Rapid Reverse Transcription-polymerase Chain Reaction Research Articles

Comparing lateral flow testing with a rapid RT-PCR method for SARS-CoV-2 detection in the United Kingdom-A retrospective diagnostic accuracy study.

Background and AimsIn late 2019, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) emerged in Wuhan, China. Rapid global spread led to the coronavirus disease 2019 (Covid‐19) pandemic. Accurate detection of SARS‐CoV‐2 has become a vitally important tool in controlling the spread of the virus. Lateral flow devices (LFDs) offer the potential advantage of speed and on‐site testing. The sensitivity of these devices compared to reverse transcription‐polymerase chain reaction (RT‐PCR) has been questioned.MethodsWe compared the sensitivity of the Innova LFD, used widely in the United Kingdom, with our rapid RT‐PCR method using stored positive samples. Samples with a range of viral loads (original Ct values 18.9–36.5) were tested.ResultsThe Innova LFD was found to be 6000–10,000 times less sensitive than RT‐PCR for SARS‐CoV‐2 detection. Overall, the LFD detected 46.2% of the positives detected by RT‐PCR, with 50% of these observed to be weak LFD positives. At lower viral loads, such as 10,000–100,000 RNA copies/ml, the LFD detected 22.2% of positives. In addition, two strong positives (3 and 1.5 million RNA copies/ml) were not detected by the LFD.ConclusionThe argument for use of LFD kits is that they detect infectious virus and hence contagious individuals. However, there is a lack of conclusive evidence supporting this claim. The Innova LFD has been subject to a Class I recall by the US Food and Drug Administration, but is still approved and widely used in the United Kingdom.

Progression of LAMP as a Result of the COVID-19 Pandemic: Is PCR Finally Rivaled?

Reflecting on the past three years and the coronavirus disease 19 (COVID-19) pandemic, varying global tactics offer insights into the most effective public-health responses. In the US, specifically, rapid and widespread testing was quickly prioritized to lower restrictions sooner. Essentially, only two types of COVID-19 diagnostic tests were publicly employed during the peak pandemic: the rapid antigen test and reverse transcription polymerase chain reaction (RT-PCR). However, neither test ideally suited the situation, as rapid antigen tests are far too inaccurate, and RT-PCR tests require skilled personnel and sophisticated equipment, leading to long wait times. Loop-mediated isothermal amplification (LAMP) is another exceptionally accurate nucleic acid amplification test (NAAT) that offers far quicker time to results. However, RT-LAMP COVID-19 tests have not been embraced as extensively as rapid antigen tests or RT-PCR. This review will investigate the performance of current RT-LAMP-based COVID-19 tests and summarize the reasons behind the hesitancy to embrace RT-LAMP instead of RT-PCR. We will also look at other LAMP platforms to explore possible improvements in the accuracy and portability of LAMP, which could be applied to COVID-19 diagnostics and future public-health outbreaks.

Validation of rapid SARS-COV-2 antigen detection test as a screening tool for detection of Covid-19 infection at district hospital in northern India

Background: Testing of samples from suspected SARS-CoV-2 patients with reverse transcription polymerase chain reaction (RT-PCR) may result in delayed detection of infection. Given the number of people affected in pandemic, it is important to use a test that gives faster results and can be used on large number of sample size to cope with the increased testing capacity. Rapid Antigen detection test fulfills both the criteria. Aims and Objectives: The study aims to evaluate the diagnostic accuracy of rapid antigen detection test compared to RT-PCR on the same patients in District Hospital. Materials and Methods: Subjects were tested by both Rapid antigen detection testing and RT-PCR in District Hospital. The present study compared RAT and RT-PCR for detection of SARS-CoV-2 in nasopharyngeal specimen.756 Nasopharyngeal samples were obtained from suspected cases of SARS-CoV-2, Contacts of CoV2 patients, pre-operative patients in District Hospital. Results: Of 756 nasopharyngeal samples, 81 (10.71%) were positive and 675 (89.28%) were negative for SARS-CoV-2 by RT-PCR. The rapid SARS-CoV-2 antigen detection test’s sensitivity 55.04% and specificity was 99.2%, respectively. Positive predictive value 93.42%, Negative predictive value 91.47%, Diagnostic Accuracy 91.67%, Cohen’s Kappa Coefficient 0.6482, 95% CI: 0.5801–0.7163. Conclusion: The rapid antigen detection test showed comparable sensitivity and specificity with RT-PCR assay. These results support the fact that RAT is an accurate alternative to RT-PCR in areas where there is increased testing burden, for screening of asymptomatic carriers, in areas that lack suitable laboratories to perform RT-PCR, in areas such as airport, train stations, and bus stands.

