scholarly journals Establishment of a New PNA-FISH Method for Aspergillus fumigatus Identification: First Insights for Future Use in Pulmonary Samples

2020 ◽  
Vol 8 (12) ◽  
pp. 1950
Author(s):  
Laura Cerqueira ◽  
Sara Moura ◽  
Carina Almeida ◽  
Maria João Vieira ◽  
Nuno Filipe Azevedo
Keyword(s):  
Aspergillus Fumigatus ◽  
False Negative ◽  
Clinical Samples ◽  
Suitable Alternative ◽  
Fish Method ◽  
Negative Results ◽  
Specificity And Sensitivity ◽  
Pure Cultures ◽  
Low Sensitivity ◽  
Pna Fish

Aspergillus fumigatus is the main causative agent of Invasive Aspergillosis. This mold produces conidia that when inhaled by immunocompromized hosts can be deposited in the lungs and germinate, triggering disease. In this paper, the development of a method using peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) is described. The PNA-FISH probe was tested in several strains and a specificity and sensitivity of 100% was obtained. Detection of A. fumigatussensu stricto was then achieved in artificial sputum medium (ASM) pre-inoculated with 1 × 102 conidia·mL−1–1 × 103 conidia·mL−1, after a germination step of 24 h. The PNA-FISH method was evaluated in 24 clinical samples (10 sputum, 8 bronchoalveolar lavage (BAL), and 6 bronchial lavage (BL)) that were inoculated with 1 × 104 conidia·mL−1 in sputum; 1 × 103 conidia·mL−1 in BL and BAL, for 24 h. Despite a specificity of 100%, the sensitivity was 79%. This relatively low sensitivity can be explained by the fact that hyphae can yield “fungal ball“ clusters, hindering pipetting procedures and subsequent detection, leading to false negative results. Nonetheless, this study showed the potential of the PNA-FISH method for A. fumigatussensu stricto detection since it takes only 1 h 30 m to perform the procedure with a pre-enrichment step of 6 h (pure cultures) and 24 h (clinical samples), and might provide a suitable alternative to the existing methods for studies in pure cultures and in clinical settings.

scholarly journals Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

2012 ◽  
Vol 19 (8) ◽  
pp. 1193-1198 ◽  
Author(s):  
Vijai Pal ◽  
Subodh Kumar ◽  
Praveen Malik ◽  
Ganga Prasad Rai
Keyword(s):  
Sensitivity And Specificity ◽  
Recombinant Proteins ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Burkholderia Mallei ◽  
Content Type ◽  
Whole Cells ◽  
Negative Results ◽  
Elisa Format ◽  
Low Sensitivity

ABSTRACTGlanders is a contagious disease caused by the Gram-negative bacillusBurkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins ofB. malleiwere used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

scholarly journals UTILITY OF ANTIGEN DETECTION TEST AND POLYMERASE CHAIN REACTION IN THE DIFFERENTIATION OF TUBERCULOUS AND NON-TUBERCULOUS MYCOBACTERIA

2017 ◽  
Vol 10 (6) ◽  
pp. 289
Author(s):  
Yogita Singh ◽  
Raji Vasanth ◽  
Shrikala Baliga ◽  
Dhanashree B
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Diagnostic Methods ◽  
Clinical Samples ◽  
Chain Reaction ◽  
College Hospital ◽  
Medical College ◽  
Negative Results ◽  
Polymerase Chain ◽  
Mycobacterium Spp

Objectives: Cultivation and identification of mycobacteria to species level remains difficult and time-consuming. Hence, easy and rapid diagnostic methods are necessary for the differentiation of Mycobacterium tuberculosis (MTB) from non-tuberculous mycobacteria (NTM). The present study aims to detect and differentiate MTB from NTM isolated from clinical samples by immunochromatographic test (ICT) and polymerase chain reaction (PCR). Methods: Over a period of 1 year, clinical samples (n=496) received from suspected cases of TB, at the Department of Microbiology, Kasturba Medical College Hospital, Mangalore were cultured to isolate Mycobacterium spp. Identification of all the isolates was done by conventional biochemical technique, ICT, and PCR. Results: Among the 496 samples processed, 49 (9.87%) were acid-fast bacilli smear positive and 59 (11.89%) samples showed the growth of Mycobacterium spp. Among these, 10 were rapid growers, 49 were slow-growing mycobacteria, out of which 30 were MTB as identified by conventional biochemical reaction. Out of 59 Mycobacterial isolates subjected to ICT for the detection of MPT 64 antigen, only 28 were identified as MTB. However, all the 30 isolates were correctly identified as MTB by PCR. Conclusion: Hence, PCR is essential for rapid differentiation of non-tuberculous Mycobacterium from MTB. False negative results seen with immunochromatographic MPT 64 antigen assay could be due to mutations within the mpt64 gene. Further studies are necessary to characterize these PCR-positive and immunochromatographic assay negative MTB isolates.

