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

Performance of the ImmuView and BinaxNOW assays for the detection of urine and cerebrospinal fluid Streptococcus pneumoniae and Legionella pneumophila serogroup 1 antigen in patients with Legionnaires' disease or pneumococcal pneumonia and meningitis.

The performances of the ImmuView Streptococcus pneumoniae (Sp) and Legionella pneumophila (Lp) urinary antigen test were compared to that of the BinaxNOW Sp and Lp assays, using frozen urine from 166 patients with Legionnaires' disease (LD) and 59 patients with pneumococcal pneumonia. Thirty Sp-positive or contrived cerebrospinal fluids (CSF) were also tested. Test specimens were collected and tested at different sites, with each site testing unique specimens by technologists blinded to expected results. No significant differences in test concordances were detected for the ImmuView and BinaxNOW assays for the Sp or Lp targets for urine from patients with pneumococcal pneumonia or LD when performance from both sites were combined. At one of two test sites the ImmuView Lp assay was more sensitive than the BinaxNOW assay, with no correlation between test performance and Lp serogroup 1 monoclonal type. Urines from six of seven patients with LD caused by Legionella spp. bacteria other than Lp serogroup 1 were negative in both assays. Both tests had equivalent performance for Sp-positive CSF. The clinical sensitivities for pneumococcal pneumonia were 88.1 and 94.4% for the ImmuView and Binax assays, and 87.6 and 84.2% for the Lp assays, respectively. Test specificities for pneumococcal pneumonia were 96.2 and 97.0% for the ImmuView and Binax assays, and 99.6 and 99.1% for the Lp assays. Both assays were highly specific for Sp in pediatric urines from children with nasopharyngeal colonization by the bacterium. ImmuView and BinaxNOW assay performance was equivalent in these studies

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

Three-Year Trend in Antimicrobial Resistance and Genotypes among Salmonella in Swine and Humans

The aim of this study was to determine antimicrobial resistance among Salmonella isolated from swine and humans in North Carolina, compare genotypes among isolates from humans originated from pig-producing areas and characterize important genes. Resistance to 9 and 11 of the 12 antimicrobial agents tested was detected among isolates from swine and humans respectively. Frequency of resistance to tetracycline and b-lactams was significantly higher among isolates from swine than humans (p \u3c0.05). Two common multi-drug resistance (MDR) patterns were found among isolates from apparently healthy swine: AmKmStSuTe and AmCmStSuTe. However, the former MDR pattern was rare among clinical isolates. Genotyping revealed that two predominant genotypes, one composed of clinical isolates and the other non-clinical were noticed. Further characterization using Salmonella plasmid virulence; spvA gene also revealed that this gene is absent among the most common MDR pattern, AmKmStSuTe, in swine

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.