ,SARS-CoV-2/chemistry ,Specimen Handling/methods ,RNA/genetics ,Computational biology ,Real-Time Polymerase Chain Reaction/methods ,Biomolecular isolation ,Real-Time Polymerase Chain Reaction ,Diagnostic system ,Microbiology ,Sensitivity and Specificity ,Virus ,Specimen Handling ,03 medical and health sciences ,Extraction techniques ,Diagnostic Medicine ,Humans ,Pandemics ,Medicine and health sciences ,Clinical Laboratory Techniques/methods ,Phenol/chemistry ,Chromatography ,Molecular Diagnostic Techniques/methods ,Biology and life sciences ,Phenol ,Clinical Laboratory Techniques ,SARS-CoV-2 ,fungi ,Organisms ,Viral pathogens ,COVID-19 ,RNA ,Covid 19 ,Microbial pathogens ,Research and analysis methods ,body regions ,Molecular biology techniques ,030104 developmental biology ,Paramyxoviruses - Abstract
The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2. The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2.
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- 2020
- Full Text
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3. Added diagnostic value of 16S rRNA gene pan-mycobacterial PCR for nontuberculous mycobacterial infections: a 10-year retrospective study
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Andenmatten Simon, Laurent P. Nicod, Jesica Mazza-Stalder, Jaton Katia, Opota Onya, and Greub Gilbert
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Microbiology (medical) ,medicine.medical_specialty ,Genes, rRNA ,Humans ,Microscopy/methods ,Molecular Diagnostic Techniques/methods ,Mycobacterium Infections, Nontuberculous/diagnosis ,Nontuberculous Mycobacteria/genetics ,Nontuberculous Mycobacteria/isolation & purification ,Polymerase Chain Reaction/methods ,Predictive Value of Tests ,RNA, Ribosomal, 16S/genetics ,Retrospective Studies ,Sensitivity and Specificity ,Time Factors ,16S rRNA gene ,Acid-fast bacilli ,Auramine staining ,Extra-pulmonary infection ,Infection ,Microscopy ,Molecular diagnostics ,Mycobacteria ,Mycobacterial culture ,Nontuberculous mycobacteria ,Pan-mycobacterial PCR ,Polymerase chain reaction ,Pulmonary infection ,Mycobacterium Infections, Nontuberculous ,Gastroenterology ,Polymerase Chain Reaction ,law.invention ,Medical microbiology ,law ,Positive predicative value ,Internal medicine ,RNA, Ribosomal, 16S ,Medicine ,Gene ,biology ,business.industry ,Retrospective cohort study ,Nontuberculous Mycobacteria ,General Medicine ,16S ribosomal RNA ,biology.organism_classification ,Infectious Diseases ,Molecular Diagnostic Techniques ,Original Article ,business - Abstract
The diagnosis of mycobacterial infections has been dramatically improved by the introduction of molecular methods aimed to reduce the time to diagnosis as compared with culture. The broad range pan-mycobacterial PCR can detect all the mycobacterial species directly from clinical specimens. We aimed to evaluate its usefulness and its clinical added value for the diagnosis of nontuberculous mycobacterial (NTM) infections. We performed a retrospective study (2003–2013) including 952 samples taken from 639 patients with clinical suspicion of NTM infection. The performance of smear microscopy, PCR and culture was established using clinical data to investigate discrepant results. We also compared the time to microbial diagnosis between the direct PCR and culture. The sensitivity, specificity, positive and negative predictive values of the PCR were 61.6% (53.5–69.1), 99.1% (98.2–99.6), 92.8% (85.8–96.5) and 93.4% (91.6–94.9), respectively, when considering all specimens. When considering smear-positive specimens and smear-negative specimens, the sensitivity was 81.6% and 40%, respectively. The sensitivity for pulmonary and extra-pulmonary smear-positive specimens was 85.2% versus 72.7%. The median time to identification at species level was 35 days (SD, 17.67) for culture and 6 days (SD, 2.67) for the PCR (when positive), which represents a 29-day shorter time to results (p
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- 2019
4. Are we approaching the end of pediatric culture-negative osteoarticular infections?
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Christina Steiger, Dimitri Ceroni, and Romain Dayer
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Microbiological Techniques ,Microbiology (medical) ,Arthritis, Infectious ,ddc:618 ,Microbiological Techniques/methods ,Molecular Diagnostic Techniques/methods ,business.industry ,Arthritis ,Pcr assay ,High-Throughput Nucleotide Sequencing ,Osteomyelitis ,Microbiology ,Virology ,DNA sequencing ,Molecular Diagnostic Techniques ,Medicine ,Humans ,Osteomyelitis/diagnosis/microbiology ,Culture negative ,business ,Child ,Nucleic Acid Amplification Techniques ,Infectious/diagnosis/microbiology - Published
- 2019
5. Evaluation of three consecutive versions of a commercial rapid PCR test to screen for methicillin-resistant Staphylococcus aureus
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Laurence Senn, Dominique S. Blanc, E. Bulliard, Gilbert Greub, and Bruno Grandbastien
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Methicillin-Resistant Staphylococcus aureus ,0301 basic medicine ,Microbiology (medical) ,medicine.medical_specialty ,030106 microbiology ,medicine.disease_cause ,Polymerase Chain Reaction ,Sensitivity and Specificity ,03 medical and health sciences ,0302 clinical medicine ,Pcr test ,Internal medicine ,medicine ,Humans ,Mass Screening ,Bacteriological Techniques/methods ,Mass Screening/methods ,Methicillin-Resistant Staphylococcus aureus/genetics ,Methicillin-Resistant Staphylococcus aureus/isolation & purification ,Molecular Diagnostic Techniques/methods ,Polymerase Chain Reaction/methods ,Staphylococcal Infections/diagnosis ,Staphylococcal Infections/microbiology ,Diagnostic test ,Methicillin-resistant Staphylococcus aureus ,Performance ,Rapid PCR test ,Rapid test ,Screening ,030212 general & internal medicine ,Bacteriological Techniques ,business.industry ,General Medicine ,Staphylococcal Infections ,Infectious Diseases ,Molecular Diagnostic Techniques ,Staphylococcus aureus ,business ,Mrsa screening - Abstract
Objectives Screening for methicillin-resistant Staphylococcus aureus (MRSA) is part of many recommendations to control MRSA. Several rapid PCR tests are available commercially and updated versions are constantly released. We aimed to evaluate the performance of three consecutive versions (G3, Gen3 and NxG) of the XpertMRSA test. Methods Routine samples for MRSA screening were simultaneously tested by culture and rapid PCR. The three versions of XpertMRSA were used successively and compared with culture. Results A total of 3512, 2794 and 3288 samples were analysed by culture and by the G3, Gen3 and NxG XpertMRSA versions, respectively. The rates of positive-by-culture in the three groups were 5.0%, 4.7% and 4.3%, respectively. The sensitivity improved over time (71.4, 95% CI 64.0–77.9; 82.3, 95% CI 74.4–88.2; and 84.3%, 95% CI 77.0–89.7, respectively), but not significantly. The specificity (98.4, 95% CI 97.9–98.8; 96.8, 95% CI 96.0–97.4; and 99.1, 95% CI 98.7–99.4, respectively) and the positive likelihood ratios (45.7, 95% CI 34.4–60.8; 25.6, 95% CI 20.5–32.0; and 97.1, 95% CI 66.3–142.4) were significantly lower in the Gen3 version (p Conclusions These significant differences in performance show the importance of evaluating each new version of a commercial test.