A prospective clinical evaluation of the diagnostic accuracy of the SARS-CoV-2 rapid antigen test using anterior nasal samples

IntroductionThe diagnostic accuracy of antigen testing of anterior nasal (AN) samples for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has not been evaluated in the Japanese population. This study assessed the diagnostic accuracy of the Roche SARS-CoV-2 rapid antigen test (rapid antigen test) using AN samples. MethodsTwo AN samples and one nasopharyngeal (NP) sample were collected from individuals undergoing screening for SARS-CoV-2 infection. The results of the rapid antigen test and the reverse-transcription polymerase chain reaction (RT-PCR) test using AN samples were compared to those of RT-PCR tests using NP samples. ResultsSamples were collected from 800 participants, 95 and 110 of whom tested positive for SARS-CoV-2 on RT-PCR tests of AN and NP samples, respectively. The overall sensitivity/specificity of the AN rapid antigen test and AN RT-PCR were 72.7%/100% and 86.4%/100%, respectively. In symptomatic cases, the sensitivities of the AN rapid antigen test and AN RT-PCR were 84.7% and 94.9%, respectively. In asymptomatic cases, the sensitivities of the AN rapid antigen test and AN RT-PCR were 58.8% and 76.5%, respectively. The sensitivity of the AN rapid antigen test was over 80% in cases with cycle threshold (Ct) values < 25; it significantly decreased with an increase in the Ct values (p < 0.001). ConclusionThe rapid antigen test with AN samples had a favorable sensitivity, especially in symptomatic cases or in cases with Ct values < 25. It gave no false-positive results. Compared with AN-RT PCR, the AN rapid antigen test had a modestly lower sensitivity in asymptomatic cases.

Validation of rapid antibody (IgG-IgM) test kit for SARS-CoV-2 infection in Qatar.

Background: In response to the growing coronavirus disease 2019 (COVID-19) pandemic and the shortage of laboratory based molecular testing capacity and reagents, multiple diagnostic test manufacturers have developed rapid and easy to use devices to facilitate testing outside laboratory settings. These kits are either based on detection of proteins from SARS-CoV-2 virus or detection of antigen or human antibodies generated in response to the infection. However, it is important to understand their performance characteristics and they must be validated in the local population setting.Design and methods: The objective is to assess the validity of the rapid test for IgG and IgM immunoglobulins compared to the current gold standard reverse transcription polymerase chain reaction (RT-PCR) test. A total of 16951 asymptomatic individuals were tested by the Ministry of Public Health track-and-trace team using both rapid immunodiagnostic test and RT-PCR as part of screening across various random settings with potential risk of community interaction prior to gradual lifting of restrictions in Qatar. Rapid test was considered to be posiive if both IgG and IgM are positive, while only IgG/IgM positive was considered as rapid test negative. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated.Results: The sensitivity of rapid test kit was found to be 0.9%, whereas the specificity was found to be 97.8%. the PPV was found to be 0.3% whereas the NPV was found to be 99.4%.Conclusions: Based on the outcome and results of the study, it appears that the sensitivity and PPV of the rapid antibody test are low. As such, this test is not recommended for use to assist in taking clinic-based decisions or decisions related to quarantine/isolation. Significance for public health It is important to understand the performance characteristics of the Rapid Antibody (IgG-IgM) kits and they must be validated in the local population setting before they can be recommended for use.

