Performance of real-time PCR and immunofluorescence assay for diagnosis of Pneumocystis pneumonia in real-world clinical practice

Background PCR is more sensitive than immunofluorescence assay (IFA) for detection of Pneumocystis jirovecii. However, PCR cannot always distinguish infection from colonization. This study aimed to compare the performance of real-time PCR and IFA for diagnosis of P. jirovecii pneumonia (PJP) in a real-world clinical setting. Methods A retrospective cohort study was conducted at a 1,300-bed hospital between April 2017 and December 2018. Patients whose respiratory sample (bronchoalveolar lavage or sputum) were tested by both Pneumocystis PCR and IFA were included. Diagnosis of PJP was classified based on multicomponent criteria. Sensitivity, specificity, 95% confidence intervals (CI), and Cohen's kappa coefficient were calculated. Results There were 222 eligible patients. The sensitivity and specificity of PCR was 91.9% (95% CI, 84.0%–96.7%) and 89.7% (95% CI, 83.3%–94.3%), respectively. The sensitivity and specificity of IFA was 7.0% (95% CI, 2.6%–14.6%) and 99.2% (95% CI, 95.6%–100.0%), respectively. The percent agreement between PCR and IFA was 56.7% (Cohen's kappa -0.02). Among discordant PCR-positive and IFA-negative samples, 78% were collected after PJP treatment. Clinical management would have changed in 14% of patients using diagnostic information, mainly based on PCR results. Conclusions PCR is highly sensitive compared with IFA for detection of PJP. Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.

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Development and Validation of aPneumocystis jiroveciiReal-time Polymerase Chain Reaction Assay for Diagnosis ofPneumocystisPneumonia

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2015/138787 ◽

2015 ◽

Vol 26 (5) ◽

pp. 263-267 ◽

Cited By ~ 8

Author(s):

Deirdre L Church ◽

Anshula Ambasta ◽

Amanda Wilmer ◽

Holly Williscroft ◽

Gordon Ritchie ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Gold Standard ◽

Pathogenic Fungus ◽

Immunofluorescence Assay ◽

Immunocompromised Patients ◽

Chain Reaction ◽

Pcr Assay ◽

Predictive Values ◽

Sensitivity Specificity

BACKGROUND:Pneumocystis jirovecii(PJ), a pathogenic fungus, causes severe interstitialPneumocystispneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients.

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744. Clinical Performance of Real-time PCR in the Diagnosis of Pneumocystis jirovecii Pneumonia compared with Immunofluorescence Assay

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.934 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S420-S420

Author(s):

Cristina Veintimilla ◽

Ana Alvarez-Uria ◽

Pablo Martin-Rabadan ◽

Luis Alcala ◽

Patricia Muñoz ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Subunit ◽

Immunofluorescence Assay ◽

Pneumocystis Jirovecii ◽

Pneumocystis Jirovecii Pneumonia ◽

Rrna Gene ◽

Sample Type ◽

Sensitivity Specificity ◽

Pcr Targeting

Abstract Background The laboratory diagnosis of Pneumocystis jirovecii pneumonia (PJP) has been traditionally based on microscopy techniques, which have suboptimal sensitivity and depends on the experience and skills of the microbiologist. Molecular detection assays based in PCR (Polymerase chain reaction) could improve sensitivity. Our aim was to evaluate the utility of real-time PCR in the diagnosis of PJP compared with IFA (Immunofluorescence assay) performed in different respiratory samples of patients with PJP suspicion for routine use in a clinical laboratory setting. Methods From September 2015 to April 2018, we studied by a real-time PCR targeting the large subunit of rRNA gene of P. jirovecii (PJ-PCR RealCycler PJIR kit Progenie Molecular) and Immunofluorescence assay (MONOFLUO P. carinii IFA BioRad) in all respiratory samples received for microbiological diagnosis of PJP. The definite clinical diagnosis of PJP was established by infectious disease physicians considering symptoms, radiological and laboratory findings. Results Overall, 302 samples were included (182 bronchoalveolar lavage, 67 sputum, 53 tracheal aspirates). PJ-PCR was positive in 51 (16.9%) and IFA in 11 (3.6%) of the patients with PJP. There were not IFA positive/PCR negative samples. Sensitivity, specificity, PPV and NPV for IFA were 26% (95%CI 15.9-39.6%), 100% (95%CI 98.5-100%), 100% (95% CI 77.2-100%) and 87.2% (95% CI 82.6-90.6%). Whereas, sensitivity, specificity, PPV and NPV for PCR was 92% (95%CI 81.2-96.8%), 98% (95% CI 95.4-99.2%), 90.2% (95% CI 79.0-95.7%) and 98.4% (95% CI 96.0-99.4%). PJ-PCR had sensitivity > 80% and specificity > 90% in all type of samples included. A definitive diagnosis of PJP was considered in 50 (16.6%) patients, including 4 (1.3%) cases with negative PJ-PCR. Five cases (9.8%) with positive PJ-PCR were considered as colonization. Conclusion P. jirovecii PCR improves the sensitivity and NPV of PJP diagnosis respecting to IFA, regardless of respiratory sample type. Our results suggest that Microbiology laboratories should use PCR techniques to diagnose PJP better than IFA. Disclosures All Authors: No reported disclosures