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- 2019
6. Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR
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Onya Opota, Burkard Springer, Karoline Wagner, Gilbert Greub, Frank Imkamp, Peter M. Keller, University of Zurich, and Keller, Peter M
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Male ,0301 basic medicine ,Mycoplasma pneumoniae ,Antibiotics ,medicine.disease_cause ,2726 Microbiology (medical) ,Serology ,0302 clinical medicine ,Multiplex ,030212 general & internal medicine ,Respiratory Tract Infections ,biology ,10179 Institute of Medical Microbiology ,2404 Microbiology ,General Medicine ,Chlamydophila pneumoniae ,Streptococcus pneumoniae ,Infectious Diseases ,Molecular Diagnostic Techniques ,Female ,DNA, Bacterial ,Microbiology (medical) ,Fastidious organism ,Adolescent ,Legionella ,medicine.drug_class ,030106 microbiology ,610 Medicine & health ,Sensitivity and Specificity ,Microbiology ,Bacteria/genetics ,Bacteria/isolation & purification ,Bacteria/pathogenicity ,Chlamydophila pneumoniae/genetics ,Chlamydophila pneumoniae/isolation & purification ,Chlamydophila pneumoniae/pathogenicity ,DNA, Bacterial/genetics ,High-Throughput Screening Assays/methods ,Humans ,Legionella/genetics ,Legionella/isolation & purification ,Legionella/pathogenicity ,Molecular Diagnostic Techniques/methods ,Multiplex Polymerase Chain Reaction/economics ,Multiplex Polymerase Chain Reaction/methods ,Mycoplasma pneumoniae/genetics ,Mycoplasma pneumoniae/isolation & purification ,Mycoplasma pneumoniae/pathogenicity ,Pneumonia, Bacterial/diagnosis ,Pneumonia, Bacterial/microbiology ,Pneumonia, Mycoplasma/diagnosis ,Pneumonia, Mycoplasma/microbiology ,Reagent Kits, Diagnostic ,Respiratory Tract Infections/diagnosis ,Respiratory Tract Infections/microbiology ,Retrospective Studies ,Streptococcus pneumoniae/genetics ,Streptococcus pneumoniae/isolation & purification ,Atypical pneumonia ,High throughput screening ,Lightmix(®) kit ,Molecular detection ,Multiplex RT-PCR ,Respiratory pathogens ,03 medical and health sciences ,Pneumonia, Mycoplasma ,Pneumonia, Bacterial ,medicine ,Bacteria ,2725 Infectious Diseases ,biology.organism_classification ,medicine.disease ,High-Throughput Screening Assays ,570 Life sciences ,Multiplex Polymerase Chain Reaction - Abstract
Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.
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- 2018
7. Accuracy of different Xpert MTB/Rif implementation strategies in programmatic settings at the regional referral hospitals in Uganda: Evidence for country wide roll out
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Achilles Katamba, Rogers Sekibira, Willy Ssengooba, Moses Joloba, Winters Muttamba, Bruce Kirenga, University of St Andrews. School of Medicine, and University of St Andrews. Infection and Global Health Division
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Referral and consultation/standards ,Male ,Pathology and Laboratory Medicine ,Fluorescence Microscopy ,0302 clinical medicine ,Immunodeficiency Viruses ,Molecular diagnostic techniques/methods ,lcsh:Science ,Referral and Consultation ,Aged, 80 and over ,education.field_of_study ,Light Microscopy ,QR Microbiology ,Evidence-based practice ,Medical Microbiology ,Viral Pathogens ,Evidence-Based Practice ,Cross-sectional studies ,medicine.medical_specialty ,Tuberculosis ,030106 microbiology ,Microbiology ,Sensitivity and Specificity ,03 medical and health sciences ,SDG 3 - Good Health and Well-being ,Humans ,education ,Microbial Pathogens ,Secondary Care Centers ,Tuberculosis, Pulmonary ,Aged ,lcsh:R ,Uganda/epidemiology ,Organisms ,Sputum ,Disease notification/methods ,Biology and Life Sciences ,Tropical Diseases ,medicine.disease ,bacterial infections and mycoses ,QR ,Mucus ,Cross-Sectional Studies ,Africa ,lcsh:Q ,Government Laboratories ,Reproducibility of results ,RNA viruses ,Bacterial Diseases ,0301 basic medicine ,Research Facilities ,Physiology ,Human immunodeficiency virus (HIV) ,lcsh:Medicine ,medicine.disease_cause ,Geographical Locations ,Limited access ,Secondary care centers/statistics & numerical data ,RA0421 ,RA0421 Public health. Hygiene. Preventive Medicine ,Medicine and Health Sciences ,Uganda ,030212 general & internal medicine ,Middle aged ,Microscopy ,Multidisciplinary ,HIV diagnosis and management ,Middle Aged ,Laboratory results ,Body Fluids ,Sensitivity and specificity ,Infectious Diseases ,Mycobacterium tuberculosis/genetics ,Molecular Diagnostic Techniques ,Viruses ,Tuberculosis Diagnosis and Management ,Female ,Health plan implementation/standards ,Pathogens ,Anatomy ,medicine.symptom ,Research Laboratories ,Research Article ,Adult ,Adolescent ,Referral ,Population ,Reference laboratory ,Research and Analysis Methods ,Young Adult ,Diagnostic Medicine ,Internal medicine ,Retroviruses ,medicine ,Disease Notification ,business.industry ,Lentivirus ,Health Plan Implementation ,HIV ,Reproducibility of Results ,DAS ,Mycobacterium tuberculosis ,Young adult ,People and Places ,Tuberculosis, pulmonary/diagnosis ,business - Abstract
Funding: This study was funded by the World Bank under the East Africa Public Health Laboratory Networking Project (EAPHLNP). Background: Xpert MTB/RIF assay is a highly sensitive test for TB diagnosis, but still costly to most low-income countries. Several implementation strategies instead of frontline have been suggested; however with scarce data. We assessed accuracy of different Xpert MTB/RIF implementation strategies to inform national roll-out. Methods: This was a cross-sectional study of 1,924 adult presumptive TB patients in five regional referral hospitals of Uganda. Two sputum samples were collected, one for fluorescent microscopy (FM) and Xpert MTB/RIF examined at the study site laboratories. The second sample was sent to the Uganda Supra National TB reference laboratory for culture using both Lowenstein Jensen (LJ) and liquid culture (MGIT). We compared the sensitivities of FM, Xpert MTB/RIF and the incremental sensitivity of Xpert MTB/RIF among patients negative on FM using LJ and/or MGIT as a reference standard. Results: A total 1924 patients were enrolled of which 1596 (83%) patients had at least one laboratory result and 1083 respondents had a complete set of all the laboratory results. A total of 328 (30%) were TB positive on LJ and /or MGIT culture. The sensitivity of FM was n (%; 95% confidence interval) 246 (63.5%; 57.9-68.7) overall compared to 52 (55.4%; 44.1-66.3) among HIV positive individuals, while the sensitivity of Xpert MTB/RIF was 300 (76.2%; 71.7-80.7) and 69 (71.6%; 60.5-81.1) overall and among HIV positive individuals respectively. Overall incremental sensitivity of Xpert MTB/RIF was 60 (36.5%; 27.7-46.0) and 20 (41.7%; 25.5-59.2) among HIV positive individuals. Conclusion: Xpert MTB/RIF has a higher sensitivity than FM both in general population and HIV positive population. Xpert MTB/RIF offers a significant increase in terms of diagnostic sensitivity even when it is deployed selectively i.e. among smear negative presumptive TB patients. Our results support frontline use of Xpert MTB/RIF assay in high HIV/TB prevalent countries. In settings with limited access, mechanisms to refer smear negative sputum samples to Xpert MTB/RIF hubs are recommended. Publisher PDF
- Published
- 2018
8. Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection
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April C. Pettit, Andrew J.M. Nel, David W. Wright, Carlos Zamudio, Frederick R. Haselton, Mark L. Baglia, Megan E. Pask, Keersten M. Ricks, Keertan Dheda, Hali Bordelon, Francesca Solinas, Patricia K. Russ, Tatiana Cáceres, Philip A. Short, and Jonathan Michael Blackburn
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0301 basic medicine ,Urine/microbiology ,Polymerase Chain Reaction/methods ,HIV Infections ,Urine ,Polymerase Chain Reaction ,law.invention ,Mice ,chemistry.chemical_compound ,South Africa ,law ,Polymerase chain reaction ,Tuberculosis/complications/diagnosis/microbiology/urine ,Coinfection ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Molecular Diagnostic Techniques ,Gene Targeting ,Microbiology (medical) ,Genetic Markers ,Tuberculosis ,030106 microbiology ,HIV Infections/complications ,Gene Targeting/methods ,Mice/genetics ,Biology ,Mycobacterium tuberculosis/genetics/isolation & purification ,Microbiology ,Sensitivity and Specificity ,Article ,03 medical and health sciences ,Tuberculosis diagnosis ,medicine ,Humans ,Animals ,Glyceraldehyde-3-Phosphate Dehydrogenases/genetics ,Peptide Fragments/genetics ,purl.org/pe-repo/ocde/ford#1.06.01 [https] ,Molecular Biology ,Retrospective Studies ,Base Sequence ,Molecular Diagnostic Techniques/methods ,DNA ,Mycobacterium tuberculosis ,DNA/isolation & purification ,medicine.disease ,DNA extraction ,Virology ,Molecular biology ,Peptide Fragments ,030104 developmental biology ,chemistry ,Genetic marker ,Nucleic acid - Abstract
Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1 milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5 mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found a increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N=18, p=0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types.