A Field Evaluation of the Covid-19 Antigen Testing Regimen at a Primary Hospital in Ghana

Background: Accurate, reliable and rapid diagnostic tools for COVID-19 are essential for tackling the ongoing pandemic, especially in the developing world. This study aimed to assess the performance and viability of the testing regimen at a district-level hospital testing centre where both nasopharyngeal and oropharyngeal samples are taken simultaneously but the samples are tested separately. The nasopharyngeal samples are tested using the SD Biosensor Standard F Covid-19 FIA antigen test kit whiles RT-PCR is conducted on the oropharyngeal samples. Methods: A retrospective analysis was conducted on 314 paired samples taken from March to August 2021 whereby the dual testing scheme was applied. The field diagnostic performance of the Standard F Antigen test was compared with Reverse Transcription Polymerase Chain Reaction (RT-PCR) results. The sensitivity, specificity and expected predictive values were then evaluated. The RT-PCR is the gold standard for Covid-19 testing. Results: A total of 314 matched Covid-19 Antigen Rapid Diagnostic Test (Ag-RDT) and RT-PCR samples were analysed retrospectively. The sensitivity and specificity of the Ag-RDT was 80.85% (95% Confidence interval of 66.74% – 90.85%) and 98.9% (95% Confidence interval of 96.74% – 99.77%) respectively. The overall accuracy was determined to be 96.2% with a Cohen’s Kappa score of 0.84 indicating an almost perfect agreement. Conclusion: The results of samples taken from the different anatomical sites have an almost perfect agreement. Therefore, the performance of the Standard F Covid-19 antigen test kit is optimal and the testing regimen at the hospital is adequate. It can thus be replicated at other testing sites. However, adequate training in the sampling procedure, storage, transport as well as stringent monitoring and supervision is required.

A man in his eighties with gait problems and elevated CRP.

Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in hospitals is essential for early isolation and treatment. However, false positive test results can have adverse consequences for patient safety. A man in his eighties was admitted to hospital with fatigue and new-onset gait and balance difficulties, without fever or symptoms of focal infection, but with elevated C-reactive protein. On admission, he tested positive on screening for SARS-CoV-2 using a fully automated rapid reverse transcription polymerase chain reaction (RT-PCR) test. He was placed together with two patients with confirmed COVID-19 infection in cohort isolation. Due to very low exposure risk and nonspecific symptoms, the primary and multiple subsequent test swabs were analysed using RT-PCR analyses guided by laboratory personnel, and all gave negative results. The patient had several risk factors for developing severe COVID-19 illness, but fortunately he remained COVID-19 negative on repeated tests. The case presentation highlights the danger of false-positive SARS-CoV-2 test results, and the importance of interpreting a diagnostic test in the context of pretest probability and test accuracy. It also underlines the risk of using cohort isolation instead of individual isolation.

2553. Individual Patient-Level Data Meta-Analysis of Live Attenuated and Inactivated Influenza Vaccine Effectiveness Among US Children, 2013–2014 Through 2015–2016

