DEVELOPMENT OF IN-HOUSE MULTIPLEX AND QUANTITATIVE PCR ASSAYS FOR DETECTION OF PNEUMOCYSTIS JIROVECII IN BRONCHOALVEOLAR LAVAGE AND SPUTUM SPECIMENS

Patsharaporn Techasintana Sarasombath1, Sirirat Wijit1, Pisith Chinabut1, Jerawan Ongrotchanakun1, Ladawan Khowawisetsut1, Suporn Foongladda2, Patimaporn Wongprompitak3 and Darawan Wanachiwanawin1

Abstract

A definite diagnosis of Pneumocystis pneumonia requires laboratory confirmation of the presence of Pneumocystis jirovecii in clinical specimens. In-house multiplex and quantitative (q)PCR assays were developed to detect P. jirovecii mtLSU rRNA in bronchoalveolar lavage (BAL) and sputum specimens in comparison with three standard cytological assays [Gomori’s methenamine silver (GMS) staining, Giemsa staining and immunofluorescence assay (IFA)] and conventional PCR. Both in-house multiplex and qPCR assays demonstrated specific of P. jirovecii without cross-amplification with other organisms. In BAL samples (n = 131), median copies/µl of P. jirovecii mtLSU rRNA DNA (measured by in-house qPCR) in samples positive by GMS staining (37%), Giemsa staining (37%) and IFA samples (42%) were 4.5x105, 3.8x105 and 3.8x105, respectively; and in sputum samples (n = 54), median value in 4, 4 and 7 % positives was 5.5x105, 5.5x105 and 2.2x105 copies/µl, respectively. Using conventional PCR median copies/µl P. jirovecii mtLSU rRNA DNA in BAL (49% positive) samples compared to in-house multiplex PCR (43% positive) were 2.5x105 and 3.1x105 respectively, while in sputum samples (17 and 13% positives) were 4.3x104 and 4.3x104 respectively. PCR methods showed higher sensitivity in P. jirovecii detection in both type of specimens compared to the classical staining methods. Overall, qPCR was considered the most promising diagnostic method due to its sensitivity and quantitative ability.