Distinct RT-PCR diagnosis profiles of father and son patients of COVID-19 using nasopharyngeal and alveolar lavage fluid samples

BackgroundMany acute respiratory illness surveillance systems collect and test nasopharyngeal (NP) and/or oropharyngeal (OP) swab specimens, yet there are few studies assessing the relative measures of performance for NP versus OP specimens.MethodsWe collected paired NP and OP swabs separately from pediatric and adult patients with influenza-like illness or severe acute respiratory illness at two respiratory surveillance sites in Kenya. The specimens were tested for eight respiratory viruses by real-time reverse transcription-polymerase chain reaction (qRT-PCR). Positivity for a specific virus was defined as detection of viral nucleic acid in either swab.ResultsOf 2,331 paired NP/OP specimens, 1,402 (60.1%) were positive for at least one virus, and 393 (16.9%) were positive for more than one virus. Overall, OP swabs were significantly more sensitive than NP swabs for adenovirus (72.4% vs. 57.6%, p<0.01) and 2009 pandemic influenza A (H1N1) virus (91.2% vs. 70.4%, p<0.01). NP specimens were more sensitive for influenza B virus (83.3% vs. 61.5%, p = 0.02), parainfluenza virus 2 (85.7%, vs. 39.3%, p<0.01), and parainfluenza virus 3 (83.9% vs. 67.4%, p<0.01). The two methods did not differ significantly for human metapneumovirus, influenza A (H3N2) virus, parainfluenza virus 1, or respiratory syncytial virus.ConclusionsThe sensitivities were variable among the eight viruses tested; neither specimen was consistently more effective than the other. For respiratory disease surveillance programs using qRT-PCR that aim to maximize sensitivity for a large number of viruses, collecting combined NP and OP specimens would be the most effective approach.

BackgroundRapid and accurate detection of viral infections is crucial in clinical settings, yet traditional quantitative polymerase chain reaction (qPCR) methods, though highly accurate, are typically laboratory-based and require significant time and resources, limiting their effectiveness in point-of-care environments. In this study, we applied a rapid qPCR method, originally developed for severe acute respiratory syndrome coronavirus 2, to a range of viral infections. The primary aim was to establish a novel diagnostic platform that combines mobile qPCR with multiplex detection for rapid, chairside application in clinical settings, and to explore its potential for detecting environmental contamination.MethodsThe viral infections targeted for rapid diagnosis included herpes simplex, herpes zoster, herpesviruses 4 or 5 -related diseases, hand-foot-and-mouth disease, herpangina, and respiratory syncytial virus infections. Specific primers and probes were designed to enable multiplex detection. The detection of viral nucleic acids using both benchtop and mobile qPCR devices was evaluated. Furthermore, viral synthetic nucleic acids were spread into the clinical environment, and swabs of the environmental surfaces were used as samples to examine whether this rapid method could be employed to detect contamination of environmental surfaces. Clinical samples from patients suspected of having labial herpes were also tested to examine whether viral detection is possible using this method.ResultsViral synthetic nucleic acids could be detected on both benchtop and mobile qPCR devices. This method can also detect environmental contamination, and differences in the detection of viral synthetic nucleic acids were observed before and after cleaning the clinical environment.ConclusionThis study introduces a novel approach combining mobile qPCR with multiplex detection for the rapid, chairside diagnosis of multiple viral infections. Furthermore, it demonstrates potential use in detecting environmental contamination in the clinical environment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12879-025-11823-4.