BackgroundQuadrivalent live attenuated influenza vaccine (LAIV4) was not recommended for use in the United States for the 2016–2017 and 2017–2018 influenza seasons based on US observational studies of vaccine effectiveness (VE) from 2013–2014 to 2015–2016. We pooled individual patient data on children aged 2–17 years enrolled in 5 US studies during these 3 influenza seasons to further investigate VE by vaccine type.MethodsAnalyses included 17,173 children enrolled in the US Department of Defense Global Laboratory-based Influenza Surveillance Program, US Influenza Vaccine Effectiveness Network, Influenza Incidence Surveillance Project, Influenza Clinical Investigation for Children, and a Louisiana State University study. Participants’ specimens were tested for influenza by reverse transcription-polymerase chain reaction (RT-PCR), culture, or a combination of rapid antigen testing and RT-PCR. VE was calculated by comparing odds of vaccination with either inactivated influenza vaccine (IIV) or LAIV4 among influenza-positive cases to test-negative controls and calculated as 100 × (1 − odds ratio) in logistic regression models with age, calendar time, influenza season, and study site (random effect). Patients were stratified by prior season vaccination status in a subanalysis.ResultsOverall, 38% of patients (N = 6,558) were vaccinated in the current season, of whom 30% (N = 1,979) received LAIV4. Pooled VE of IIV against any influenza virus was 51% (95% CI: 47, 54) versus 26% (95% CI: 15, 36) for LAIV4. Point estimates for pooled VE against any influenza by age group ranged from 45% to 58% for IIV and 19% to 34% for LAIV4 during the 3 seasons (Figures 1 and 2). Pooled VE against influenza A(H1N1)pdm09 was 67% (95% CI: 62, 72) for IIV versus 20% (95% CI: −6, 39) for LAIV4. Pooled VE against influenza A(H3N2) was 29% (95% CI: 14, 42) for IIV versus 7% (95% CI: −11, 23) for LAIV4, and VE against influenza B was 52% (95% CI: 42, 60) for IIV and 66% (95% CI: 47, 77) for LAIV4. VE against influenza A(H1N1)pdm09 was lower for LAIV4 versus IIV across all strata of prior season vaccination (Figure 3).ConclusionConsistent with individual studies, our pooled analyses found that LAIV4 effectiveness was reduced for all age groups against influenza A(H1N1)pdm09 compared with IIV. This result did not vary based on prior vaccination status. Disclosures H. Caspard, AstraZeneca: Employee, Salary.

DETECTION OF RESPIRATORY SYNCYTIAL VIRUS IN INFANTS AND YOUNG CHILDREN WITH CHEST INFECTION: A COMPARISON OF REVERSE TRANSCRIPTION-PCR TECHNIQUE TO CHROMATOGRAPHIC IMMUNOASSAY AND ENZYME LINKED IMMUNOSORBENT ASSAY

Background: Human respiratory syncytial virus (hRSV) is a major cause of viral lower respiratory tract infection among infants and young children less than 2 years old. Multiple methods are used for the laboratory diagnosis of hRSV infections, including chromatographic immunoassay, enzyme linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) technique for detection hRSV-antigens, hRSV-antibodies and hRSV-RNA, respectively. Objective: To compare the efficiency of three diagnostic methods in detection of hRSV in infants and young children with chest infection. Methods: This study included 100 hospitalized infants and young children (39 females and 61 males) aged from (1) month to (24) months, their mean age (6.87 ± 6.03) months, who required hospital admission at the Pediatric Department in Al-Imamein AL-Kadhimein Medical City Hospital, Central Teaching Pediatric Hospital, and Al-Kadhimiya Pediatric Hospital in Baghdad-Iraq. Samples were collected over a three-month winter period from January 2017 to April 2017. Fresh nasal swab specimens were collected and testes for hRSV antigens by using chromatographic immunoassay as a rapid test, in addition, nasopharyngeal/throat swabs specimens were processed for detection of hRSV-RNA by RT-PCR, both for direct detection. Also, ELISA was done to measure anti-hRSV IgM antibodies in serum for indirect detection of RSV infection. Results: hRSV was found to be positive in (27%), (56%) and (44%) of specimens by rapid chromatographic immunoassay, ELISA and RT-PCR technique, respectively. Comparing with RT-PCR, the sensitivity of rapid test was (59.09%) ranged from (44.41) to (72.31) and the specificity was (98.21%) ranged from (90.55) to (99.91) with likelihood ratio equal to (33.09), while the sensitivity of ELISA test was (75.61%) ranged from (60.66) to (86.17) and specificity was (59.62%) ranged from (46.07) to (71.84) with likelihood ratio equals to (1.87). Conclusion: The RT-PCR technique was more sensitive than antigen or antibody detection methods for the diagnosis of hRSV Keywords: hRSV, rapid chromatographic immunoassay, ELISA, RT-PCR Citation: Al-Shuwaikh AMA, Ali SH, Arif HS. Detection of respiratory syncytial virus in infants and young children with chest infection: a comparison of reverse transcription-PCR technique to chromatographic immunoassay and enzyme linked immunosorbent assay. Iraqi JMS. 2018; 16(3): 319-326. doi: 10.22578/IJMS.16.3.11

