Dataset Information

Development and Evaluation of Rapid and Accurate CRISPR/Cas13-Based RNA Diagnostics for Pneumocystis jirovecii Pneumonia.

ABSTRACT:

Background

Pneumocystis jirovecii can result in a serious pulmonary infection, Pneumocystis jirovecii pneumonia, in immunocompetent hosts. The diagnosis of Pneumocystis jirovecii pneumonia has long been a major clinical concern, and there are limitations with the currently utilized immunostaining and polymerase chain reaction diagnosis/detection technologies (e.g., insufficient sensitivity and accuracy). Hence, we sought to establish a rapid and RNA-specific transcription mediated amplification and CRISPR/Cas13a-based diagnostics targeted P. jirovecii-mitochondrial large subunit ribosomal RNA.

Methods

The procedure of the diagnostics included amplification of the extracted RNA samples by transcription mediated amplification, followed by CRISPR/Cas13 detection, and ultimately, the judgment of the results after 30 minutes of fluorescence signal. Later, the diagnostic performance of the CRISPR/Cas13-based diagnostics were tested on the 62 surplus clinical samples.

Results

This CRISPR/Cas13-based diagnostics achieved limits of detection of approximately 2 copies/µL transcribed RNA templates, with no cross reaction to other respiratory pathogens, including bacteria and fungi. Similar to in-house quantitative real-time polymerase chain reaction, CRISPR/Cas13-based diagnostics was still positive in 243-fold diluted bronchial alveolar lavage fluid. A preliminary evaluation of 62 surplus bronchial alveolar lavage fluid samples from patients suspected of Pneumocystis jirovecii pneumonia showed that CRISPR/Cas13-based diagnostics achieved a 78.9% sensitivity and a 97.7% specificity in the diagnosis of Pneumocystis jirovecii pneumonia.

Conclusion

Our study demonstrates that the CRISPR/Cas13-based diagnostics technique has good performance for the accurate and specific diagnosis of Pneumocystis jirovecii pneumonia.

SUBMITTER: Zhan Y

PROVIDER: S-EPMC9240425 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Objectives: Pneumocystis jirovecii pneumonia (PCP) is an AIDS-defining illness. In patients with HIV, the benefit of PCP prophylaxis is well-defined when the CD4 T-cell count decreases below 200 cells/?L. In other immunocompromised patients, the value of PCP prophylaxis is not always as well-established. This study aimed to describe the epidemiology of PCP in recent years and assess how many patients with PCP did or did not receive prophylaxis in the month preceding the infection. Material and Methods: A multicenter retrospective study was performed in 3 tertiary care hospital. A list of patients that underwent broncho-alveolar lavage sampling and Pneumocystis jirovecii (PJ) PCR testing was retrieved from the microbiology laboratories. An in-house PJ quantitative PCR (qPCR) was used in each center. A cycle threshold (Ct) value of ? 28.5-30 was considered a probable PCP. For patients with a positive PJ qPCR but above this threshold, a predefined case definition of possible PCP was defined as a qPCR Ct value ? 34-35 and both of the following criteria: 1. Clinical and radiological features compatible with PCP and 2. The patient died or received PCP therapy and survived. Patient files from those with a qPCR Ct value ? 35 were reviewed to determine whether the patient fulfilled the case definition and if PCP prophylaxis had been used in the weeks preceding the PCP. Disease-specific guidelines, as well as hospital-wide guidelines, were used to evaluate if prophylaxis could be considered indicated. Results: From 2012 to 2018, 482 BAL samples were tested. Two hundred and four had a qPCR Ct value ? 35 and were further evaluated: 90 fulfilled the definition of probable and 63 of possible PCP while the remaining 51 were considered colonized. Seventy-four percentages of the patients with PCP were HIV-negative. Only 11 (7%) of the 153 patients had received prophylaxis, despite that in 133 (87%) cases prophylaxis was indicated according to guidelines. Conclusion: In regions where HIV testing and treatment is available without restrictions, PCP is mainly diagnosed in non-HIV immunocompromised patients. More than four out of five patients with PCP had not received prophylaxis. Strategies to improve awareness of antimicrobial prophylaxis guidelines in immunocompromised patients are urgently needed.