Ultrasensitive detection and quantification of viral nucleic acids with Raindance droplet digital PCR (ddPCR)

The dynamic changes in CO concentration [CO] during a single breath could be influenced by topographic inhomogeneity in the lung or by peripheral inhomogeneity due to a gas mixing resistance in the gas phase of the lung or to serial gradients in gas diffusion. Ten healthy subjects performed single-breath maneuvers by slowly inhaling test gas from functional residual capacity to one-half inspiratory capacity and slowly exhaling to residual volume with target breath-hold times of 0, 1.5, 3, 6, and 9 s. We calculated the three-equation single-breath diffusing capacity of the lung for CO (DLSBCO-3EQ) from the mean [CO] in both the entire alveolar gas sample and in four successive equal alveolar gas samples. DLSBCO-3EQ from the entire alveolar gas sample was independent of breath-hold time. However, with 0 s of breath holding, from early alveolar gas samples DLSBCO-3EQ was reduced and from late alveolar gas samples it was increased. With increasing breath-hold time, DLSBCO-3EQ from the earliest alveolar gas sample rapidly increased, whereas from the last alveolar gas sample it rapidly decreased such that all values from the small alveolar gas samples approached DLSBCO-3EQ from the entire alveolar sample. These changes correlated with ventilation inhomogeneity, as measured by the phase III He concentration slope and the mixing efficiency, and were larger for maneuvers with inspired volumes to one-half inspiratory capacity vs. total lung capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Early diagnosis of coronavirus disease 2019 (COVID-19) is crucial to early treatment and quarantine measures. In this narrative review, diagnostic tools for COVID-19 diagnosis and their main critical issues were reviewed. The COVID-19 real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test is considered the gold standard test for the qualitative and quantitative detection of viral nucleic acid. In contrast, tests can be used for epidemiological surveys on specific communities, including occupational cohorts, but not for clinical diagnosis as a substitute for swab tests. Computed tomography (CT) scans can be useful for the clinical diagnosis of COVID-19, especially in symptomatic cases. The imaging features of COVID-19 are diverse and depend on the stage of infection after the onset of symptoms. CT sensitivity seems to be higher in patients with positive RT-PCR. Conventional chest sensitivity shows a lower sensitivity. An important diagnostic screening tool is ultrasounds, whose specificity and sensitivity depend on disease severity, patient weight, and operator skills. Nevertheless, ultrasounds could be useful as a screening tool in combination with clinical features and molecular testing to monitor disease progression. Clinical symptoms and non-specific laboratory findings may be useful if used in combination with RT-PCR test and CT-scanning.

Detection of Viral Nucleic Acid in Specimens Spotted on Commercial Filter Papers: A Review and Meta-Analysis.

The Chase for the RIG-I Ligand—Recent Advances

Though the 2009 worldwide influenza A (H1N1) pandemic has been declared to have ended, the influenza virus is expected to continue to circulate from some years as a seasonal influenza. A rapid antigen test (RAT) can aid in rapid diagnosis and allow for early antiviral treatment. We evaluated the clinical usefulness of RAT using SD Bioline Influenza Antigen Test® kit to detect the influenza virus, considering various factors. From August 1, 2009 to October 10, 2009, a total of 938 patients who visited the outpatient clinic at Korea University Guro Hospital with influenza-like illnesses were enrolled in the study. Throat or nasopharyngeal swab specimens were obtained from each of the patients. Using these specimens, we evaluated the influenza detection rate by rapid antigen test based on the real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) method. In comparison with rRT-PCR, the sensitivity and specificity of the RAT were 44.0% and 99.9%, respectively. The cyclic threshold values of RAT negative specimens were higher than RAT positive specimens (30.1±3.1 vs. 28.3±3.9, p=0.031). The sensitivity of the RAT kit was higher in patients who visited clinics within two days of symptom onset (60.4% vs. 11.1%, p=0.026). The results of this study show that the RAT cannot be recommended for general use in all patients with influenza-like illness because of its low sensitivity. The RAT may be used, only in the settings with limited diagnostic resources, for patients who visit a clinic within two days of symptom onset.

