Evaluation of the Performance of a Multiplex Real-Time PCR Assay for the Identification of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii Simultaneously from Sputum in Multicenter

Figures & data

Table 1 Multiplex Real-Time Assay versus DNA Sequencing for Detecting Aspergillus

Gold Method Prediction Method		DNA Sequencing		Total
		Condition Positive	Condition Negative
Multiplex real-time assay	Prediction positive	Ture positive=167	False positive=5	172	PPV =167/172 =97.09%
	Prediction negative	False negative=1	True negative=364	365	NPV =364/365 =99.73%
Total		168	369	537
		Sensitivity =167/168 =99.40%	Specificity =364/369 =98.64%

Abbreviations: PPV, positive predictive value; NPV, negative predictive value.

Table 2 Multiplex Real-Time Assay versus DNA Sequencing for Detecting Cryptococcus Neoformans

Gold Method Prediction Method		DNA Sequencing		Total
		Condition Positive	Condition Negative
Multiplex real-time assay	Prediction positive	Ture positive=53	False positive=2	55	PPV =53/55 =96.36%
	Prediction negative	False negative=0	True negative=482	482	NPV =482/482 =100%
Total		53	484	537
		Sensitivity =53/53 =100%	Specificity =482/484 =99.59%

Abbreviations: PPV, positive predictive value; NPV, negative predictive value.

Table 3 Multiplex Real-Time Assay versus DNA Sequencing for Detecting Pneumocystis

Gold Method Prediction Method		DNA Sequencing		Total
		Condition Positive	Condition Negative
Multiplex real-time assay	Prediction positive	Ture positive=137	False positive=6	143	PPV =137/143 =95.80%
	Prediction negative	False negative=1	True negative=393	394	NPV =393/394 =99.75%
Total		138	399	537
		Sensitivity =137/138 =99.28%	Specificity =393/399 =98.50%

Abbreviations: PPV, positive predictive value; NPV, negative predictive value.

Figure 1 Continued.

Figure 1 PCR amplification curves of simultaneous positive for two or three pathogens. (A) Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii were all positive. (B) Aspergillus, and Cryptococcus neoformans were both positive, Pneumocystis jirovecii was negative. (C) Aspergillus and Pneumocystis jirovecii were both positive, Cryptococcus neoformans was negative. (D) Cryptococcus neoformans and Pneumocystis jirovecii were both positive, Aspergillus was negative. PCR channel: Pneumocystis jirovecii: CY5; Aspergillus: FAM; Internal control: ROX; Cryptococcus neoformans: VIC.

Table 4 The Pathogen Combinations with More Than Two Positive Results by Multiplex Real-Time Assay and DNA Sequencing

Multiplex Real-Time Assay		DNA Sequencing
Pathogens detected simultaneously	N	Different results from multiplex real-time assay
Aspergillus, Cryptococcus neoformans, and Pneumocystis	3	One sample is positive for Aspergillus and Cryptococcus neoformans but negative for Pneumocystis
Aspergillus and Cryptococcus neoformans	5	One sample is negative for both Aspergillus and Cryptococcus neoformans
Aspergillus and Pneumocystis	30	One sample is positive for Aspergillus but negative for PneumocystisTwo samples are positive for Pneumocystis but negative for Aspergillus
Cryptococcus neoformans and Pneumocystis	2	0

Abbreviation: N, number of specimens.