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- 2017
9. Treatment decisions and mortality in HIV-positive presumptive smear-negative TB in the Xpert™ MTB/RIF era: a cohort study
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Christine Sekaggya-Wiltshire, Barbara Castelnuovo, Sabine Hermans, Olive Mbabazi, Yukari C. Manabe, Robert Colebunders, Juliet Babirye, Francis Kakooza, Rosalind Parkes-Ratanshi, APH - Methodology, APH - Global Health, Global Health, Hermans, Sabine M [0000-0001-5146-2932], and Apollo - University of Cambridge Repository
- Subjects
Adult ,Male ,medicine.medical_specialty ,Tuberculosis ,030231 tropical medicine ,HIV Infections ,HIV Infections/complications ,Sensitivity and Specificity ,lcsh:Infectious and parasitic diseases ,Mycobacterium tuberculosis ,Cohort Studies ,03 medical and health sciences ,0302 clinical medicine ,Medical microbiology ,Internal medicine ,medicine ,Humans ,Molecular diagnostic techniques/methods ,lcsh:RC109-216 ,Uganda ,030212 general & internal medicine ,Prospective Studies ,Prospective cohort study ,Empirical treatment ,Microscopy ,biology ,business.industry ,Coinfection ,Tuberculosis, pulmonary/epidemiology ,Sputum ,medicine.disease ,biology.organism_classification ,3. Good health ,Surgery ,CD4 Lymphocyte Count ,Infectious Diseases ,Molecular Diagnostic Techniques ,Tropical medicine ,Female ,Human medicine ,medicine.symptom ,business ,Tuberculosis, pulmonary/diagnosis ,Cohort study ,Research Article - Abstract
Background: The Xpert (TM) MTB/RIF (XP) has a higher sensitivity than sputum smear microscopy (70% versus 35%) for TB diagnosis and has been endorsed by the WHO for TB high burden countries to increase case finding among HIV co-infected presumptive TB patients. Its impact on the diagnosis of smear-negative TB in a routine care setting is unclear. We determined the change in diagnosis, treatment and mortality of smear-negative presumptive TB with routine use of Xpert MTB/RIF (XP). Methods: Prospective cohort study of HIV-positive smear-negative presumptive TB patients during a 12-month period after XP implementation in a well-staffed and trained integrated TB/HIV clinic in Kampala, Uganda. Prior to testing clinicians were asked to decide whether they would treat empirically prior to Xpert result; actual treatment was decided upon receipt of the XP result. We compared empirical and XP-informed treatment decisions and all-cause mortality in the first year. Results: Of 411 smear-negative presumptive TB patients, 175 (43%) received an XP; their baseline characteristics did not differ. XP positivity was similar in patients with a pre-XP empirical diagnosis and those without (9/29 [17%] versus 14/142 [10%], P = 0.23). Despite XP testing high levels of empirical treatment prevailed (18%), although XP results did change who ultimately was treated for TB. When adjusted for CD4 count, empirical treatment was not associated with higher mortality compared to no or microbiologically confirmed treatment. Conclusions: XP usage was lower than expected. The lower sensitivity of XP in smear-negative HIV-positive patients led experienced clinicians to use XP as a "rule-in" rather than "rule-out" test, with the majority of patients still treated empirically.
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- 2017
10. Current use and acceptability of novel diagnostic tests for active tuberculosis: a worldwide survey
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Rosella Centis, Lorenzo ZAMMARCHI, Luis Anibarro, Giovanni Battista Migliori, Novel N Chegou, Kingsley N. Ukwaja, Maria Nikolova, Muktar Gadanya, Lorenzo Guglielmetti, Carlotta Montagnani, Adrian Rendon, Kedir Abdella Abdulsemed, Delia Goletti, Silva Tafaj, Andre Loxton, Tomas Maria Perez-Porcuna, Tuula Vasankari, Simone A Joosten, Carla Montesano, Mariya Ivanovska, Marc Lipman, Prof. Sarman Singh, Daniel Blázquez-Gamero, Lanfranco Fattorini, and IVAN PAVIĆ
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Male ,Pathology ,Mycobacterium tuberculosis/isolation & purification ,Técnicas de diagnóstico molecular/métodos ,Tuberculosis/diagnosis ,Surveys and questionnaires ,Income ,Molecular diagnostic techniques/methods ,Serologic tests/methods ,Global Health ,0302 clinical medicine ,Tuberculose/diagnóstico ,030212 general & internal medicine ,Testes sorológicos/métodos ,biology ,Diagnostic test ,Middle Aged ,Mycobacterium tuberculosis/isolamento & purificação ,Inquéritos e questionários ,Female ,Pulmonary and Respiratory Medicine ,Adult ,medicine.medical_specialty ,Tuberculosis ,Attitude of Health Personnel ,Sensitivity and Specificity ,Mycobacterium tuberculosis ,Interviews as Topic ,03 medical and health sciences ,Special Article ,Young Adult ,Tuberculosis diagnosis ,Artigo Especial ,medicine ,Humans ,Intensive care medicine ,lcsh:RC705-779 ,business.industry ,lcsh:Diseases of the respiratory system ,medicine.disease ,Active tuberculosis ,biology.organism_classification ,Renda ,030228 respiratory system ,Reagent Kits, Diagnostic ,business - Abstract
Objective: To determine the current use and potential acceptance (by tuberculosis experts worldwide) of novel rapid tests for the diagnosis of tuberculosis that are in line with World Health Organization target product profiles. Methods: A multilingual survey was disseminated online between July and November of 2016. Results: A total of 723 individuals from 114 countries responded to the survey. Smear microscopy was the most commonly used rapid tuberculosis test (available to 90.9% of the respondents), followed by molecular assays (available to 70.7%). Only a small proportion of the respondents in middle- and low-income countries had access to interferon-gamma-release assays. Serological and lateral flow immunoassays were used by more than a quarter (25.4%) of the respondents. Among the respondents who had access to molecular tests, 46.7% were using the Xpert assay overall, that proportion being higher in lower middle-income countries (55.6%) and low-income countries (76.6%). The data also suggest that there was some alignment of pricing for molecular assays. Respondents stated they would accept novel rapid tuberculosis tests if available, including molecular assays (acceptable to 86.0%) or biomarker-based serological assays (acceptable to 81.7%). Simple biomarker-based assays were more commonly deemed acceptable in middle- and low-income countries. Conclusions: Second-generation molecular assays have become more widely available in high- and low-resource settings. However, the development of novel rapid tuberculosis tests continues to be considered important by tuberculosis experts. Our data also underscore the need for additional training and education of end users.