scholarly journals Detection of LytA Genes in Streptococcus pneumoniae Isolated from sputum pneumonia patients

Biomedika ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 23-30
Author(s):  
Mustika Sari Hutabarat ◽  
Firdaus Hamid ◽  
Irawaty Djaharuddin ◽  
Alfian Zainuddin ◽  
Rossana Agus ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
False Negative ◽  
Pneumococcal Infection ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Streptococcus pneumoniae (pneumococcus) is a Gram-positive facultative anaerobic bacterium that is a major cause of morbidity and mortality worldwide. But the lack of reporting of disease by this bacterium in Indonesia, one of the causes is because the diagnosis of pneumococcal infection is often clinically not typical and conventional methods which are still the standard gold method often give false-negative results. So the purpose of this study was to evaluate the performance of culture and molecular diagnostic methods using the Polymerase Chain Reaction (PCR) technique in detecting Streptococcus pneumoniae in sputum clinical samples using the Autolysin (LytA) gene which is a virulence factor of this bacterium. 57 isolates from 60 samples were confirmed as Streptococcus sp through microscopic identification, culture, and biochemical tests. Then the sensitivity test with an optochin test of 9 (9%) compared the results descriptively with the PCR technique using the Autolysin A (LytA) gene which was obtained more sensitive by 15 (25%).

scholarly journals Low Sensitivity of NS1 Protein Tests Evidenced during a Dengue Type 2 Virus Outbreak in Santos, Brazil, in 2010

2012 ◽  
Vol 19 (12) ◽  
pp. 1972-1976 ◽  
Author(s):  
Alvina Clara Felix ◽  
Camila Malta Romano ◽  
Cristiane de Campos Centrone ◽  
Célia Lima Rodrigues ◽  
Lucy Villas-Boas ◽  
...  
Keyword(s):  
Secondary Infection ◽  
False Negative ◽  
Ns1 Protein ◽  
Negative Results ◽  
False Negative Results ◽  
Large Outbreak ◽  
Ns1 Antigen ◽  
Low Sensitivity ◽  
Dengue Type 2 Virus

ABSTRACTIn 2010, a large outbreak of dengue occurred in Santos, Brazil. The detection of the NS1 antigen was used for diagnosis in addition to the detection of IgG, IgM, and RNA. A large number of NS1 false-negative results were obtained. A total of 379 RNA-positive samples were selected for thorough evaluation. NS1 was reactive in 37.7% of cases. Most of the cases were characterized as a secondary infection by dengue 2 virus. Sequencing of NS1 positive and negative isolates did not reveal any mutation that could justify the diagnostic failure. Use of existing NS1 tests in the Brazilian population may present a low negative predictive value, and they should be used with caution, preferentially after performing a validation with samples freshly obtained during the ongoing epidemic.

scholarly journals The Use of a Mimic to Detect Polymerase Chain Reaction– Inhibitory Factors in Feces Examined for the Presence of Lawsonia Intracellularis

