Development of a Real-time PCR assay for Pneumocystis jirovecii on the Luminex ARIES® Platform

Pneumocystis pneumonia (PCP) is an opportunistic infection caused by the fungus Pneumocystis jirovecii. Infection with P. jirovecii can result in serious illness in patients with a weakened immune system, and can lead to death if it is not properly diagnosed and treated. Direct detection of P. jirovecii in lower respiratory tract specimens such as bronchoalveolar lavage (BAL) is preferred for rapid diagnosis, a laboratory service currently not available locally. We report here the development of a diagnostic real-time Polymerase Chain Reaction (PCR) assay using BAL specimens to detect P. jirovecii. By targeting the multi-copy mitochondrial large subunit ribosomal RNA gene (mtLSU rRNA) of P. jirovecii, assay sensitivity is increased. Primer pairs were designed to include a fluorescent reporter dye-labeled primer with a unique MultiCode® base pair isoC on the 5’end and one unlabeled primer. The performance characteristics were determined on the Luminex ARIES® instrument, combining DNA extraction, amplification and detection into a one-step process. The cassette contains the reagents needed to perform all of the steps including extraction, purification, amplification, and detection, plus a sample processing control. Accuracy, precision, sensitivity, specificity and stability studies were conducted to validate the assay to meet CLIA requirements. The analytical sensitivity was 89.1%, and the analytical specificity was 100%. The assay could reliably detect 200 organisms/mL, crossing thresholds (Ct) and melt temperatures (Tm) were consistent, and no cross-reactivity was observed with other pathogens known to cause respiratory infections. The results demonstrated that these primers are specific to Pneumocystis jirovecii. The real-time PCR method using the ARIES® system allowed for rapid and sensitive detection of Pneumocystis pneumonia infections with P. jirovecii using clinical respiratory specimens

Real-Time PCR Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila in Respiratory Specimens Using the ARIES® System

Background: Mycoplasma pneumoniae (Mpn), Chlamydia pneumoniae (Cpn), and Legionella pneumophila (Lpn) can cause both epidemic and endemic occurrences of acute respiratory disease and are responsible for up to 22% of cases of community acquired pneumonia. Due to the limited availability of FDA-approved molecular diagnostic assays, we developed and evaluated a multiplexed Real-time PCR assay for the detection of these agents in two respiratory specimen types on the Luminex ARIES® instrument. The instrument provides for nucleic acid extraction plus PCR amplification and target detection in the same cassette. The ARIES® instrument generates a cycle threshold value and a confirmatory melt curve value for each reaction, including results for an internal sample processing control. The limit of detection for Mpn, Cpn and Lpn, was 100 CFU/mL, 1000 CFU/mL and 100 CFU/mL, respectively. In addition, accuracy, precision, specificity and stability studies were conducted to validate the assay for diagnostic use. Between November 2016 and June 2017, a total of 836 patient specimens were processed in our reference laboratory, with six positive Mpn and two positive Lpn. No specimens were positive for Cpn during this time period. The availability of a robust multiplex PCR assay greatly enhances the ability to rapidly diagnose infections caused by these three agents causing atypical pneumonia

Detection Of Insider Attacks In Block Chain Network Using The Trusted Two Way Intrusion Detection System

For data privacy, system reliability, and security, Blockchain technologies have become more popular in recent years. Despite its usefulness, the blockchain is vulnerable to cyber assaults; for example, in January 2019 a 51% attack on Ethereum Classic successfully exposed flaws in the platform's security. From a statistical point of view, attacks represent a highly unusual occurrence that deviates significantly from the norm. Blockchain attack detection may benefit from Deep Learning, a field of study whose aim is to discover insights, patterns, and anomalies within massive data repositories. In this work, we define an trusted two way intrusion detection system based on a Hierarchical weighed fuzzy algorithm and self-organized stacked network (SOSN) deep learning model, that is trained exploiting aggregate information extracted by monitoring blockchain activities. Here initially the smart contract handles the node authentication. The purpose of authenticating the node is to ensure that only specific nodes can submit and retrieve the information. We implement Hierarchical weighed fuzzy algorithm to evaluate the trust ability of the transaction nodes. Then the transaction verification step ensures that all malicious transactions or activities on the submitted transaction by self-organized stacked network deep learning model. The whole experimentation was carried out under matlab environment. Extensive experimental results confirm that our suggested detection method has better performance over important indicators such as Precision, Recall, F-Score, overhead

Preliminary Evaluation of an lytA PCR Assay for Detection of Streptococcus pneumoniae in Urine Specimens from Hospitalized Patients with Community-Acquired Pneumonia

Community acquired pneumonia (CAP) due to Streptococcus pneumoniae still occurs in at risk populations, despite the availability of effective vaccines. Laboratory confirmation of S. pneumoniae remains challenging in cases of CAP despite advances in blood culture techniques and the availability of nucleic acid amplification tests such as PCR-based methods. Urine specimens are an attractive sample type because they are non-invasive compared to bronchial washes or whole blood specimens for patients with CAP. While urine specimens have been used successfully in antigen detection assays, they have not been extensively evaluated for PCR-based assays. In this preliminary study, we evaluated the potential for a real-time PCR assay targeting the S. pneumoniae autolysin gene (lytA) to detect in archived urine samples from patients with CAP. Results indicate that the real time lytA PCR assay on the Luminex ARIES® system shows promise as a screening tool for patients with CAP based on comparison to urine antigen detection assay results