- Published
- 2017
11. Importance of whole genome sequencing for the assessment of outbreaks in diagnostic laboratories: analysis of a case series of invasive Streptococcus pyogenes infections
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G. Praz, B. Aubert, N. Troillet, Florian Tagini, Sandra A. Asner, P. A. Crisinel, Trestan Pillonel, Gilbert Greub, and Guy Prod'hom
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0301 basic medicine ,Microbiology (medical) ,Male ,medicine.medical_specialty ,Adolescent ,Genotype ,Streptococcus pyogenes ,Virulence Factors ,Virulence ,Single-nucleotide polymorphism ,Biology ,Aged ,Bacteriological Techniques/methods ,Child ,Child, Preschool ,Disease Outbreaks ,Drug Resistance, Bacterial ,Female ,Humans ,Infant ,Middle Aged ,Molecular Diagnostic Techniques/methods ,Molecular Typing/methods ,Streptococcal Infections/diagnosis ,Streptococcal Infections/epidemiology ,Streptococcus pyogenes/isolation & purification ,Switzerland/epidemiology ,Virulence Factors/genetics ,Whole Genome Sequencing/methods ,Emm-typing ,Group a streptococcus ,MLST ,Outbreak ,Whole genome sequencing ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,Medical microbiology ,Streptococcal Infections ,medicine ,Bacteriological Techniques ,Whole Genome Sequencing ,General Medicine ,Resistome ,Molecular Typing ,030104 developmental biology ,Infectious Diseases ,Molecular Diagnostic Techniques ,Multilocus sequence typing ,Original Article ,Switzerland - Abstract
Outbreaks of Streptococcus pyogenes hypervirulent clones are constant public health threats. In western Switzerland, an increase of severe cases of S. pyogenes invasive infections was observed between December 2015 and March 2016. Our aim was (i) to investigate these cases by the use of Whole Genome Sequencing (WGS) and (ii) to determine the specific virulome and resistome of each isolate in order to undertake adequate public health measures. Eleven Streptococcus pyogenes strains isolated from 11 patients with severe invasive infections between December 13, 2015 and March 12, 2016 were included in our study. Practically, emm-typing, MLST and WGS were used to investigate the relatedness between the isolates. The presence of virulence and antibiotic resistance genes as well as mutations in transcriptional regulators of virulence and in genes encoding for antibiotic targets were assessed. Three and two groups of isolates shared the same emm-type and ST type, respectively. Single Nucleotide Polymorphism (SNP) analysis revealed 14 to 32 SNPs between the strains of the same emm-type group, ruling out the possibility of a clonal outbreak. Mutations found in covS and rocA could partially explain an increased virulence. As these reassuring results were obtained in less than 10 days, no specific hospital hygiene and no dedicated public health measures had to be undertaken. WGS is a powerful technique to discriminate between closely related strains, excluding an outbreak in less than 10 days. Moreover, WGS provided extensive data on the virulome and resistome of all these strains. Electronic supplementary material The online version of this article (doi:10.1007/s10096-017-2905-z) contains supplementary material, which is available to authorized users.
- Published
- 2016
12. The detection of microbial DNA but not cultured bacteria is associated with increased mortality in patients with suspected sepsis-a prospective multi-centre European observational study
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J-L Vincent, Martin Wilks, N Libert, David J. Ecker, Kai Zacharowski, Mervyn Singer, David Brealey, M H Starczewska, Jacques Schrenzel, Michael J. O’Dwyer, and Ranga Sampath
- Subjects
Male ,Polymerase Chain Reaction/methods ,Polymerase Chain Reaction ,law.invention ,0302 clinical medicine ,law ,Blood culture ,030212 general & internal medicine ,Prospective Studies ,Young adult ,Prospective cohort study ,Polymerase chain reaction ,DNA, Bacterial/blood ,ddc:616 ,Aged, 80 and over ,medicine.diagnostic_test ,Bacteria/isolation & purification ,General Medicine ,Middle Aged ,Prognosis ,Europe ,Infectious Diseases ,Blood ,Molecular Diagnostic Techniques ,Female ,Risk assessment ,Microbiology (medical) ,Adult ,DNA, Bacterial ,medicine.medical_specialty ,Spectrometry, Mass, Electrospray Ionization ,Adolescent ,Critical Illness ,Spectrometry, Mass, Electrospray Ionization/methods ,Risk Assessment ,Sepsis ,03 medical and health sciences ,Young Adult ,Intensive care ,Internal medicine ,medicine ,Humans ,Sepsis/diagnosis/mortality ,Survival analysis ,Aged ,Bacteriological Techniques ,Molecular Diagnostic Techniques/methods ,Bacteria ,business.industry ,030208 emergency & critical care medicine ,medicine.disease ,Survival Analysis ,Surgery ,Blood/microbiology ,Early Diagnosis ,Bacteriological Techniques/methods ,business - Abstract
Objectives Blood culture results inadequately stratify the mortality risk in critically ill patients with sepsis. We sought to establish the prognostic significance of the presence of microbial DNA in the bloodstream of patients hospitalized with suspected sepsis. Methods We analysed the data collected during the Rapid Diagnosis of Infections in the Critically Ill (RADICAL) study, which compared a novel culture-independent PCR/electrospray ionization-mass spectrometry (ESI-MS) assay with standard microbiological testing. Patients were eligible for the study if they had suspected sepsis and were either hospitalized or were referred to one of nine intensive care units from six European countries. The blood specimen for PCR/ESI-MS assay was taken along with initial blood culture taken for clinical indications. Results Of the 616 patients recruited to the RADICAL study, 439 patients had data on outcome, results of the blood culture and PCR/ESI-MS assay available for analysis. Positive blood culture and PCR/ESI-MSI result was found in 13% (56/439) and 40% (177/439) of patients, respectively. Either a positive blood culture (p 0.01) or a positive PCR/ESI-MS (p 0.005) was associated with higher SOFA scores on enrolment to the study. There was no difference in 28-day mortality observed in patients who had either positive or negative blood cultures (35% versus 32%, p 0.74). However, in patients with a positive PCR/ESI-MS assay, mortality was significantly higher in comparison to those with a negative result (42% versus 26%, p 0.001). Conclusions Presence of microbial DNA in patients with suspected sepsis might define a patient group at higher risk of death.
- Published
- 2016
13. Added value of molecular assay Xpert MTB/RIF compared to sputum smear microscopy to assess the risk of tuberculosis transmission in a low-prevalence country
- Author
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Laurence Senn, Gilbert Greub, Katia Jaton, Onya Opota, Frederic Tissot, Guy Prod'hom, and Jesica Mazza-Stalder
- Subjects
Tuberculosis diagnosis ,0301 basic medicine ,Male ,Smear microscopy ,law.invention ,0302 clinical medicine ,Molecular POCT ,law ,Medicine ,030212 general & internal medicine ,Microscopy ,Adult ,Female ,Humans ,Microscopy/methods ,Middle Aged ,Molecular Diagnostic Techniques/methods ,Mycobacterium tuberculosis/cytology ,Mycobacterium tuberculosis/genetics ,Mycobacterium tuberculosis/isolation & purification ,Point-of-Care Systems ,Predictive Value of Tests ,Sensitivity and Specificity ,Sputum/microbiology ,Switzerland ,Tuberculosis, Pulmonary/diagnosis ,Young Adult ,biology ,General Medicine ,Infectious Diseases ,Transmission (mechanics) ,Molecular Diagnostic Techniques ,Predictive value of tests ,medicine.symptom ,Microbiology (medical) ,medicine.medical_specialty ,Tuberculosis ,Xpert MTB/RIF ,030106 microbiology ,Acid-fast bacilli staining ,Molecular diagnostic ,Mycobacterium tuberculosis ,03 medical and health sciences ,Pulmonary tuberculosis ,Internal medicine ,Tuberculosis, Pulmonary ,Transmissibility ,business.industry ,Sputum ,bacterial infections and mycoses ,medicine.disease ,biology.organism_classification ,Virology ,business ,Real-time PCR - Abstract
Airborne precautions are required at hospital admission for patients with suspected pulmonary tuberculosis. The isolation is maintained until 3 serially collected sputum smears are acid-fast bacilli negative, a time- and labor-intensive method with limited sensitivity and specificity, which has a great impact on patient flow management. We evaluated the possibility of replacing the result of microscopy by the semiquantitative result of the molecular point-of-care test Xpert MTB/RIF to assess patients' transmission risk to quickly guide airborne isolation decisions in low-endemic countries. The performance of the Xpert MTB/RIF, used as a first-line test, was compared to the results of microscopy for specimens (n=242) collected from May 2010 to December 2014 in Lausanne, Switzerland. The sensitivity and specificity of Xpert MTB/RIF were 91.5% (65/71) and 99.6% (170/171), respectively, vs. 64.8% (46/71) and 94.2% (161/171) for microscopy. Samples with negative Xpert MTB/RIF were all smear negative for Mycobacterium tuberculosis (negative predictive value, 100%). The semiquantitative results of Xpert MTB/RIF—high, medium, low or very low—were found to correlate with acid-fast bacilli detection: positive predictive value of 100% (6/6), 96.5% (27/28), 52.2% (12/23) and 11.1% (1/9) respectively. Finally, when including clinical criteria, we identified 11 smear-negative but Xpert MTB/RIF–positive patients with a significant transmission potential. In conclusion, our data support the introduction of an Xpert MTB/RIF–based strategy as a replacement of smear microscopy for a faster and more accurate management of tuberculosis patients' transmission risk in a low-prevalence country.
