scholarly journals 2181. Yield and Impact of Molecular Diagnostics for Pathogen Detection in Pediatric Patients: 16/18S rRNA PCR and Noninvasive Assays

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S740-S741
Author(s):  
Angela Chun ◽  
Taylor Heald-Sargent ◽  
Michael Malczynski ◽  
William J Muller ◽  
Chao Qi ◽  
...  
Keyword(s):  
Molecular Diagnostics ◽  
18S Rrna ◽  
Pediatric Patients ◽  
Clinical Care ◽  
High Yield ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Molecular Testing ◽  
Rrna Gene ◽  
Fungal Culture

Abstract Background Molecular diagnostic tests can identify bacterial and fungal pathogens from clinical samples. Nucleic acid detection tests include 16S and 18S rRNA gene PCR (16/18S PCR) and plasma next-generation sequencing (NGS). Other assays (fungal galactomannan and 1,3-β-d-glucan) detect structural factors. Our objective was to assess the utilization, yield, and impact of molecular diagnostics in pediatric patients who had samples sent for 16/18S PCR. Methods Sterile site fluid or tissue specimens were collected as part of standard care at Lurie Children’s Hospital, cultured, and sent to Northwestern Memorial Hospital for 16/18S PCR as clinically indicated. Medical records were reviewed for diagnostics, antibiotics, and clinical course. Results From 1/2016–8/2018, 236 samples were sent for 16 and/or 18S PCR from 183 patients. 83% had a concurrent ID consult. 16S PCR was done on 215 samples, 42 (20%) were positive, and 36 yielded species identification (Table 1). Antibacterial agents were administered prior to specimen collection in 73% and did not affect likelihood of positive 16S PCR. 18S PCR was sent on 163 samples; 12 (7.4%) were positive (Table 2) of which 10 were from immunocompromised hosts. 40% of patients were on antifungals prior to sample acquisition. 16/18S PCR impacted antimicrobial decision-making in 70 cases (30%). A pathogenic fungus was detected by PCR but not culture in 2 cases. Time to positivity of fungal culture was 1–15 days. Fungal culture was positive in 5 cases with-negative 18S PCR. Seventeen patients had positive serum 1,3-ß-D-glucan and/or galactomannan: 3 of which had positive 18S PCR, 5 with fungal growth, 5 presumed infection based on imaging, 1 Nocardia, and 3 noninfectious etiology. Plasma NGS was sent on 45 cases, was positive in 34, and affected clinical management in 10. Conclusion 16S PCR can identify bacterial pathogens in the setting of negative culture and impact clinical care. Abscess, bronchial/pleural fluid, and brain/organ tissue were high yield specimens. 18S PCR can provide expeditious fungal identification in cases of suspected invasive disease, but fungal culture and serum molecular testing increase diagnostic yield. No single fungal test is comprehensive. Plasma NGS had relatively high yield and clinical impact in selected patients. Disclosures All authors: No reported disclosures.

scholarly journals Toward harmonization of clinical molecular diagnostic reports: findings of an international survey

2018 ◽  
Vol 0 (0) ◽  
Author(s):  
Deborah A. Payne ◽  
Katarina Baluchova ◽  
Graciela Russomando ◽  
Parviz Ahmad-Nejad ◽  
Cyril Mamotte ◽  
...  
Keyword(s):  
Molecular Diagnostics ◽  
Clinical Decision Making ◽  
Clinical Chemistry ◽  
Clinical Decision ◽  
Molecular Diagnostic ◽  
Molecular Testing ◽  
End User ◽  
Iso 15189 ◽  
Reporting Practices ◽  
Reporting Phase

