Multiplex Polymerase Chain Reaction

PurposeNearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy.Materials and methodsA novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board-approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings.ResultsHierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P v not reached; P v 12 months; P v 40.6 months; P ConclusionWe demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.

A multiplex reverse transcription quantitative PCR (RT-qPCR)-based method was designed for the simultaneous detection of different SARS-CoV-2 genes. In this study, we used three target genes encoding for the nucleocapsid 1 and 3 (N1, N3), and the spike (S) proteins, all commonly used in the detection of SARS-CoV-2 in human and environmental samples. The performance of the multiplex assay, compared to the single assay was assessed for the standard calibration curve, required for absolute quantification, and then, for the real environmental samples to detect SARS-CoV-2. For this latter, four environmental samples were collected at a local wastewater treatment plant (WWTP). The results showed that the cycle threshold (Ct) values of the multiplex were comparable to the values obtained by the singleplex PCR. The amplification of the three target genes indicated the presence of SARS-CoV-2 in the four water samples with an increasing trend in February and these results were confirmed in the multiplex approach, showing the robustness of this method and its applicability for the relative abundance analysis among the samples. Overall, both the laboratory and field work results demonstrated that the multiplex PCR assay developed in this study could provide a method for SARS-CoV-2 detection as robust as the single qPCR, but faster and cost-effective, reducing by three times the number of reactions, and consequently the handling time and reagents.

Quantitative PCR (qPCR) is the gold standard for detection and quantitation of known DNA targets, but the scarcity of spectrally distinct fluorophores and filter sets limits the number of detectable targets. Here, we introduce color cycle multiplex amplification (CCMA) to significantly increase the number of detectable DNA targets in a single qPCR reaction using standard instrumentation. In CCMA, presence of one DNA target species results in a pre-programmed pattern of fluorescence increases. This pattern is distinguished by cycle thresholds (Cts) through rationally designed delays in amplification. For example, we design an assay wherein Staphylococcus aureus sequentially induces FAM, then Cy5.5, then ROX fluorescence increases with more than 3 cycles between each signal. CCMA offers notably higher potential for multiplexing because it uses fluorescence permutation rather than combination. With 4 distinct fluorescence colors, CCMA theoretically allows the detection of up to 136 distinct DNA target sequences using fluorescence permutation. Experimentally, we demonstrated a single-tube qPCR assay screening 21 sepsis-related bacterial DNA targets in samples of blood, sputum, pleural effusion and bronchoalveolar lavage fluid, with 89% clinical sensitivity and 100% clinical specificity, showing its potential as a powerful tool for advanced quantitative screening in molecular diagnostics.

Lower respiratory tract infections (LRTIs) place a substantial burden on emergency departments (EDs) during winter outbreaks. Their microbiological diagnosis is currently based on either broad-spectrum molecular techniques performed in laboratory, with turnaround times incompatible with ED workflows, or point-of-care systems, restricted to SARS-CoV-2, influenza A/B, and RSV (quadriplex panel). The Spotfire combines rapid result and syndromic approach (10 respiratory viruses and 4 atypical bacteria), but its added value in EDs remains unassessed. During the 2023 winter, trained nurses collected nasopharyngeal swabs from adult patients with LRTI symptoms and operated the Spotfire in the ED of the Poitiers University Hospital. We described the viral epidemiology and evaluated the impact and efficacy of this rapid broad-spectrum diagnosis by comparing the management of patients with or without respiratory pathogen detected. From 15 December 2023 to 15 March 2024, 1,320 samples were analyzed with Spotfire, with a median turnaround time of 37 min and 10 uninterpretable results. A total of 540 (41%) were positive. Influenza A (30.9%), rhinovirus/enterovirus (21.1%), and SARS-CoV-2 (18.7%) were the main viruses detected, while atypical bacteria represented 8% of all pathogens. Overall, a pathogen not represented on the quadriplex panel was detected in 43% samples. Positive test results were associated with faster medical decision (380 ± 234 vs 431 ± 238 min; P P P = 0.006) than negative test results. Antibiotic therapy was administered to 42 of 43 of Mycoplasma pneumoniae-positive patients (98%) compared with 110 of 1,265 (9%) negative patients. This study validated the feasibility of this new diagnostic tool in high-volume EDs improving patient flow, antimicrobial decisions, and isolation strategies.IMPORTANCEThis study provides the first real-world evidence supporting the use of a broad multiplex PCR platform for respiratory pathogens directly at the point of care in a high-volume emergency department. By enabling the simultaneous detection of 14 viruses and atypical bacteria within 20 min, this system bridges a critical gap between laboratory diagnostics and bedside clinical decision-making. Its implementation proved feasible and reliable, improving patient flow, antimicrobial stewardship, and infection control measures. Nearly half of the pathogens identified would have been missed by conventional quadriplex assays, highlighting the added diagnostic value of broader syndromic coverage. These findings are of interest to both clinicians and microbiologists, as they provide pragmatic evidence to guide the integration of advanced molecular diagnostics into acute-care workflows and to optimize patient management during respiratory infection surges.