2003 ◽  
Vol 15 (3) ◽  
pp. 268-273 ◽  
Author(s):  
Magdalena Jacobson ◽  
Stina Englund ◽  
András Ballagi-Pordány
Keyword(s):  
Polymerase Chain Reaction ◽  
False Negative ◽  
Clinical Samples ◽  
Lawsonia Intracellularis ◽  
Chain Reaction ◽  
Fecal Samples ◽  
Wild Boars ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Lawsonia intracellularis is an intracellular organism that causes proliferative enteritis in pigs. This bacterium is difficult to culture, and antemortem demonstration of the microbe is therefore often performed on fecal samples by polymerase chain reaction (PCR). Polymerase chain reaction is sensitive and specific, but inhibitory factors in feces might cause false-negative results. This article describes the construction and use of an internal standard, a mimic. The mimic is amplified by the same primers as those used for L. intracellularis DNA and thus could indicate false-negative results in clinical samples. The amplicon was clearly visible when as few as 10 mimic molecules were added per amplification reaction and when no inhibitors were present. When fecal samples were spiked with the mimic, the detection limit was 102 molecules per PCR. Sixty clinical samples, 20 from wild boars, 20 from growing pigs with diarrhea, and 20 from pigs without diarrhea, were prepared by a boiling procedure and subjected to PCR together with 103 mimic molecules. Nine samples were positive, of which 7 originated from pigs with diarrhea and 2 from pigs without diarrhea. In 14 samples from wild boars, in 8 samples from pigs without diarrhea, and in 3 samples from pigs with diarrhea, neither the mimic nor the target DNA was visible. This indicated the presence of inhibitors in these samples. It is concluded that the mimic can be used as an internal control in the diagnosis of L. intracellularis to indicate inhibition of PCR.

scholarly journals SARS-CoV-2 RNA detection using pooling of self-collected samples: Simple protocol may foster asymptomatic surveillance

2020 ◽  
Author(s):  
Giselle Ibette Lopez-Lopes ◽  
Rita de Cassia Compagnoli Carmona ◽  
Valéria Oliveira Silva ◽  
Cintia Mayumi Ahagon ◽  
Lincoln Spinazola do Prado ◽  
...  
Keyword(s):  
False Negative ◽  
Clinical Samples ◽  
Rna Detection ◽  
Negative Results ◽  
False Negative Results ◽  
Throat Wash ◽  
Asymptomatic Volunteers ◽  
Simple Protocol ◽  
Antigen Tests ◽  
Public Health Institute

1AbstractBackgroundSurveillance of COVID infection and isolation of infected individuals is one of the available tools to control the spread of SAR-CoV-2. Asymptomatic and pre symptomatic are responsible for substantial transmission. RNA or antigen tests are necessary to identify non-symptomatic individuals. We tested the feasibility of using samples pooling offering different collection alternatives (swab/throat wash/saliva) to volunteers of a public health institute.MethodsWe evaluated pool samples from frozen material from previously tested samples and a prospective collection from asymptomatic volunteers. Some collections were paired for comparison. Pools and some individual samples were extracted with QIAamp Viral RNA Mini Kit (Qiagen, USA) and/or Lucigen Quick Extract DNA extraction solution (BioSearch, USA) and submitted to rtPCR (Allplex, Seegene, Korea).ResultsA total of 240 samples from 130 new collections and 37 samples with known result were evaluated. Pool CT was generally higher than individual samples. Lucigen extraction showed higher CT, including false negative results for samples with high CT at Qiagen extraction. Paired Swab and TW samples showed comparable results. No volunteer from negative pools reported any symptom in the 2-3 days after collection.ConclusionsClinical samples pooling to detect SARS-CoV-2 RNA is feasible and an economical way to test for COVID-19, especially in surveillance strategies targeting more infectiousness, higher viremia individuals. The use of Lucigen reagents show lower sensibility that may lead to false negative results with lower viremia samples. Combining throat wash with saliva may provide and interesting self-collection alternative, but more comparative work is needed.

scholarly journals Impact of false negative results of RT-PCR test for SARS-CoV-2 in COVID-19 case count as applied to Mexico

2020 ◽  
Author(s):  
Isaac J. Núñez ◽  
Pablo F. Belaunzarán-Zamudio ◽  
Yanink Caro-Vega
Keyword(s):  
False Negative ◽  
Open Data ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Negative Results ◽  
Case Count ◽  
False Negative Results ◽  
True Number ◽  
High Prevalence ◽  
Polymerase Chain