- Published
- 2016
14. Feasibility and Operational Performance of Tuberculosis Detection by Loop-Mediated Isothermal Amplification Platform in Decentralized Settings: Results from a Multicenter Study
- Author
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Doris Hillemann, Pratibha Narang, Achilles Katamba, Moses Joloba, Frank Cobelens, C. N. Paramasivan, Jorge Giraldo, Danielle Amisano, Rahul Narang, Pamela Nabeta, Carlos Zamudio, Catharina Boehme, David Alland, Christen Gray, AII - Amsterdam institute for Infection and Immunity, APH - Amsterdam Public Health, and Global Health
- Subjects
0301 basic medicine ,Microbiology (medical) ,Adult ,Male ,medicine.medical_specialty ,Operational performance ,Tuberculosis ,Adolescent ,030106 microbiology ,Loop-mediated isothermal amplification ,Tuberculosis/diagnosis ,India ,Sensitivity and Specificity ,03 medical and health sciences ,Young Adult ,Tuberculosis diagnosis ,Internal medicine ,parasitic diseases ,Peru ,Medicine ,Humans ,Uganda ,Nucleic Acid Amplification Techniques/methods ,purl.org/pe-repo/ocde/ford#1.06.01 [https] ,High humidity ,Aged ,Aged, 80 and over ,Molecular Diagnostic Techniques/methods ,business.industry ,Mycobacteriology and Aerobic Actinomycetes ,Nucleic acid amplification technique ,Middle Aged ,medicine.disease ,Confidence interval ,Surgery ,030104 developmental biology ,Molecular Diagnostic Techniques ,Multicenter study ,Female ,business ,Nucleic Acid Amplification Techniques - Abstract
Currently available nucleic acid amplification platforms for tuberculosis (TB) detection are not designed to be simple or inexpensive enough to implement in decentralized settings in countries with a high burden of disease. The loop-mediated isothermal amplification platform (LAMP) may change this. We conducted a study in adults with symptoms suggestive of TB in India, Uganda, and Peru to establish the feasibility of using TB-LAMP (Eiken Chemical Co.) in microscopy laboratories compared with using smear microscopy against a reference standard of solid and liquid cultures. Operational characteristics were evaluated as well. A total of 1,777 participants met the eligibility criteria and were included for analysis. Overall, TB-LAMP sensitivities among culture-positive samples were 97.2% (243/250; 95% confidence interval [CI], 94.3% to 98.2%) and 62.0% (88/142; 95% CI, 53.5% to 70.0%) for smear-positive and smear-negative TB, respectively, but varied widely by country and operator. Specificities ranged from 94.5% (446/472; 95% CI, 92.0% to 96.4%) to 98.0% (350/357; 95% CI, 96.0% to 99.2%) by country. A root cause analysis identified high temperatures, high humidity, and/or low reaction volumes as possible causes for false-positive results, as they may result in nonspecific amplification. The study was repeated in India with training focused on vulnerable steps and an updated protocol; 580 participants were included for analysis. Specificity in the repeat trial was 96.6% (515/533; 95% CI, 94.7% to 97.9%). To achieve acceptable performance of LAMP at the microscopy center level, significant training and infrastructure requirements are necessary.
- Published
- 2016
15. An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp Infections
- Author
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Peter C. Melby, Renato Porrozzi, Nancy G. Saravia, Maxy De los Santos, Erika M Costa, Bruno L. Travi, Olga Lucía Fernández, Gerald C. Baldeviano, Andres G. Lescano, Omar A. Saldarriaga, and Alejandro Castellanos-Gonzalez
- Subjects
0301 basic medicine ,Leishmaniasis, Cutaneous/diagnosis ,Recombinase Polymerase Amplification ,Artificial Gene Amplification and Extension ,Pathology and Laboratory Medicine ,Polymerase Chain Reaction ,Geographical locations ,Chromatography, Affinity ,law.invention ,0302 clinical medicine ,Filter Paper ,law ,Zoonoses ,Peru ,Medicine and Health Sciences ,Leishmaniasis ,Polymerase chain reaction ,Protozoans ,Leishmania ,Oligonucleotide Probes/genetics ,DNA, Kinetoplast ,lcsh:Public aspects of medicine ,3. Good health ,Laboratory Equipment ,Infectious Diseases ,Real-time polymerase chain reaction ,Molecular Diagnostic Techniques ,Kinetoplast ,Engineering and Technology ,DNA, Protozoan/genetics ,Nucleic Acid Amplification Techniques ,Brazil ,purl.org/pe-repo/ocde/ford#3.03.06 [https] ,Research Article ,Neglected Tropical Diseases ,lcsh:Arctic medicine. Tropical medicine ,lcsh:RC955-962 ,Point-of-Care Systems ,030231 tropical medicine ,Equipment ,Leishmaniasis, Cutaneous ,Biology ,Research and Analysis Methods ,DNA, Kinetoplast/genetics ,Sensitivity and Specificity ,03 medical and health sciences ,Signs and Symptoms ,Cutaneous leishmaniasis ,Diagnostic Medicine ,Parasitic Diseases ,medicine ,Humans ,Molecular Biology Techniques ,Molecular Biology ,DNA Primers ,DNA Primers/genetics ,Protozoan Infections ,Molecular Diagnostic Techniques/methods ,Organisms ,Public Health, Environmental and Occupational Health ,Biology and Life Sciences ,lcsh:RA1-1270 ,Nucleic acid amplification technique ,South America ,DNA, Protozoan ,Tropical Diseases ,medicine.disease ,biology.organism_classification ,Virology ,Parasitic Protozoans ,Leishmania/genetics/isolation & purification ,030104 developmental biology ,Immunology ,Lesions ,People and places ,Oligonucleotide Probes - Abstract
Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis., Author Summary Cutaneous leishmaniasis is a parasitic disease transmitted by the bite of sandflies that produces skin ulcers. The severe, disfiguring form of the disease is characterized by parasite dissemination to the mucosa of the nose and palate. Current diagnosis is based on microscopy which has low sensitivity in chronic cases. Molecular methods (PCR) that detect parasite DNA are highly sensitive but costs and personnel training make it impossible to implement it in resource-limited areas. We developed a novel molecular method (RPA-LF) that could be applied in the field because it does not require sophisticated equipment. It is also very sensitive and specific to detect the principal Leishmania species that produce cutaneous leishmaniasis in Latin America. Future field implementation of RPA-LF could have a positive impact on disease management and control.
