A rapid multiplex assay of human malaria parasites by digital PCR. Clinica chimica acta; international journal of clinical chemistry BACKGROUND:Blood smear examination through traditional optical microscopy is the gold standard for malaria diagnosis. However, it imposes strict requirements for operational staff and its sensitivity cannot perfectly satisfy the needs of clinical requirements. More sensitive and accurate modern technologies should be applied to this field. Digital PCR (dPCR), as an absolute quantification detection method, can serve as an effective tool to facilitate the diagnosis and classification of different malaria species. OBJECTIVE:We aimed to establish a new multiplex dPCR detection system for four main Plasmodium species: P. vivax, P. falciparum, P. ovale and P. malariae, which can distinguish exact species of malaria by one PCR reaction. METHODS:A total of 39 patients were identified as malaria-positive by microscopic examination in Huashan Hospital from 2016 to 2021; seventy blood samples from these patients were collected. Additionally, 20 healthy individuals, 20 patients with fever and 6 patients with other types of blood parasites infection were also included in this study. Each blood sample was subjected to examination by both blood smears and dPCR. By optimizing four different fluorescence-labeled probes in one reaction system, dPCR permitted the performance of accurate quantitation and working out the exact number of copies of malaria DNA per microliter in whole blood. Rapid diagnostic tests were also conducted to verify part of the results obtained by dPCR. RESULTS:The dPCR system was able to make rapid diagnosis and quantification of malaria DNA samples. The analytical sensitivity of multiplex dPCR was as low as 0.557 copies/μL (95% CI 0.521 to 0.607), and it had a sensitivity of 98.0% and a specificity of 100% in clinical samples. Additionally, three multiple malaria co-infection samples have been detected by this dPCR system, including one triple malaria infection case. By testing consecutive daily blood samples of Patient 39, dPCR facilitated monitoring the efficacy of drug treatment. It showed that the DNA concentrations of P. falciparum ranged from 5474 copies/μL to 0 copies/μL, which can reflect the efficacy of antimalarials in real time. This study also found that haemocyte samples (plasma removed) rather than whole blood had higher malaria detection capability and an enhanced positive rate. CONCLUSION:The multiplex dPCR system newly established here made a substantial contribution in detecting malaria infection at low concentrations. It is suitable for mixed-infection diagnosis and multi-sample continuous monitoring, and presents a promising candidate as an absolute quantitative tool in clinical practice. 10.1016/j.cca.2022.12.001

A rapid, multiplex digital PCR assay to detect gene variants and fusions in non-small cell lung cancer. Molecular oncology Digital PCR (dPCR) is emerging as an ideal platform for the detection and tracking of genomic variants in cancer due to its high sensitivity and simple workflow. The growing number of clinically actionable cancer biomarkers creates a need for fast, accessible methods that allow for dense information content and high accuracy. Here, we describe a proof-of-concept amplitude modulation-based multiplex dPCR assay capable of detecting 12 single-nucleotide and insertion/deletion (indel) variants in EGFR, KRAS, BRAF, and ERBB2, 14 gene fusions in ALK, RET, ROS1, and NTRK1, and MET exon 14 skipping present in non-small cell lung cancer (NSCLC). We also demonstrate the use of multi-spectral target-signal encoding to improve the specificity of variant detection by reducing background noise by up to an order of magnitude. The assay reported an overall 100% positive percent agreement (PPA) and 98.5% negative percent agreement (NPA) compared with a sequencing-based assay in a cohort of 62 human formalin-fixed paraffin-embedded (FFPE) samples. In addition, the dPCR assay rescued actionable information in 10 samples that failed to sequence, highlighting the utility of a multiplexed dPCR assay as a potential reflex solution for challenging NSCLC samples. 10.1002/1878-0261.13523