Comparative Evaluation of Norovirus Infection in Children with Acute Gastroenteritis by Rapid Immunochromatographic Test, RT-PCR and Real-time RT-PCR.

Immunochromatographic (IC) test for norovirus detection is a rapid and simple detection method. This study evaluated the sensitivity and specificity of a recent version of R-Biopharm RIDA®QUICK Norovirus IC assay for norovirus detection in fecal specimens from children hospitalized with acute gastroenteritis. Fecal specimens were tested by IC kit in comparison with gold standard reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. The IC kit showed high sensitivity and specificity comparable with PCR-based methods. None of false positive and false negative was found and the assay did not cross-react with other gastroenteritis viruses. The IC assay could detect genogroup I.5 (GI.5) and a wide range of genotypes in the GII noroviruses including GII.3, GII.4, GII.6, GII.7, GII.14, GII.15, GII.21, and also newly emerging GII.17 norovirus. In conclusion, this norovirus IC kit could be an alternative choice for rapid screening or a quick diagnostic tool for norovirus detection in fecal specimens of acute gastroenteritis patients.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

A Reliable and Rapid Multiplex RT-PCR Assay for Detection of Two Potyviruses and a Pararetrovirus Infecting Canna Plants.

Canna plants are subject to serious virus diseases. The three most common viruses identified in canna plants are Bean yellow mosaic virus, Canna yellow mottle virus, and Canna yellow streak virus. Recent studies indicate that canna plants are commonly infected with more than one virus. Thus, the efficient control of these viruses in canna plants requires the availability of a reliable method for detecting mixed virus infection. This report presents a two-step multiplex reverse-transcription polymerase chain reaction (RT-PCR) that was developed to simultaneously detect two potyviruses and one pararetrovirus genome. We optimized the method for nucleic acid isolation for managing a large population of samples, and the primer concentrations to ensure sensitivity and reliability of the assay, and determined the detection limit in simplex and multiplex RT-PCR assays using plasmid controls and nucleic acids isolated from virus-infected plants. Combined with an automated method for total nucleic acid isolation, this multiplex RT-PCR procedure could be routinely used for virus detection in research and diagnostic laboratories.

The Association between Cerebrospinal Fluid Pleocytosis and Clinical Manifestations of Enteroviral Meningitis in Children

Objective: Enteroviruses are major causes of aseptic meningitis in children. This study aimed to describe the clinical manifestations of enteroviral meningitis according to the presence of cerebrospinal fluid (CSF) pleocytosis, and to investigate the factors influencing the CSF pleocytosis in children with this condition. Methods: Eighty children with enteroviral meningitis treated at Soonchunhyang University Hospitals in Seoul and Bucheon between July 2012 and August 2013 were enrolled. The patients were diagnosed by reverse transcription-polymerase chain reaction (RT-PCR), and the clinical variables were compared according to the presence of CSF pleocytosis. Results: Of the 80 patients, 54 (67.5%) and 26 (32.5%) patients had and did not have CSF pleocytosis, respectively. Forty-eight (60%) patients were male, and the median age was 63 months (range, 2 to 192 months). Seventy-six (95%), 67 (83.7%), 51 (63.7), and 2 (2.5%) patients presented with fever, headache, vomiting, and seizure, respectively. Increased CSF protein and pressure were associated with CSF pleocytosis. However, age, peripheral white blood cell count, C-reactive protein, CSF glucose, CSF/serum glucose ratio, and onset-puncture time interval were not associated with the presence of CSF pleocytosis. Conclusion: This study demonstrated a high proportion of non-pleocytic enteroviral meningitis in children, and identified several clinical manifestations that were associated with CSF pleocytosis. The findings of this study may help us better understand the characteristics of the disease and facilitate early diagnosis and treatment of enteroviral meningitis. During the outbreak seasons of enteroviral meningitis, the importance of continuous surveillance of enteroviruses and rapid RT-PCR testing should be emphasized.

