Evaluation of a ddPCR Commercial Assay for the Absolute Quantification of the Monkeypox Virus West Africa in Clinical Samples

Abstract

Background: Monkeypox virus (MPXV) is a double-stranded DNA virus belonging to the orthopoxvirus genus in the family Poxviridae. Distinct clades are identified: the clade I belonging to the Central African (or Congo Basin) clade and the subclades IIa and IIb belonging to the West African clade. Here, a commercial droplet digital PCR (ddPCR) assay was evaluated for the quantification of the MPXV West Africa clade in clinical samples.

Methods: The ddPCR reaction was assessed as a duplex assay using RPP30 as an internal amplification control. A total of 60 clinical specimens were tested, 40 positives (skin lesions, n=10; rectal swabs, n = 10; pharyngeal swabs, n = 10; and whole blood, n = 10), and 20 negatives (n = 5 for each biological matrix) were found at the routine molecular diagnostics (orthopoxvirus qPCR followed by confirmation with Sanger sequencing). To evaluate the analytical sensitivity, the ddPCR reaction was first analyzed on serial dilutions of synthetic DNA spiked in water and in negative biological matrices, achieving a limit of detection of 3.5 copy/µL.

Results: Regarding the clinical samples, compared to routine molecular diagnostics, the ddPCR duplex assay showed 100% of specificity for all biological matrices and 100% sensitivity (10/10) for lesions, 100% (10/10) for rectal swabs, 90% (9/10) for pharyngeal swabs, and 60% (6/10) for whole blood.

Conclusion: Overall, our data showed that the commercial ddPCR assay allowed the DNA detection of MPXV in 87.5% (35/40) of our cohort, highlighting useful technical indications for the different specimens with a potential greatest performance for skin lesions and rectal swabs.

Keywords: DNA; ddPCR; monkeypox virus; quantification.

Figure 1

Evaluation of the ddPCR duplex assay (MPXV as Channel 1 and RPP30 as Channel 2) performance using MPXV synthetic amplicon Gblock Sequence as reference DNA. Standard curves were obtained from 10-fold serial dilutions of the reference DNA (3.5 × 10⁴ copies/µL) in four biological matrices in two different transport media (UTM and eSwab) (A–G). Positive samples (up) and negative samples (below) (H).

Figure 2

Evaluation of the ddPCR assay performance using MPXV synthetic amplicons and Gblock sequences as reference DNA. Standard curves were obtained from 10-fold serial dilutions of the reference DNA (3.5 × 10⁴ copies/µL) in four biological matrices in two different transport media (UTM and eSwab). The curves also include the negative point (no DNA). (A–G). The error bar represented the standard deviation of the mean. Pearson statistics were used.