Impact of Pooling Samples on Analytic Sensitivity of a Real-Time Reverse Transcriptase PCR Assay for SARS CoV-2

During the COVID-19 pandemic, laboratories experienced periods of shortages for certain critical materials required to meet the high demand for SARS-CoV-2 testing. The U.S. Food & Drug Administration provided a template for molecular diagnostic testing, including guidance for a specimen pooling process in order to evaluate performance of the SARS-CoV-2 nucleic acid amplification assay. This study aimed to evaluate the testing of pooled specimens consisting of four nasopharyngeal swab specimens using the Luminex ARIES® nucleic acid amplification platform. Results indicated that there was a loss of analytic sensitivity with pooled nasopharyngeal swab samples, demonstrating that this approach should be balanced against material shortages and the clinical utility of a less sensitive assay

Association of Urine Levels of C-Reactive Protein with Clinical Outcomes in Patients with Pneumonia: A Pilot Study

Finding relevant biomarkers as a potential predictor of severity for patients hospitalized with community acquired pneumonia (CAP), in addition to the clinical scoring system, could advance progress towards more effective patient management. The inflammatory marker, C-reactive protein (CRP), which is elevated in the pathogenesis of many infectious diseases, may be a key biomarker target for CAP. Previous studies have shown that serum CRP may be a useful diagnostic marker for pneumonia in hospitalized patients with acute respiratory symptoms. The main aims of this study were to determine the correlation between serum and urine CRP levels in hospitalized patients with CAP, and any correlation with patient outcomes. Our laboratory employed a commercially available human high sensitive CRP ELISA kit to check the level of CRP in the corresponding patient urine sample. The results showed that there was a positive correlation between patient serum and urine CRP levels. In addition, we showed the correlation of urine CRP levels with certain patient comorbidities, time to clinical stability, length of patient hospital stay, and mortality

Development of a real-time Reverse-Transcription PCR for SARS CoV-2 on the Luminex ARIES® Platform

The University of Louisville Infectious Diseases Laboratory followed the US Food and Drug Administration (FDA) Emergency Use Authorization (EUA) guidance for developing a molecular diagnostic test for SARS CoV-2 to help address the novel coronavirus pandemic. As a Clinical Laboratory Improvement Amendment ‘88 (CLIA) certified, high-complexity clinical laboratory, the Infectious Diseases Laboratory chose to use the Luminex ARIES® platform to evaluate a laboratory developed test. This instrument was already familiar to the Infectious Diseases Laboratory and in use for molecular diagnostic testing for pathogens causing atypical pneumonia and two tick-borne pathogens. The FDA EUA guidance for molecular diagnostic tests recommended limit of detection studies, inclusivity and exclusivity (specificity) analysis, and validation with clinical samples to ensure the performance of the assay was acceptable for use as a molecular diagnostic tool. Data obtained from these experiments demonstrated acceptable performance per FDA guidance, as well as for CLIA requirements. Thus, the real-time Reverse Transcription PCR assay was implemented for diagnostic use on March 27, 2020 and was a great benefit to the local community in responding to the pandemic.

Performance of a SARS-CoV-2 RT-PCR Assay with Non-Traditional Specimen Types

During the first two years of the coronavirus disease 2019 (COVID-19) pandemic, nasopharyngeal (NP) specimens were the gold standard for clinical diagnostic testing. As information about the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the pandemic continued to be shared, it was clear that the virus could be detected in other specimen types during an active infection. The University of Louisville Infectious Diseases Laboratory accepted non-traditional specimen types, most without a paired, positive NP result, for research purposes only to support local epidemiology efforts. A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay originally validated for NP specimens was used for non-traditional specimen types using a variety of specimen preparation methods. Limit of detection (LOD) studies allowed for direct comparison between NP, sputum, and breast milk specimen types. The primary aim of the study was to determine whether SARS-CoV-2 RNA could be detected in different human specimen types. The results showed that the non-traditional specimens were not inherently inhibitory since SARS-CoV-2 RNA was detected in 36 (14.5%) out of 249 non-traditional specimens, and the limit of detection for SARS-CoV-2 in breast milk and sputum was the same as for NP specimens. SARS-CoV-2 was not detected in 15 breast milk specimens from mothers with positive SARS-CoV-2 NP results. In addition, a direct comparison study showed that NP specimens performed better than paired nasal specimens. In conclusion, by analyzing real-time RT-PCR test results for these non-traditional specimen types, two benefits were realized. Health care providers gained additional epidemiologic information (since information was not to be used for managing or treating patients), and the laboratory gathered important information about specimen types for which complete method validation studies could be pursued in the future