Abstract Background: The International Organization for Standardization (ISO) 15189 standard provides recommendations for the postexamination reporting phase to enhance quality in clinical laboratories. The purpose of this study was to encourage a broad discussion on current reporting practices for molecular diagnostic tests by conducting a global survey of such practices. Methods: The International Federation of Clinical Chemistry and Laboratory Medicine’s Committee for Molecular Diagnostics (IFCC C-MD) surveyed laboratories on selected ISO 15189 recommendations and topics. The survey addressed the following aspects: (1) laboratory demographics, (2) report format, (3) result reporting/layout, (4) comments in report and (5) interpretation and clinical decision-making information. Additionally, participants indicated categories needing standardization. Results: Sixteen responses from laboratories located in Asia, Europe, the Middle East, North America and South America were received. Several categories yielded 100% agreement between laboratories, whereas other categories had less than or equal to 50% concordance. Participants scored “nomenclature” and “description of methodologies” as the two most frequently cited aspects needing standardization. Conclusions: The postexamination phase requires extensive and consistent communication between the laboratory, the healthcare provider and the end user. Surveyed laboratories were most likely to follow explicit ISO 15189 recommendations vs. recommendations when the term(s) “where appropriate or where applicable” was used. Interpretation and reporting of critical values varied among participants. Although the outcome of this study may not fully represent the practices of all molecular testing laboratories in countries around the world, the survey identified and specified several recommendations that are requirements for harmonized reporting in molecular diagnostics.

scholarly journals Barcoding in trypanosomes

Parasitology ◽  
2017 ◽  
Vol 145 (5) ◽  
pp. 563-573 ◽  
Author(s):  
RACHEL HUTCHINSON ◽  
JAMIE R. STEVENS
Keyword(s):  
Genetic Markers ◽  
Molecular Diagnostics ◽  
Potential Application ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Detection And Identification ◽  
Potential Alternative ◽  
Genetic Barcoding ◽  
The Ideal

SUMMARYTrypanosomes (genus Trypanosoma) are parasites of humans, and wild and domestic mammals, in which they cause several economically and socially important diseases, including sleeping sickness in Africa and Chagas disease in the Americas. Despite the development of numerous molecular diagnostics and increasing awareness of the importance of these neglected parasites, there is currently no universal genetic barcoding marker available for trypanosomes. In this review we provide an overview of the methods used for trypanosome detection and identification, discuss the potential application of different barcoding techniques and examine the requirements of the ‘ideal’ trypanosome genetic barcode. In addition, we explore potential alternative genetic markers for barcoding Trypanosoma species, including an analysis of phylogenetically informative nucleotide changes along the length of the 18S rRNA gene.

Identification of Acanthamoeba genotype T4 and Paravahlkampfia sp. from two clinical samples

2008 ◽  
Vol 57 (3) ◽  
pp. 392-396 ◽  
Author(s):  
Soykan Ozkoc ◽  
Sema Tuncay ◽  
Songul Bayram Delibas ◽  
Ciler Akisu ◽  
Zeynep Ozbek ◽  
...  
Keyword(s):  
18S Rrna ◽  
Single Agent ◽  
18S Rrna Gene ◽  
Maximum Temperature ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Free Living ◽  
History Of ◽  
Simplex Virus ◽  
Corneal Trauma

In this study, two free-living amoebae strains, Acanthamoeba genotype T4 and Paravahlkampfia sp., which were isolated from keratitis cases are presented. While the Acanthamoeba strain was isolated as a single agent, the Paravahlkampfia strain was found together with herpes simplex virus. Neither of the patients were contact lens wearers, but they did have a history of minor corneal trauma. Amoebae were detected on non-nutrient agar covered with Escherichia coli. Based on PCR-amplified 18S rRNA-gene analysis the first isolate was identified as Acanthamoeba genotype T4 and the second as Paravahlkampfia sp. In thermotolerance tests, the maximum temperature at which trophozoites continued to divide was determined as 37 °C for this Acanthamoeba strain and 35 °C for the Paravahlkampfia strain. To the best of our knowledge, the Acanthamoeba strain described herein is the second molecularly identified Acanthamoeba strain in an Acanthamoeba keratitis patient in Turkey. However, the Paravahlkampfia isolate is believed to be the first strain that has been isolated from a keratitis patient and has been molecularly differentiated from Vahlkampfia.

scholarly journals Rapid Detection of Babesia motasi Responsible for Human Babesiosis by Cross-priming Amplification combined with a vertical flow

2020 ◽  
Author(s):  
Jinming Wang ◽  
Shandian Gao ◽  
Shangdi Zhang ◽  
Xin He ◽  
Junlong Liu ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Clinical Performance ◽  
Clinical Care ◽  
Epidemiological Studies ◽  
Gansu Province ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Vertical Flow ◽  
Accurate Identification ◽  
Human Babesiosis

