Analytical and clinical validation of multiplex droplet digital PCR assay for detecting pathogenic fungal infection in lungs

Figures & data

Figure 1. Detection of invasive pulmonary fungal infection by droplet digital PCR (ddPCR).

Table 1. Target pathogens detection for droplet digital PCR (ddPCR).

Fluorescence channel	Genus	Target pathogens
FAM	Pneumocystis	Pneumocystis jiroveci
ROX	Aspergillus	Aspergillus fumigatus / Aspergillus flavus / Aspergillus niger / Aspergillus terreus / Aspergillus nidulans
CY5	Cryptococcus	Cryptococcus neoformans / Cryptococcus gattii

Table 2. The result of limit of blank (LoB) and limit of detection (LoD).

Genus	LoB (copies/test)	LoD (copies/test)
Pneumocystis	0	3
Aspergillus	10	35
Cryptococcus	0	3

Figure 2. Specificity of droplet digital PCR (ddPCR) in detecting the target pathogen DNA. (a) Strong positive. (b) Weak positive.

Table 3. The coincidence rate of detection results detected by Droplet digital PCR (ddPCR) and quantitative real-time PCR (qPCR).

	Positive coincidence (%)	Negative coincidence (%)	Κ (%)
Aspergillus	80.00	100.00	87.59
Pneumocystis	66.67	100.00	85.11

Table 4. The inconsistent results between droplet digital PCR (ddPCR) and quantitative real-time PCR (qPCR) were verified by metagenomics next generation sequencing (mNGS).

Specimen No.	ASP DNA for ddPCR (copies/test)	PJ DNA for ddPCR (copies/test)	ASP DNA for qPCR (Ct value)	PJ DNA for qPCR (Ct value)	mNGS
Patient 1	51	0	Undetermined	Undetermined	Aspergillus-positive
Patient 2	42	0	Undetermined	Undetermined	Aspergillus-positive
Patient 3	46.5	0	Undetermined	Undetermined	Aspergillus-positive
Patient 4	43.5	0	Undetermined	Undetermined	Aspergillus-positive
Patient 5	3.5	31.5	Undetermined	Undetermined	Pneumocystis-positive
Patient 6	2.75	27	Undetermined	Undetermined	Pneumocystis-positive

Figure 3. Dynamic target pathogen DNA detection range of droplet digital PCR (ddPCR). (a) Aspergillus. (b) Pneumocystis jirovecii. (c) Cryptococcus.

Figure 4. The ROC curve for target pathogen DNA detection by droplet digital PCR (ddPCR). (a) Aspergillus. (b) Cryptococcus. (c) Pneumocystis jirovecii.