Following the first reports of coronavirus disease-19 (COVID-19) by China to the World Health Organization (WHO) on 31st December 2019, more than 4,302,774 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cases have been reported by authorities in 212 countries and territories by 12th May 2020. The outbreak and spread of COVID-19 worldwide, highlights the critical need for developing rapid and accurate diagnostic testing methods for emerging human coronavirus (CoV) infections. Testing is crucial to track the spread of disease during a pandemic, and to swiftly permit public health interventions including isolation, quarantine, and appropriate clinical management of afflicted individuals. The key components of viral diagnostic tests are (1) collection of the appropriate sample (blood, nasal swab, and throat swab), (2) availability of the genetic and proteomic sequences of the novel virus for analysis, and (3) rapid and accurate laboratory testing methods. The current gold standard for the molecular diagnosis of SARS-CoV-2 infection is the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the qualitative and quantitative detection of viral nucleic acids. Other relevant laboratory methods include enzyme-linked immunoassays (EIA) for viral antibody and antigen detection, and serum viral neutralization (SVN) assays for antibody neutralization determination. The challenges faced in developing a diagnostic test for a novel pathogen are the ability to measure low viral loads for early detection, to provide low or no cross-reactivity with other viral strains and to deliver results rapidly. Several point-of-care molecular devices are currently being integrated for fast and accurate diagnosis of SARS-CoV-2 infections. This review discusses the current laboratory methods available to test for coronaviruses by focusing on the present COVID-19 outbreak.

Herein, we report a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated chemiluminescence assay (CRISPR-Cas-CLA) for the ultrasensitive detection of viral nucleic acids of HPV18 and HPV16. The CRISPR-Cas-CLA comprises a CRISPR/Cas12a system that specifically recognizes the target nucleic acid, a signal-conducting nanoconjugate (MB-ssDNA-PSNP) formed by coupling singlet-oxygen (1O2)-generating photosensitive nanoparticles (PSNPs) to magnetic beads (MBs) via a single-stranded DNA (ssDNA) linker, and 1O2-activated chemiluminescence nanoparticles (CLNPs). In the presence of the target nucleic acid, the ssDNA linker of the nanoconjugate is cleaved by the target-activated CRISPR/Cas12a system, and the PSNPs are dissociated from the MBs. The PSNP-containing supernatant obtained by magnetic separation is added to the CLNP-coated detection plate. Upon light irradiation of the CLNP-PSNP mixture in the well, strong chemiluminescence is generated with the subsequent addition of hydrogen peroxide, enabling the detection of the target nucleic acids. The proposed CRISPR-Cas-CLA system offers ultrahigh sensitivity (∼1.04 aM), simple operation, and low cost, providing a new direction for the development of PCR-free detection strategies for ultralow abundance nucleic acids.

Saliva Samples for Detection of SARS-CoV-2 in Mildly Symptomatic and Asymptomatic Patients

Atlantic salmon Salmo salar L. pre-smolts were experimentally infected with 2 different isolates of salmonid alphavirus (SAV): a Subtype 1 isolate from Ireland and a Subtype 3 isolate from Norway. Sequential samples of tissue and blood were collected during a period of 20 wk post injection and subjected to virus isolation from kidney tissue and serum, detection of viral nucleic acid in heart tissue and serum by real-time RT-PCR, detection of specific antibodies by virus neutralisation assay, and histopathological examination. Successful reproduction of pancreas disease (PD) was obtained by intraperitoneal (i.p.) injection of both isolates. No mortality was observed post infection in either group, but typical PD histopathological lesions in heart and pancreas tissue were observed with both isolates. The prevalence and severity of lesions in the pancreas, heart, skeletal muscle and brain were similar in both groups with only subtle differences recorded. Re-isolation of virus from kidney tissue was performed at 7 and 14 d post infection (d p.i.) only and was positive for both test groups at both sampling points. Isolation of virus from sera from both groups was positive at 4 to 14 d p.i., but was negative at later sampling points when antibody production had begun. Virus may be detected only during the acute phase using both methods. Specific neutralising antibodies could be detected for both test groups from Day 21 p.i. until the end of the experiment at 140 d p.i. Peak antibody titres were seen 70 d p.i. Using real-time RT-PCR, pancreas disease virus (PDV)-specific RNA was detected frequently in serum samples up to 14 d p.i. and occasionally thereafter. In contrast, viral RNA could still be detected in the heart tissue of fish from both groups for at least 140 d p.i.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) kits have been reliably employed for the diagnosis of coronavirus disease 2019 (COVID-19) by the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA since the beginning of the disease outbreak. In consideration of reliable diagnosis, apart from RT-PCR, the isothermal nucleic acid amplification-based point-of-care automated kits have also been tagged as a simpler and rapid alternative to the conventional techniques. Currently, the availability of a better diagnostic method for COVID-19 when compared to RT-PCR is nil. The most important step in the detection of SARS-CoV-2 in a RT-PCR diagnostic laboratory is to identify and employ RT-PCR kits with higher sensitivity as well as specificity. This study aimed to study commercially available RT-PCR kits for the detection of SARS-CoV-2 infections. The performance of seven different RT-PCR kits from different manufacturers used for diagnosis of COVID-19 in Govt Theni Medical College and Hospital, Theni, Tamil Nadu were analysed. Nasopharyngeal and oropharyngeal swabs were collected from patients and subjected to RT-PCR using these kits. The sensitivities and batch effects of the assessed kits were slightly different for different targets, for SARS-CoV-2 detection in nasopharyngeal swab specimens. Examination of COVID-19 kits should be done using currently employed kits in routine diagnosis for better efficiency.