Abstract Background: Babesia motasi is known as an etiological agent of human and ovine babesiosis. Diagnosis of babesiosis is traditionally performed by microscopy, examining Giemsa-stained thin peripheral blood smears. Rapid detection and accurate identification of species are desirable for clinical care and epidemiological studies. Methods: An easy to operate molecular method, which requires less capital equipment and incorporates cross priming amplification combined with a vertical flow (CPA-VF) visualization strip for rapid detection and identification of B. motasi. Results: The CPA-VF targets the 18S rRNA gene and has a detection limit of 50 fg per reaction; no cross reaction was observed with other piroplasms infective to sheep or Babesia infective to humans. CPA-VF and real-time (RT)-PCR had sensitivities of 95.2% (95% confidence interval [CI], 78.1-99.4%) and 90.5% (72-97.6%) and specificities of 95.8 (80.5-99.5%) and 97.9 (83.5-99.9%), respectively, versus microscopy and nested (n) PCR combined with gene sequencing. The clinical performance of the CPA-VF assay was evaluated with field blood samples from sheep (n = 240) in Jintai county, Gansu Province, and clinical specimens (n = 492) obtained from patients bitten by ticks. Conclusions: Our results indicate that the CPA-VF is a rapid, accurate, nearly instrument-free molecular diagnostic approach for identification of B. motasi. Therefore, it could be an alternative technique for epidemiological investigations and diagnoses of ovine and/or human babesiosis caused by B. motasi, especially in resource-limited regions.Keywords: Ovine babesiosis, human babesiosis, Babesia motasi, cross priming amplification, vertical flow visualization strip, detection, identification

Immunohistochemistry should undergo robust validation equivalent to that of molecular diagnostics

2015 ◽  
Vol 68 (10) ◽  
pp. 766-770 ◽  
Author(s):  
Kelly Elliott ◽  
Stephen McQuaid ◽  
Manuel Salto-Tellez ◽  
Perry Maxwell
Keyword(s):  
Molecular Diagnostics ◽  
Clinical Care ◽  
Treatment Options ◽  
Subjective Assessment ◽  
Molecular Diagnostic ◽  
Evidence Based ◽  
Uncertainty Of Measurement ◽  
Prognostic Information ◽  
Iso 15189 ◽  
Diagnostic Histopathology

Immunohistochemistry (IHC) is a widely available and highly utilised tool in diagnostic histopathology and is used to guide treatment options as well as provide prognostic information. IHC is subjected to qualitative and subjective assessment, which has been criticised for a lack of stringency, while PCR-based molecular diagnostic validations by comparison are regarded as very rigorous. It is essential that IHC tests are validated through evidence-based procedures. With the move to ISO15189 (2012), not just of the accuracy, specificity and reproducibility of each test need to be determined and managed, but also the degree of uncertainty and the delivery of such tests. The recent update to ISO 15189 (2012) states that it is appropriate to consider the potential uncertainty of measurement of the value obtained in the laboratory and how that may impact on prognostic or predictive thresholds. In order to highlight the problems surrounding IHC validity, we reviewed the measurement of Ki67and p53 in the literature. Both of these biomarkers have been incorporated into clinical care by pathology laboratories worldwide. The variation seen appears excessive even when measuring centrally stained slides from the same cases. We therefore propose in this paper to establish the basis on which IHC laboratories can bring the same level of robust validation seen in the molecular pathology laboratories and the principles applied to all routine IHC tests.

scholarly journals 18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

2003 ◽  
Vol 69 (9) ◽  
pp. 5389-5397 ◽  
Author(s):  
Zhihong Wu ◽  
Yoshihiko Tsumura ◽  
Göran Blomquist ◽  
Xiao-Ru Wang
Keyword(s):  
18S Rrna ◽  
Airborne Fungi ◽  
18S Rrna Gene ◽  
Rrna Genes ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Oligonucleotide Probes ◽  
Gene Variation ◽  
Species Specific ◽  
18S Rrna Genes

ABSTRACT In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring.

