Detection of Pneumocystis jirovecii and Toxoplasma gondii in patients with lung infections by a duplex qPCR assay

Abstract

Pneumocystis pneumonia (PCP) and pulmonary toxoplasmosis (PT) are caused by Pneumocystis jirovecii and Toxoplasma gondii. The clinical symptoms and imaging of PCP and PT are indistinguishable. A duplex qPCR was developed to differentiate between these two pathogens. In testing 92 clinical samples to validate the performance of this method for P. jirovecii detection, it identified 31 positive samples for P. jirovecii infection, consistent with clinical diagnosis. Among the remainder of the 61 clinical samples with suspected PCP, yet showing as negative by the conventional PCR diagnosis approach, 6 of them proved positive using our new assay. Our new approach also produced similar results in identification of T. gondii infections, giving a result of 2 positive and 20 negative in clinical samples. An investigation was undertaken on the prevalence of P. jirovecii and T. gondii infections using 113 samples from lung infection patients. 9% (10/113) were shown to be positive with infections of P. jirovecii, 2% with T. gondii (2/113) and 5% (6/113) were co-infected with both pathogens. Although this duplex qPCR can detect individual P. jirovecii and T. gondii infection, and co-infection of both pathogens, further large-scale investigations are needed to validate its performance, especially in T. gondii detection. Our assay provides a rapid and accurate tool for PCP and PT diagnosis in immunocompromised population and clinical surveillance of these infections in patients with no immune defects.

Fig 1. Specificity of the primers designed for P. jirovecii mtSSU and T. gondii Rep-529 amplification.

The duplex PCR method showed no cross-reaction to Mycobacterium tuberculosis, alpha hemolytic Streptococcus, Neisseria, Mycoplasma or Klebsiella pneumoniae.

Fig 2. Quantitative correlation between gene copy number and threshold cycle of the duplex qPCR assay.

(A) P. jirovecii mtSSU plasmid was serially diluted from 10¹ to 10⁸ copies/reaction and subjected to qPCR. (B) Linear regression of Cq vs. lg copy number of mtSSU plasmid using singleplex qPCR and duplex qPCR. (C) T. gondii Rep-529 plasmid was serially diluted from 10¹ to 10⁸ copies/reaction and subjected to qPCR. (D) Linear regression of Cq vs. lg copy number of Rep-529 plasmid using singleplex qPCR and duplex qPCR. ΔRn = Rn (normalized reporter)-baseline. Ct, Cycle threshold.

Fig 3

The detected P. jirovecii (A) and T. gondii (B) DNA copy number comparison between samples from sputum and BALF.

Fig 4. Detection of P. jirovecii and T. gondii infection using duplex qPCR assay on clinical samples from 113 lung infection patients.

(A) Number of P. jirovecii, P. jirovecii + T. gondii and T. gondii patients detected by the duplex qPCR. (B) P. jirovecii and T. gondii positive patients detected with duplex qPCR in infant and elder groups. (C) DNA load of P. jirovecii and T. gondii detected in lung infection patients.