- Published
- 2016
16. Target Product Profile for a Diagnostic Assay to Differentiate between Bacterial and Non-Bacterial Infections and Reduce Antimicrobial Overuse in Resource-Limited Settings: An Expert Consensus
- Author
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Francis Moussy, Anne-Laure Page, Iveth J. González, John A. Crump, Birkneh Tilahun Tadesse, Sabine Dittrich, Valérie D'Acremont, Arlene Chua, Thomas Tängdén, David L. Dolinger, Quique Bassat, Paul N. Newton, Anna Zorzet, Timothy J. Rodwell, Yoel Lubell, and Norbert Heinrich
- Subjects
Bacterial Diseases ,Bacterial Infections/diagnosis ,Consensus ,Developing Countries ,Diagnosis, Differential ,Fever/diagnosis ,Humans ,Molecular Diagnostic Techniques/economics ,Molecular Diagnostic Techniques/methods ,Molecular Diagnostic Techniques/standards ,Practice Guidelines as Topic ,Reagent Kits, Diagnostic/economics ,Reagent Kits, Diagnostic/standards ,Sensitivity and Specificity ,Pathology ,Medical laboratory ,lcsh:Medicine ,Fevers ,Infektionsmedicin ,Pathology and Laboratory Medicine ,Global Health ,Biochemistry ,0302 clinical medicine ,Global health ,Medicine and Health Sciences ,Public and Occupational Health ,030212 general & internal medicine ,lcsh:Science ,Multidisciplinary ,Antimicrobials ,Biochemical markers ,Drugs ,Bacterial Infections ,3. Good health ,Test (assessment) ,Infectious Diseases ,Molecular Diagnostic Techniques ,Community health ,Marcadors bioquímics ,Sample collection ,Research Article ,medicine.medical_specialty ,Infectious Medicine ,Fever ,030231 tropical medicine ,Bacterial diseases ,MEDLINE ,Malària ,Microbiology ,03 medical and health sciences ,Signs and Symptoms ,Diagnostic Medicine ,Microbial Control ,medicine ,Parasitic Diseases ,Intensive care medicine ,Pharmacology ,Health economics ,Malalties bacterianes ,business.industry ,lcsh:R ,Biology and Life Sciences ,Tropical Diseases ,Malaria ,New product development ,lcsh:Q ,Antimicrobial Resistance ,Reagent Kits, Diagnostic ,business ,Biomarkers - Abstract
Acute fever is one of the most common presenting symptoms globally. In order to reduce the empiric use of antimicrobial drugs and improve outcomes, it is essential to improve diagnostic capabilities. In the absence of microbiology facilities in low-income settings, an assay to distinguish bacterial from non-bacterial causes would be a critical first step. To ensure that patient and market needs are met, the requirements of such a test should be specified in a target product profile (TPP). To identify minimal/optimal characteristics for a bacterial vs. non-bacterial fever test, experts from academia and international organizations with expertise in infectious diseases, diagnostic test development, laboratory medicine, global health, and health economics were convened. Proposed TPPs were reviewed by this working group, and consensus characteristics were defined. The working group defined non-severely ill, non-malaria infected children as the target population for the desired assay. To provide access to the most patients, the test should be deployable to community health centers and informal health settings, and staff should require 90% and >80% for sensitivity and specificity, respectively. Other key characteristics, to account for the challenging environment at which the test is targeted, included: i) time-to-result
- Published
- 2016
17. Development of a duplex real time PCR for the detection ofRickettsiaspp. and typhus group rickettsia in clinical samples
- Author
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Katia Jaton, Laurence T. Trellu, A. Cometta, Stefano Giulieri, and Gilbert Greub
- Subjects
Male ,Scrub typhus ,Citrate (si)-Synthase/genetics ,RNA, Ribosomal, 16S ,Immunology and Allergy ,Rickettsia ,Oligonucleotide Probes/genetics ,RNA, Ribosomal, 16S/genetics ,General Medicine ,Rickettsia prowazekii ,RNA, Bacterial ,Infectious Diseases ,Molecular Diagnostic Techniques ,Rickettsia/isolation & purification ,Female ,Adult ,Microbiology (medical) ,animal structures ,Multiplex Polymerase Chain Reaction/methods ,Immunology ,Citrate (si)-Synthase ,Real-Time Polymerase Chain Reaction/methods ,Biology ,Real-Time Polymerase Chain Reaction ,Murine typhus ,Sensitivity and Specificity ,Microbiology ,Bacterial Proteins ,Rickettsia typhi ,medicine ,Humans ,Rickettsia Infections/diagnosis ,DNA Primers ,DNA Primers/genetics ,Molecular Diagnostic Techniques/methods ,Bacterial Proteins/genetics ,Rickettsia Infections ,bacterial infections and mycoses ,medicine.disease ,biology.organism_classification ,Virology ,Spotted fever ,Rickettsiosis ,bacteria ,Oligonucleotide Probes ,Multiplex Polymerase Chain Reaction ,RNA, Bacterial/genetics ,Typhus - Abstract
Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.
- Published
- 2012
18. Which anatomical sites should be sampled for screening of methicillin-resistant Staphylococcus aureus carriage by culture or by rapid PCR test?
- Author
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Patrick Basset, Dominique S. Blanc, Giorgio Zanetti, I. Nahimana, and Laurence Senn
- Subjects
Methicillin-Resistant Staphylococcus aureus ,Microbiology (medical) ,Culture ,Nose ,Groin ,medicine.disease_cause ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Specimen Handling ,Microbiology ,rapid test ,stomatognathic system ,Throat ,otorhinolaryngologic diseases ,medicine ,Humans ,Mass Screening ,MRSA screening ,Bacteriological Techniques ,business.industry ,extranasal screening ,screening sites ,General Medicine ,Staphylococcal Infections ,Methicillin-resistant Staphylococcus aureus ,Infectious Diseases ,medicine.anatomical_structure ,Anatomical sites ,Carriage ,Molecular Diagnostic Techniques ,Staphylococcus aureus ,Nasal Swab ,Carrier State ,Pharynx ,Bacteriological Techniques/methods ,Carrier State/diagnosis ,Carrier State/microbiology ,Groin/microbiology ,Mass Screening/methods ,Methicillin-Resistant Staphylococcus aureus/isolation & purification ,Molecular Diagnostic Techniques/methods ,Nose/microbiology ,Pharynx/microbiology ,Polymerase Chain Reaction/methods ,Specimen Handling/methods ,Staphylococcal Infections/diagnosis ,Staphylococcal Infections/microbiology ,business - Abstract
The nose is the anatomical site usually recommended for methicillin-resistant Staphylococcus aureus (MRSA) screening. Other sites are also recommended, but are more controversial. We showed that the sensitivities of MRSA detection from nasal swabs alone were 48% and 62% by culture or by rapid PCR test, respectively. These percentages increased to 79% and 92% with the addition of groin swabs, and to 96% and 99% with the addition of groin and throat swabs. In conclusion, neither by culture nor by rapid PCR test is nose sampling alone sufficient for MRSA detection. Additional anatomical sites should include at least the groin and throat.
- Published
- 2012
19. MRSA screening by the Xpert MRSA PCR assay: pooling samples of the nose, throat, and groin increases the sensitivity of detection without increasing the laboratory costs
- Author
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I. Nahimana, Giorgio Zanetti, Gilbert Greub, and Dominique S. Blanc
- Subjects
Microbiology (medical) ,Methicillin-Resistant Staphylococcus aureus ,medicine.medical_specialty ,Carrier State/diagnosis ,Polymerase Chain Reaction/methods ,Pcr assay ,Specimen Handling/methods ,Nose ,Groin ,Methicillin-Resistant Staphylococcus aureus/isolation & purification ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Nose/microbiology ,Specimen Handling ,Mass Screening/methods ,Internal medicine ,Throat ,medicine ,Groin/microbiology ,Polymerase Chain Reaction/economics ,Specimen Handling/economics ,Humans ,Mass Screening ,Molecular Diagnostic Techniques/economics ,Bacteriological Techniques ,Mass Screening/economics ,Molecular Diagnostic Techniques/methods ,business.industry ,Staphylococcal Infections/diagnosis ,Bacteriological Techniques/economics ,General Medicine ,Staphylococcal Infections ,Surgery ,Staphylococcal Infections/microbiology ,Infectious Diseases ,medicine.anatomical_structure ,Molecular Diagnostic Techniques ,Pharynx/microbiology ,Carrier State ,Bacteriological Techniques/methods ,Costs and Cost Analysis ,Carrier State/microbiology ,Pharynx ,Detection rate ,business ,Mrsa screening - Abstract
The performance of the Xpert MRSA polymerase chain reaction (PCR) assay on pooled nose, groin, and throat swabs (three nylon flocked eSwabs into one tube) was compared to culture by analyzing 5,546 samples. The sen- sitivity (0.78, 95 % confidence interval (CI) 0.73-0.82) and specificity (0.99, 95 % CI 0.98-0.99) were similar to the results from published studies on separated nose or other specimens. Thus, the performance of the Xpert MRSA assay was not affected by pooling the three specimens into one assay, allowing a higher detection rate without increasing laboratory costs, as compared to nose samples alone.