Genotypic heterogeneity based on 18S-rRNA gene sequences among Acanthamoeba isolates from clinical samples in Italy

2014 ◽  
Vol 145 ◽  
pp. S46-S49 ◽  
Author(s):  
David Di Cave ◽  
Rossella D' Alfonso ◽  
Kodjo A. Dussey Comlavi ◽  
Carlo D' Orazi ◽  
Rosa Monno ◽  
...  
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Gene Sequences

scholarly journals Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax

Diagnostics ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1950
Author(s):  
Woong Sik Jang ◽  
Da Hye Lim ◽  
YoungLan Choe ◽  
Hyunseul Jee ◽  
Kyung Chul Moon ◽  
...  
Keyword(s):  
Internal Control ◽  
Point Of Care ◽  
Lamp Assay ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Loop Mediated Isothermal Amplification

Malaria, caused by the parasite Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene (P. falciparum), 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.

scholarly journals Rapid Detection of Babesia motasi Responsible for Human Babesiosis by Cross-priming Amplification Combined with a Vertical Flow

2020 ◽  
Author(s):  
Jinming Wang ◽  
Shandian Gao ◽  
Shangdi Zhang ◽  
Xin He ◽  
Junlong Liu ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Clinical Performance ◽  
Clinical Care ◽  
Epidemiological Studies ◽  
Gansu Province ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Vertical Flow ◽  
Accurate Identification ◽  
Human Babesiosis

Abstract Background: Babesia motasi is known as an etiological agent of human and ovine babesiosis. Diagnosis of babesiosis is traditionally performed by microscopy, examining Giemsa-stained thin peripheral blood smears. Rapid detection and accurate identification of species are desirable for clinical care and epidemiological studies. Methods: An easy to operate molecular method, which requires less capital equipment and incorporates cross priming amplification combined with a vertical flow (CPA-VF) visualization strip for rapid detection and identification of B. motasi. Results: The CPA-VF targets the 18S rRNA gene and has a detection limit of 50 fg per reaction; no cross reaction was observed with other piroplasms infective to sheep or Babesia infective to humans. CPA-VF and real-time (RT)-PCR had sensitivities of 95.2% (95% confidence interval [CI], 78.1-99.4%) and 90.5% (72-97.6%) and specificities of 95.8 (80.5-99.5%) and 97.9 (83.5-99.9%), respectively, versus microscopy and nested (n) PCR combined with gene sequencing. The clinical performance of the CPA-VF assay was evaluated with field blood samples from sheep (n = 340) in Jintai county, Gansu Province, and clinical specimens (n = 492) obtained from patients bitten by ticks. Conclusions: Our results indicate that the CPA-VF is a rapid, accurate, nearly instrument-free molecular diagnostic approach for identification of B. motasi. Therefore, it could be an alternative technique for epidemiological investigations and diagnoses of ovine and/or human babesiosis caused by B. motasi, especially in resource-limited regions.

scholarly journals Rapid Detection of Babesia motasi Responsible for Human Babesiosis by Cross-priming Amplification Combined with a Vertical Flow

2020 ◽  
Author(s):  
Jinming Wang ◽  
Shandian Gao ◽  
Shangdi Zhang ◽  
Xin He ◽  
Junlong Liu ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Clinical Performance ◽  
Clinical Care ◽  
Epidemiological Studies ◽  
Gansu Province ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Vertical Flow ◽  
Accurate Identification ◽  
Human Babesiosis

Abstract Background Babesia motasi is known as an etiological agent of human and ovine babesiosis. Diagnosis of babesiosis is traditionally performed by microscopy, examining Giemsa-stained thin peripheral blood smears. Rapid detection and accurate identification of species are desirable for clinical care and epidemiological studies. Methods An easy to operate molecular method, which requires less capital equipment and incorporates cross priming amplification combined with a vertical flow (CPA-VF) visualization strip for rapid detection and identification of B. motasi. Results The CPA-VF targets the 18S rRNA gene and has a detection limit of 50 fg per reaction; no cross reaction was observed with other piroplasms infective to sheep or Babesia infective to humans. CPA-VF and real-time (RT)-PCR had sensitivities of 95.2% (95% confidence interval [CI], 78.1-99.4%) and 90.5% (72-97.6%) and specificities of 95.8 (80.5-99.5%) and 97.9 (83.5-99.9%), respectively, versus microscopy and nested (n) PCR combined with gene sequencing. The clinical performance of the CPA-VF assay was evaluated with field blood samples from sheep (n = 240) in Jintai county, Gansu Province, and clinical specimens (n = 492) obtained from patients bitten by ticks. Conclusions Our results indicate that the CPA-VF is a rapid, accurate, nearly instrument-free molecular diagnostic approach for identification of B. motasi. Therefore, it could be an alternative technique for epidemiological investigations and diagnoses of ovine and/or human babesiosis caused by B. motasi, especially in resource-limited regions.

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