Real-Time Polymerase Chain Reaction

BackgroundDengue and Chikungunya are Aedes-borne diseases that are predominantly prevalent in tropical and subtropical regions, affecting public health globally. Dengue is caused by multiple antigenically different Dengue virus (DENV) serotypes (DENV-1 to DENV 4) in the Flaviviridae family and Chikungunya (CHIKV) in the Togaviridae family. The overlapping clinical presentation of both diseases, particularly in early infection, complicates timely and differential diagnosis. In India, diagnosis primarily relies on rapid antigen-based or ELISA-based tests, which are prone to false negatives, leading to underreported disease burden. In resource-limited settings, the absence of confirmatory diagnostics often leads to reliance on clinical symptoms and epidemiological data, increasing the risk of misdiagnosis and undetected co-infections.MethodsTo address these diagnostic limitations, we developed DENCHIK, a multiplex, quantitative real-time PCR (qRT-PCR) assay for the simultaneous detection of DENV serotypes and CHIKV. Between July and December 2022, a total of 903 serum samples from febrile patients across 161 public health centers in Bengaluru were analyzed. The performance of the DENCHIK assay was compared with ELISA-based tests (NS1 antigen and IgM antibody detection) and two commercially available qRT-PCR assays for DENV and CHIKV.FindingsUsing the DENCHIK assay, 36% of samples were tested positive for DENV, 17% for CHIKV and 8% were tested positive for co-infections. In contrast, ELISA detected 29.90% of DENV and 22.92% of CHIKV infections. We observed a 9% DENV infection using NS1 ELISA and 24% by IgM ELISA, highlighting discrepancies between antigen-and antibody-based tests. Among DENV serotypes, DENV-1 was the most prevalent serotype followed by DENV-2, DENV-3, and DENV-4. A seasonal increase in cases was observed from June to September 2022, coinciding with the monsoon season. No significant difference in prevalence was noted across gender and age groups. DENCHIK demonstrated a sensitivity of 62.82% and specificity of 66.45% for DENV detection compared to NS1 ELISA. When evaluated against commercial qRT-PCR assays, DENCHIK exhibited superior performance with 99% sensitivity and 98% specificity for DENV detection. For CHIKV, DENCHIK showed 26% sensitivity, and 86% specificity compared to IgM ELISA, while achieving 98% sensitivity and specificity relative to commercial qRT-PCR assays.ConclusionDENCHIK assay successfully enabled simultaneous amplification of all four DENV serotypes and Chikungunya, from clinical samples. DENCHIK assay detected 7.6% additional Dengue infections and 6.65% fewer Chikungunya infections in clinical samples, demonstrating enhanced diagnostic accuracy. With higher sensitivity and specificity, DENCHIK allows for early detection from day one of symptom onset, improving the estimation of true disease prevalence and mitigating misdiagnosis associated with ELISA-based methods. The integration and surveillance of molecular assays, such as DENCHIK, will enhance epidemiological monitoring of circulating DENV serotypes, CHIKV, and co-infections. These advancements will provide critical insights for public health authorities, enabling them to prioritize treatment, implement effective control measures, and mitigate the transmission of arboviral infections.

BackgroundTargeted treatments for non-communicable chronic inflammatory skin diseases like eczema and psoriasis offer significant potential for effective therapy. However, therapeutic success requires an accurate diagnosis, which is challenging due to their overlapping clinical and histological features.ObjectivesWe aimed at assessing the diagnostic performance of both a manual (MC) and fully automated (PsorX-LabDisk) quantitative real-time polymerase chain reaction (RT-qPCR) test based on the expression of NOS2 and CCL27 compared with conventional dermatopathological evaluation in differentiating psoriasis from eczema.MethodsSeventy-three formalin-fixed, paraffin-embedded skin samples of psoriasis and eczema were randomly selected and evaluated histopathologically (H&E-stained sections) by 14 dermatopathologists to assess inter-observer variability, quantified using Cohen's and Fleiss' κ. To confirm that the observed variability was not cohort- or rater-specific, a validation cohort (n = 72) from an independent institution was assessed by three dermatopathologists under identical conditions. For molecular analysis, both manual (MC) and automated NOS2/CCL27-based RT-qPCR (PsorX-LabDisk) workflows were applied. Diagnostic performance (sensitivity, specificity, accuracy) of histopathological and molecular analyses was determined against reference diagnoses.ResultsDermatopathological evaluation demonstrated only fair agreement (Fleiss' κ = 0.31) in both study and validation cohort. The mean diagnostic accuracy of dermatopathology was 76.9%, with a sensitivity of 70% and specificity of 81.6%. In comparison, MC and the PsorX-LabDisk achieved sensitivities of both 92.9%, specificities of 82.2% and 84.4% and accuracies of 87.7% and 86.3%, respectively. In diagnostically ambiguous cases, molecular testing maintained high accuracy (>86%), clearly outperforming dermatopathology, which showed near-random agreement and low accuracy (61.7%).ConclusionsBoth MC and PsorX-LabDisk provide a reliable, examiner-independent complement to dermatopathology for differentiating psoriasis and eczema. By reducing diagnostic ambiguity, it enhances clinical confidence and supports more precise and timely therapeutic decisions in inflammatory skin disease management.

A sustained outbreak of H5N1 influenza virus among wild fowl and domestic livestock has caused more than 70 zoonotic infections in humans in North America, including two deaths. The United States Centers for Disease Control and Prevention has recommended rapid H5 subtyping for all hospitalized cases with influenza A virus infection to enable prompt initiation of antiviral treatment, as well as infection prevention and implementation of public health measures to control spread. To address these needs, we developed a qualitative multiplex RT-qPCR assay to subtype H5 influenza virus in nasal, nasopharyngeal, and conjunctival specimens with a limit of detection of 250 copies/mL. No cross-reactivity was observed with other common respiratory viruses, including seasonal H3N2 and H1N1 influenza A viruses. We retrospectively subtyped 590 influenza A virus-positive clinical specimens with Ct values less than 31 processed by University of Washington labs between March 2024 and February 2025, including 512 specimens collected during the 2024-2025 influenza season, and detected no H5 positives. After clinical implementation, we performed 150 clinically ordered H5 subtyping tests between February and April 2025 and again detected no positives. This work enhances clinical pandemic preparedness activities and highlights the exceedingly low prevalence of H5N1 influenza virus during the 2024-2025 respiratory season.IMPORTANCEThe spread of H5N1 influenza virus in the United States has led to the culling of almost 200 million birds, infected cow herds across 17 states, and resulted in 70 human infections as of July 2025. Rapid PCR subtyping of H5 influenza virus is critical to inform hospital infection prevention and public health to enable containment of viral transmission. Here, we report the design, validation, and clinical implementation of a qualitative multiplex H5-subtyping RT-qPCR assay for nasopharyngeal, nasal, and conjunctival swab specimens. Additionally, we offer the largest reported study of H5 subtyping of influenza A virus-positive specimens in the United States to date. No H5 infections were detected in 740 samples collected between March 2024 and April 2025 from patients with confirmed influenza A virus infection in a large academic medical system in Seattle, WA.