- Published
- 2013
20. Comparative genomics of Neisseria meningitidis strains: new targets for molecular diagnostics
- Author
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Katia Jaton, Antony Croxatto, Trestan Pillonel, Nicolas Jacquier, Gilbert Greub, S.M. Diene, and Claire Bertelli
- Subjects
DNA, Bacterial ,0301 basic medicine ,Microbiology (medical) ,Sequence analysis ,ctrA ,Single-nucleotide polymorphism ,Neisseria meningitidis ,Biology ,Real-Time Polymerase Chain Reaction ,medicine.disease_cause ,Sensitivity and Specificity ,Genome ,molecular diagnostics ,03 medical and health sciences ,medicine ,Humans ,quantitative real-time PCR ,Comparative genomics ,Genetics ,Whole genome sequencing ,Child, Preschool ,DNA, Bacterial/chemistry ,DNA, Bacterial/genetics ,Genes, Bacterial ,Genome, Bacterial ,Meningococcal Infections/diagnosis ,Meningococcal Infections/microbiology ,Molecular Diagnostic Techniques/methods ,Neisseria meningitidis/classification ,Neisseria meningitidis/genetics ,Neisseria meningitidis/isolation & purification ,Real-Time Polymerase Chain Reaction/methods ,Sequence Analysis, DNA ,General Medicine ,metA ,Molecular diagnostics ,tauE ,Meningococcal Infections ,genomic DNA ,030104 developmental biology ,Infectious Diseases ,Molecular Diagnostic Techniques ,shlA ,Genome sequence - Abstract
In 2010, Jaton et al . (False-negative PCR result due to gene polymorphism: the example of Neisseria meningitidis . J Clin Microbiol 2010;48:4590–2) reported an isolate of Neisseria meningitidis serogroup B that was not detected by the ctrA quantitative real-time PCR (qRT-PCR) used in our diagnostic laboratory. Sequence analysis of ctrA revealed several single nucleotide polymorphisms responsible for the negative qRT-PCR. Therefore, we sequenced the genome of this isolate and performed comparative genomics to propose new gene targets for the specific detection of N. meningitidis from clinical specimens. We identified 11 genes as specific to N. meningitidis genomes and common to at least 177 (97%) of the 183 genomes available. Among them, three genes ( metA, tauE and shlA ) were selected to develop new qRT-PCRs for the detection of N. meningitidis DNA. The three qRT-PCRs were highly sensitive and specific, and they exhibited a good reproducibility when tested on plasmidic positive controls and genomic DNA extracted from strains of N. meningitidis and other relevant bacterial species. The clinical sensitivity and specificity of metA and tauE qRT-PCRs were both 100% based on a testing of cerebrospinal fluid samples positive for N. meningitidis or other clinically relevant bacteria. Despite a 100% specificity, the sensitivity of the shlA qRT-PCR was only 70%. We thus recommend using the metA and/or tauE qRT-PCRs developed here. To prevent PCR failure in the presence of new polymorphic strains, the detection of dual targets by duplex qRT-PCR would be more accurate and suitable for the diagnosis of N. meningitidis from clinical specimens.
- Published
- 2016
21. Intrapartum Group B streptococcus detection by rapid polymerase chain reaction assay for the prevention of neonatal sepsis
- Author
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Gesuele Renzi, Olivier Irion, Riccardo Pfister, Jacques Schrenzel, Michel Boulvain, B Martinez de Tejada, and Patrice Francois
- Subjects
Polymerase Chain Reaction/methods ,neonatal sepsis ,Perineum ,Polymerase Chain Reaction ,Group B ,Pregnancy ,Streptococcus agalactiae/isolation & purification ,Medicine ,Mass Screening ,Prospective Studies ,Antibiotic prophylaxis ,Pregnancy Complications, Infectious ,Prospective cohort study ,Carrier State/diagnosis/microbiology ,reproductive and urinary physiology ,Vagina/microbiology ,ddc:616 ,ddc:618 ,Neonatal sepsis ,General Medicine ,Infectious Diseases ,PCR ,Molecular Diagnostic Techniques ,Carrier State ,Vagina ,Gestation ,Female ,Microbiology (medical) ,Adult ,medicine.medical_specialty ,Rectum/microbiology ,Group B streptococcus ,Sensitivity and Specificity ,Streptococcus agalactiae ,Mass Screening/methods ,Sepsis ,Streptococcal Infections ,Humans ,Gynecology ,Bacteriological Techniques ,Molecular Diagnostic Techniques/methods ,business.industry ,Pregnancy Complications, Infectious/diagnosis/microbiology ,intrapartum antibiotic prophylaxis ,Perineum/microbiology ,Rectum ,Sepsis/prevention & control ,intrapartum screening ,Assay sensitivity ,medicine.disease ,Carriage ,Bacteriological Techniques/methods ,Streptococcal Infections/diagnosis/microbiology/prevention & control ,business - Abstract
Group B streptococcus (GBS) is a leading cause of infectious neonatal morbidity and mortality. Timely and accurate identification of colonized mothers is imperative so that antibioprophylaxis can be implemented during labour to reduce the risk of neonatal sepsis. We planned our study to analyse the diagnostic accuracy of an intrapartum PCR assay to identify GBS-colonized women and to allow the implementation of correct (i.e. at least 4 h) intrapartum antibiotic prophylaxis based on the PCR results. We included 695 women in labour who were tested for rectovaginal GBS carriage by culture and PCR. Women were also screened at 35–37 weeks of gestation. Intrapartum GBS colonization was 19.3%. Assay sensitivity was 81.0% for antenatal culture and 85.0% for intrapartum PCR; p 0.72. GBS colonization (n = 107) was known at least 4 h before delivery in 68 (64%) and 73 (68%) women based on antenatal culture and intrapartum PCR, respectively. Among 43 women delivering preterm, correct status was known at least 4 h before delivery in 10 (23%) and 32 (74%) women according to antenatal culture and intrapartum PCR, respectively. These results support the concept that GBS screening can be performed routinely during labour in a clinical setting. The intrapartum approach is at least as accurate as the antenatal screening, with the additional advantage of identifying women delivering preterm or not followed during pregnancy.
- Published
- 2010
22. Correction of underquantification of Human Immunodeficiency Virus Type 1 Load with the Second Version of the Roche Cobas AmpliPrep/Cobas TaqMan Assay
- Author
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Corinne Liesnard, Denis Pierard, S. Van den Wijngaert, D. Marissens, Sabine Lauwers, Laurent Debaisieux, A. De Bel, Immunology and Microbiology, and Clinical Biology
- Subjects
Microbiology (medical) ,Adult ,Male ,Molecular Diagnostic Techniques/methods ,Cobas taqman ,Human immunodeficiency virus (HIV) ,HIV Infections ,Bacteriology ,Biology ,Viral Load ,medicine.disease_cause ,Molecular biology ,Sensitivity and Specificity ,Mean difference ,Molecular Diagnostic Techniques ,medicine ,Cobas amplicor ,HIV-1 ,HIV-1/isolation & purification ,Humans ,young adult ,Female ,HIV Infections/virology ,Viral Load/methods ,Viral load - Abstract
Initial evaluations of the Cobas AmpliPrep/Cobas TaqMan human immunodeficiency virus type 1 (HIV-1) test (CAP/CTM) demonstrated good performance but, afterwards, reports about underquantification were published. We investigated whether the problem was solved with a second version of this assay, the Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0 (CAP/CTM v2.0). The remaining plasma of 375 consecutive HIV-1 positive samples with a viral load of ≥4,000 copies/ml was collected in three laboratories. The samples were diluted and retested with our routine method Cobas AmpliPrep/Cobas Amplicor HIV-1 monitor test v1.5 in ultrasensitive mode (CAP/CA PHS), as well as with the CAP/CTM and CAP/CTM v2.0 tests. An absolute difference between the results of two methods of ≥0.71 log10copies/ml was defined as moderately discrepant, and an absolute difference of ≥0.93 log10copies/ml was defined as severely discrepant. In addition, criteria for considering the new methods equivalent to the routine method were formulated. (i) For CAP/CTM compared to CAP/CA PHS, 36 (9.5%) and 20 (5.3%) samples were, respectively, considered moderately and severely underquantified by CAP/CTM. The mean difference between CAP/CTM and CAP/CA PHS was −0.32 log10copies/ml. Eight of nineteen of the severely underquantified samples were from patients infected with HIV-1 subtype B strain. (ii) For CAP/CTM v2.0 compared to CAP/CA PHS, no sample was moderately or severely underquantified by CAP/CTM v2.0. A mean difference of 0.08 log10copies/ml was found with CAP/CTM v2.0 compared to CAP/CA PHS. The underquantification problem of the CAP/CTM kit was clearly demonstrated. The criteria for the equivalence of CAP/CTM v2.0 to the routine test CAP/CA PHS were fulfilled.