Peptide nucleic acid (PNA) molecules are known to bind complementary nucleic acid sequences with a much stronger affinity and with more stable binding than DNA or RNA molecules. We chose parvovirus B19, which is diagnosed by detection of nucleic acids by in situ hybridization assay (ISH) and/or PCR, as an experimental model to develop an ISH assay that uses biotinylated PNA probes to detect viral genome in clinical specimens. We first optimized the PNA-ISH assay on B19-infected and mock-infected UT-7/EpoS1 cells and then tested the assay on archival B19 specimens and on consecutive specimens. All data were compared with data obtained with a standardized DNA-based ISH assay and confirmed by a PCR-ELISA. PNA-ISH detected B19 genome in a higher number of B19-infected UT-7/EpoS1 cells and with a more defined localization of viral nucleic acids than the standardized DNA-ISH assay. Moreover, PNA-ISH was able to detect B19 genome in all positive archival samples, whereas DNA-ISH failed in 5 samples. PNA-ISH detected more positive samples than DNA-ISH when consecutive specimens were analyzed, and a close agreement was found with PCR-ELISA results. The PNA-ISH assay had sensitivity and specificity comparable to a PCR assay and was more practical and quicker to perform than standard hybridization assays. The assay may be a suitable diagnostic test for the detection of viral nucleic acids in clinical specimens.

The emergence of highly pathogenic and deadly human coronaviruses, namely SARS-CoV and MERS-CoV within the past two decades and currently SARS-CoV-2, have resulted in millions of human death across the world. In addition, other human viral diseases, such as mosquito borne-viral diseases and blood-borne viruses, also contribute to a higher risk of death in severe cases. To date, there is no specific drug or medicine available to cure these human viral diseases. Therefore, the early and rapid detection without compromising the test accuracy is required in order to provide a suitable treatment for the containment of the diseases. Recently, nanomaterials-based biosensors have attracted enormous interest due to their biological activities and unique sensing properties, which enable the detection of analytes such as nucleic acid (DNA or RNA), aptamers, and proteins in clinical samples. In addition, the advances of nanotechnologies also enable the development of miniaturized detection systems for point-of-care (POC) biosensors, which could be a new strategy for detecting human viral diseases. The detection of virus-specific genes by using single-stranded DNA (ssDNA) probes has become a particular interest due to their higher sensitivity and specificity compared to immunological methods based on antibody or antigen for early diagnosis of viral infection. Hence, this review has been developed to provide an overview of the current development of nanoparticles-based biosensors that target pathogenic RNA viruses, toward a robust and effective detection strategy of the existing or newly emerging human viral diseases such as SARS-CoV-2. This review emphasizes the nanoparticles-based biosensors developed using noble metals such as gold (Au) and silver (Ag) by virtue of their powerful characteristics as a signal amplifier or enhancer in the detection of nucleic acid. In addition, this review provides a broad knowledge with respect to several analytical methods involved in the development of nanoparticles-based biosensors for the detection of viral nucleic acid using both optical and electrochemical techniques.