Underestimation of the number of cases during the COVID-19 pandemic has been a constant concern worldwide. Case confirmation is based on identification of SARS-CoV-2 RNA using real time polymerase chain reaction (RT-PCR) in clinical samples. However, these tests have suboptimal sensitivity, especially during the early and late course of infection. Using open data, we estimated that among 1 343 730 people tested in Mexico since February 27th, there were 838 377 (95% CL 734 605 - 1 057 164) cases, compared with 604 376 considering only positive tests. ICU admissions and deaths were around 16% and 9% higher than reported. Thus, we show that accounting for the sensitivity of SARS-Cov-2 RT-PCR diagnostic tests is a simple way to improve estimations for the true number of COVID-19 cases in tested people, particularly in high-prevalence populations. This could aid to better inform public health measures and reopening policies.

scholarly journals Avoiding COVID-19 Hospital Outbreaks: RT-PCR Swab and Clinical Assessment

2021 ◽  
Author(s):  
Luca Allievi ◽  
Amedeo Bongarzoni ◽  
Guido Tassinario ◽  
Stefano Carugo
Keyword(s):  
Clinical Assessment ◽  
False Negative ◽  
False Negative Rate ◽  
High Specificity ◽  
Diagnostic Process ◽  
Rt Pcr ◽  
Negative Results ◽  
High False Negative Rate ◽  
Degree Of Confidence ◽  
Low Sensitivity

Nasopharyngeal RT-PCR swab test for COVID-19 diagnosis has a high specificity but also a low sensitivity. The high false-negative rate and the overconfidence in negative results sometimes lead to hospital outbreaks. Therefore, we recommend always integrating the clinical assessment in the diagnostic process, mostly after the test, to determine what degree of confidence can be attributed to a negative result.

scholarly journals Measuring Precarious Employment: Type of Contract Can Lead to Serious Misclassification Error

2020 ◽  
Vol 64 (9) ◽  
pp. 1035-1038 ◽  
Author(s):  
Alejandra Vives ◽  
Francisca Gonzalez Lopez ◽  
Joan Benach
Keyword(s):  
False Negative ◽  
Temporary Employment ◽  
Misclassification Error ◽  
Precarious Employment ◽  
The Public ◽  
Negative Results ◽  
False Negative Results ◽  
Proxy Measure ◽  
Health Relevance ◽  
Low Sensitivity

Abstract This study aims to assess the accuracy of temporary employment as indicator or proxy measure of precarious employment. Using sensitivity and specificity analysis, we compared type of contract (temporary versus permanent) with the Chilean version of the multidimensional Employment Precariousness Scale. Temporary employment exhibited very low sensitivity (<30%) (specificity >90%), resulting in roughly 38% of false negative results. Different EPRES-Ch cut-off scores produced similar results. The main implication of these findings is that the public health relevance of precarious employment is being underestimated both in terms of prevalence and of its association with health, making it critical that valid multidimensional measures of precarious employment be implemented.

Monochrome LightCycler PCR assay for detection and quantification of five common species of Candida and Aspergillus

2005 ◽  
Vol 54 (3) ◽  
pp. 243-248 ◽  
Author(s):  
Rong Bu ◽  
Rajeev K Sathiapalan ◽  
Muna M Ibrahim ◽  
Ibrahim Al-Mohsen ◽  
Edna Almodavar ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Fungal Pathogens ◽  
Low Cost ◽  
False Negative ◽  
Common Species ◽  
Fungal Species ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Negative Results ◽  
Identification And Quantification

Invasive fungal pathogens, especially in immunocompromised hosts, can result in life-threatening infections. Current laboratory/radiological methods for fungal identification are time-consuming and lack sensitivity and specificity. A monochrome, multiplex, real-time PCR assay for the identification and quantification of Candida albicans, Candida krusei, Candida tropicalis, Aspergillus flavus and Aspergillus fumigatus is described here. Detection of each of these fungi was specific and demonstrated 100 % concordance with biochemical/culture identification in all 60 isolates tested. Samples from 16 febrile neutropenic patients with haematological malignancies were also analysed and the utility of the assay in clinical samples was reconfirmed without false-negative results. The sensitivity of this assay was 0.1 pg fungal genomic DNA, corresponding to three cells, for C. albicans, C. krusei, C. tropicalis and A. flavus, and 0.01 pg fungal genomic DNA, i.e. less than one cell, for A. fumigatus. The analysis allows a low-cost, simple, rapid and sensitive alternative for clinical identification and quantification of these five common fungal species.

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