- Published
- 2010
23. Molecular diagnosis reveals genetic heterogeneity for the overlapping MKKS and BBS phenotypes
- Author
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Sophie Hellé, Israël Nisand, Vincent Marion, Sabine Sigaudy, Corinne Stoetzel, Marie-Claire Vincent, Laurence Faivre, Jean-Louis Mandel, Alain Verloes, Bérénice Doray, Jean-Marc Danse, Pierre Bitoun, Elise Schaefer, Christian P. Hamel, Alice Goldenberg, Hélène Dollfus, Dominique Bonneau, Sonia Finck, Myriam Durand, Muriel Holder, B. Viville, Mitochondrie : Régulations et Pathologie, and Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)
- Subjects
Heart Defects, Congenital ,BBS2 ,congenital, hereditary, and neonatal diseases and abnormalities ,Genotype ,Genetic counseling ,[SDV]Life Sciences [q-bio] ,Hydrocolpos/diagnosis/genetics ,Biology ,Polydactyly/diagnosis/genetics ,MKKS ,McKusick–Kaufman syndrome ,Diagnosis, Differential ,03 medical and health sciences ,Genetic Heterogeneity ,Bardet–Biedl syndrome ,Diagnosis ,Genetics ,medicine ,Humans ,Abnormalities, Multiple ,Bardet-Biedl Syndrome/diagnosis/genetics ,Congenital/diagnosis/genetics ,Bardet-Biedl Syndrome ,Genetics (clinical) ,030304 developmental biology ,Heart Defects ,Uterine Diseases ,0303 health sciences ,Polydactyly ,Molecular Diagnostic Techniques/methods ,Genetic heterogeneity ,030305 genetics & heredity ,fungi ,Infant, Newborn ,Infant ,Hydrocolpos ,General Medicine ,Uterine Diseases/diagnosis/genetics ,medicine.disease ,Newborn ,Phenotype ,Molecular Diagnostic Techniques ,Differential ,Mutation ,BBS12 ,Abnormalities ,Multiple/diagnosis/genetics - Abstract
International audience; Hydrometrocolpos and polydactyly diagnosed in the prenatal period or early childhood may raise diagnostic dilemmas especially in distinguishing McKusick-Kaufman syndrome (MKKS) and the Bardet-Biedl syndrome (BBS). These two conditions can initially overlap. With time, the additional features of BBS appearing in childhood, such as retinitis pigmentosa, obesity, learning disabilities and progressive renal dysfunction allow clear differentiation between BBS and MKKS. Genotype overlap also exists, as mutations in the MKKS-BBS6 gene are found in both syndromes. We report 7 patients diagnosed in the neonatal period with hydrometrocolpos and polydactyly who carry mutations in various BBS genes (BBS6, BBS2, BBS10, BBS8 and BBS12), stressing the importance of wide BBS genotyping in patients with this clinical association for diagnosis, prognosis and genetic counselling.
- Published
- 2010
24. Evaluation of the prognostic and predictive value of p53 and Bcl-2 in breast cancer patients participating in a randomized study with dose-dense sequential adjuvant chemotherapy
- Author
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Sofia Markaki, Georgia Kafiri, Dimitrios Vlachodimitropoulos, Olympia Tzaida, Vassiliki Malamou-Mitsi, Chrisoula D. Scopa, E Karagianni, Stefanos Papadopoulos, Pavlos Papakostas, Kitty Pavlakis, C. Christodoulou, George Fountzilas, Maria Sotiropoulou, Helen Gogas, Vasiliki Kyriakou, Urania Dafni, A Bourli, T Fillipidis, Irini Papaspyrou, Dimitris Bafaloukos, and T. Toliou
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Oncology ,Pathology ,medicine.medical_treatment ,Breast Neoplasms/*diagnosis/drug therapy/metabolism/mortality/pathology ,Gene Expression ,Cyclophosphamide/administration & dosage ,Tumor Suppressor Protein p53/*metabolism ,Antineoplastic Combined Chemotherapy Protocols ,Methotrexate/administration & dosage ,Incidence (epidemiology) ,Carcinoma, Ductal, Breast ,Hematology ,Middle Aged ,Prognosis ,Chemotherapy regimen ,Fluorouracil/administration & dosage ,Molecular Diagnostic Techniques ,Proto-Oncogene Proteins c-bcl-2 ,Fluorouracil ,Chemotherapy, Adjuvant ,dosage/*therapeutic use ,Female ,Tumor Markers, Biological/metabolism ,medicine.drug ,Epirubicin ,Adult ,medicine.medical_specialty ,Paclitaxel ,Cyclophosphamide ,Breast Neoplasms ,Epirubicin/administration & dosage ,Proto-Oncogene Proteins c-bcl-2/*metabolism ,Breast cancer ,Predictive Value of Tests ,Internal medicine ,Paclitaxel/administration & dosage ,Biomarkers, Tumor ,medicine ,Humans ,Carcinoma, Ductal, Breast/*diagnosis/drug therapy/metabolism/mortality/pathology ,Aged ,Chemotherapy ,Dose-Response Relationship, Drug ,Molecular Diagnostic Techniques/methods ,business.industry ,medicine.disease ,Survival Analysis ,Antineoplastic Combined Chemotherapy Protocols/administration & ,Methotrexate ,Tumor Suppressor Protein p53 ,business - Abstract
PURPOSE: To assess the prognostic and predictive significance of p53 and Bcl-2 protein expression in high risk patients with breast cancer treated with dose-dense sequential chemotherapy. PATIENTS AND METHODS: From June 1997 until November 2000, 595 patients were randomized to three cycles of epirubicin (E) 110 mg/m2 followed by three cycles of paclitaxel (P) 250 mg/m2 followed by three cycles of 'intensified' CMF (cyclophosphamide 840 mg/m2, methotrexate 47 mg/m2 and fluorouracil 840 mg/m2) or to four cycles of E, followed by four cycles of CMF. p53 and Bcl-2 expression was investigated by immunohistochemistry in 392 and 397 patients respectively. RESULTS: Positive expression of p53 was detected in 104 (26.5%) patients and was significantly associated with negative hormonal status, worse histologic grade, higher incidence of disease relapse and higher rate of death. p53 positive expression was a significant negative predictor of overall survival (OS) (P = 0.002) and disease-free survival (DFS) (P = 0.001). Negative expression of Bcl-2 was detected in 203 (51%) patients and was significantly associated with negative hormonal status. Multivariate analysis revealed that, positive p53 expression, higher number of positive nodes and worse tumor grade were related to significantly poorer OS and DFS. CONCLUSIONS: For both treatments, p53 positive expression was a significant negative prognostic factor for OS and DFS while Bcl-2 was not. No predictive ability of p53 status or Bcl-2 status for paclitaxel treatment was evident. Ann Oncol
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- 2006
25. Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease
- Author
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Henrik Westh, Frank Møller Aarestrup, Katrine Grimstrup Joensen, Ole Lund, Anne Line Engsbro, Oksana Lukjancenko, and Rolf Sommer Kaas
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Diarrhea ,0301 basic medicine ,Microbiology (medical) ,Adult ,Male ,medicine.medical_specialty ,Feces/microbiology ,Pathogen detection ,Adolescent ,Denmark ,030106 microbiology ,Virulence ,Biology ,medicine.disease_cause ,DNA sequencing ,Microbiology ,Hospitals, University ,Feces ,03 medical and health sciences ,Young Adult ,Medical microbiology ,SDG 3 - Good Health and Well-being ,medicine ,Humans ,Child ,Pathogen ,Aged ,Aged, 80 and over ,Diarrhea/diagnosis ,High-Throughput Nucleotide Sequencing/methods ,Molecular Diagnostic Techniques/methods ,Diarrhoeal disease ,Infant, Newborn ,High-Throughput Nucleotide Sequencing ,Giardia ,Infant ,Pathogenic bacteria ,General Medicine ,Middle Aged ,biology.organism_classification ,3. Good health ,Infectious Diseases ,Molecular Diagnostic Techniques ,Child, Preschool ,Female ,Erratum - Abstract
The accurate microbiological diagnosis of diarrhoea involves numerous laboratory tests and, often, the pathogen is not identified in time to guide clinical management. With next-generation sequencing (NGS) becoming cheaper, it has huge potential in routine diagnostics. The aim of this study was to evaluate the potential of NGS-based diagnostics through direct sequencing of faecal samples. Fifty-eight clinical faecal samples were obtained from patients with diarrhoea as part of the routine diagnostics at Hvidovre University Hospital, Denmark. Ten samples from healthy individuals were also included. DNA was extracted from faecal samples and sequenced on the Illumina MiSeq system. Species distribution was determined with MGmapper and NGS-based diagnostic prediction was performed based on the relative abundance of pathogenic bacteria and Giardia and detection of pathogen-specific virulence genes. NGS-based diagnostic results were compared to conventional findings for 55 of the diarrhoeal samples; 38 conventionally positive for bacterial pathogens, two positive for Giardia, four positive for virus and 11 conventionally negative. The NGS-based approach enabled detection of the same bacterial pathogens as the classical approach in 34 of the 38 conventionally positive bacterial samples and predicted the responsible pathogens in five of the 11 conventionally negative samples. Overall, the NGS-based approach enabled pathogen detection comparable to conventional diagnostics and the approach has potential to be extended for the detection of all pathogens. At present, however, this approach is too expensive and time-consuming for routine diagnostics.
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