CRISPR-Based Test Diagnoses Life-Threatening Fungal Infection More Quickly

Pneumocystis jirovecii pneumonia (PJP) is a serious fungal infection that mainly affects children and those with weakened immune systems. Diagnosing PJP typically requires invasive procedures like bronchoalveolar lavage (BAL) specimens, which can be difficult to obtain. While oropharyngeal swabs and serum could offer a simpler alternative, current diagnostic methods for this leading cause of fungal pneumonia have remained largely unchanged for decades, leaving many patients without quick or definitive diagnoses. Now, a new study published in the Journal of Clinical Investigation suggests that utilizing CRISPR (clustered regularly interspaced short palindromic repeats) technology could improve diagnostic sensitivity, making it possible to accurately detect P. jirovecii using swabs and serum samples.

The research team from Tulane University (New Orleans, LA, USA) developed an ultra-sensitive RT-PCR combined with a CRISPR assay, which demonstrated high specificity for active infections in infant swabs, adult BAL, and serum samples. They used an RT-PCR CRISPR assay to analyze P. murina transcripts in lung RNA, BAL, and serum from wild-type and Rag2–/– mice at 2, 4, and 6 weeks after infection. For human studies, the team optimized the RT-PCR CRISPR assay to detect P. jirovecii transcripts in oropharyngeal swabs from infants, as well as in adult serum and BAL samples from both infected and non-infected individuals. Their findings showed that the P. murina assay was highly effective in detecting Pneumocystis RNA in the serum of infected mice during the course of the infection.

The CRISPR assay used on oropharyngeal swabs in infants identified P. jirovecii infections with significantly higher sensitivity (96.3% vs. 66.7%) and specificity (100% vs. 90.6%) compared to the traditional RT-qPCR method. Moreover, the CRISPR assay achieved greater sensitivity than RT-qPCR (93.3% vs. 26.7%) in adult serum samples. Since swabs are commonly collected from pediatric pneumonia patients and serum is easier to obtain than BAL, the researchers concluded that their CRISPR-based assay could offer a more accurate and timely diagnosis for both pediatric and adult patients with P. jirovecii infections, reducing the need for invasive BAL specimens.

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