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Deep learning algorithm performance compared to experts in visual evaluation of interior vena cava collapse on ultrasound to determine intravenous ﬂuid need in dehydration management

10.22514/sv.2021.128 ◽

2021 ◽

Keyword(s):

Deep Learning ◽

Real Time ◽

Real World ◽

Pediatric Patients ◽

Vena Cava ◽

Learning Curves ◽

Intravenous Fluid ◽

Substantial Agreement ◽

Cohen’S Kappa ◽

Cohen's Kappa

Objectives: To create a deep learning (DL) algorithm capable of analyzing real time ultrasound video of the inferior vena cava (IVC) for complete collapse in pediatric patients being evaluated for intravenous fluid (IVF) resuscitation. Methods: Researchers employed a VGG-16 based DL architecture, running inside a Long Short Term Memory algorithm design, to analyze prospectively obtained ultrasound video from pediatric patients presenting with dehydration to a busy urban ED, obtained for a prior clinical study. All videos were de-identified and no patient information was available. A total of 184 patient IVC ultrasound videos were used in the study. All videos were previously reviewed and graded by two blinded POCUS experts (PedEM fellow and PedEM attending with 20 years experience) and split into two categories, those showing complete (95 patients) and those with incomplete (89 patients) IVC collapse. Approximately 10% (9) patient videos were randomly removed from each original data groups to be used for algorithm testing after training was completed. A standard 80%/20% training and validation split was used on the remaining 166 patient videos for algorithm training. Training accuracy, losses and learning curves were tracked and various training parameters such as learning rates and batch sizes were optimized throughout training. As a final real world test, the DL algorithm was tasked with analyzing the 18 previously unseen, randomly selected IVC videos. Cohen’s kappa was calculated for each of the blinded POCUS reviewers and DL algorithm. Results: This DL algorithm completed analysis of each previously unseen real world test video and is the first such algorithm to analyze IVC collapse through visual estimation in real-time. The algorithm was able to deliver a collapse result prediction for all 18 test IVC videos and there were no failures. Algorithm agreement with PedEM POCUS attending was substantial with a Cohen’s kappa of 0.78 (95% CI 0.49 to 1.0). Algorithm agreement with PedEM Fellow was substantial with Cohen’s kappa of 0.66 (95% CI 0.31 to 1.0). The PEM fellow and PEM POCUS attending also had substantial agreement, yielding a Cohen’s kappa of 0.66 (95% CI 0.32 to 1.0). Conclusions: This DL algorithm developed on prospectively acquired IVC video data from patients being studied for an IVF resuscitation study proved accurate at identifying when the IVC collapsed completely in real time. There was substantial agreement with POCUS reviewers of the same videos. Such an algorithm could allow novice clinicians to rapidly identify complete IVC collapse in children and the need for IVF administration. This could expand patient access to point of care technology by enabling novices with little training to use the diagnostic tool at bedside and decide if patients require intravenous fluid administration.

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Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

Journal of Clinical Microbiology ◽

10.1128/jcm.01962-15 ◽

2015 ◽

Vol 53 (12) ◽

pp. 3935-3937 ◽

Cited By ~ 5

Author(s):

Daniel Golparian ◽

Stina Boräng ◽

Martin Sundqvist ◽

Magnus Unemo

Keyword(s):

Neisseria Gonorrhoeae ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Molecular Detection ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Content Type ◽

Dna Assay ◽

Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

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Accurate and Highly Sensitive Screening of DNA-binding Molecules with Primer-dimer Templates Using a Real-time PCR Analyzer

Analytical Sciences ◽

10.2116/analsci.27.461 ◽

2011 ◽

Vol 27 (5) ◽

pp. 461

Author(s):

Akira INOUE ◽

Tsunehiro KUMEDA ◽

Hiroshi NAKAMURA

Keyword(s):

Dna Binding ◽

Real Time ◽

Real Time Pcr ◽

Highly Sensitive

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A new highly sensitive single-tube nested real-time PCR assay: Clinical utility in perinatal HIV-1 diagnosis

Journal of Virological Methods ◽

10.1016/j.jviromet.2021.114273 ◽

2021 ◽

Vol 297 ◽

pp. 114273

Author(s):

Matias Moragas ◽

Marcelo D. Golemba ◽

Andrea Mangano

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Utility ◽

Pcr Assay ◽

Single Tube ◽

Perinatal Hiv ◽

Highly Sensitive ◽

Hiv 1

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A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

Journal of Food Protection ◽

10.4315/0362-028x-70.5.1080 ◽

2007 ◽

Vol 70 (5) ◽

pp. 1080-1087 ◽

Cited By ~ 63

Author(s):

V. M. BOHAYCHUK ◽

G. E. GENSLER ◽

M. E. McFALL ◽

R. K. KING ◽

D. G. RENTER

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Culture Method ◽

Pcr Assay ◽

Culture Methods ◽

The Real ◽

Conventional Culture ◽

Food Animal ◽

Highly Sensitive ◽

Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

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