Diagnosis of nervous necrosis virus in orange-spotted grouper, Epinephelus coioides, by a rapid and convenient RT-PCR method

Viral nervous necrosis (VNN) causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction (RT-PCR) was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necrosis virus (NNV), in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), spring viraemia of carp virus (SVCV), epizootic haematopoietic necrosis virus (EHNV), and large yellow croaker iridovirus (LYCIV). With this method, the orange-spotted grouper (Epinephelus coioides) fry from hatcheries with or without incidence of the VNN epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the two hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.

Usefulness of Influenza Rapid Antigen Test in Influenza A (H1N1)

Purpose : The aim of this study was to examine the sensitivity and specificity of the influenza rapid antigen test, in comparison with reverse transcription polymerase chain reaction (RT-PCR), according to the time of the test from symptom onset and the clinical manifestations in the patients tested for suspected infection of the influenza A (H1N1) at a second hospital. Methods : A total of 529 pediatric patients, aged between 6 and 12 years old, who visited the emergency department from October 1, 2009 to December 31, 2009, received the influenza rapid antigen test and RT-PCR. We examined the sensitivity and specificity of the influenza rapid antigen test in comparison with RT-PCR according to the time of the test from symptom onset (˂24 hours, 24 to 48 hours, 48 to 72 hours, ˃72 hours) and clinical manifestations (fever, cough, rhinorrhea·nasal obstruction, sore throat, gastrointestinal symptoms, and general symptoms) in a retrospective study based on hospital charts. Results : The sensitivity of the influenza rapid antigen test at elapsed times of less than 24 hours, 24 to 48 hours, and 48 to 72 hours after the onset of the symptoms was 53.9%, 61.4%, and 62.1% respectively. When the elapse time was greater than 72 hours, the sensitivity was 31.6%; thus, the sensitivity of the influenza rapid antigen test tended to decrease with elapsed time. The sensitivity of the test was 79% in patients presenting with gastrointestinal symptoms, which was the highest, but there was no statistical difference according to the clinical manifestations of the patients. Conclusions : Our study suggests that more accurate results might be gained when the influenza rapid antigen test is performed within 72 hours after symptom onset. [Pediatr Allergy Respir Dis(Korea) 2012;22:71-77]

Sentinel Node Biopsy in Early Gastric Cancer: Constant Exertion for Clinical Application

Lymph node status is the most important prognostic factor in gastric cancer. Standard surgery, therefore, is radical subtotal or total gastrectomy with wide dissection of regional lymph nodes. Recently, the proportion of early gastric cancer has increased to more than 60% in Korea and Japan because of screening gastroscopy. The rate of lymph node metastasis in early gastric cancer ranges from 5 to 20%, which means that more than 80% of patients with early gastric cancer undergo unnecessarily wide excision of the stomach with aggressive lymphadenectomy. If there is a tool to predict the exact status of lymph nodes, radical gastrectomy with lymphadenectomy can be substituted with limited resection of stomach without lymph node dissection. However, preoperative endoscopic ultrasound and computed tomography have limited accuracy for nodal staging in gastric cancer patients. The sentinel node (SN) concept has been introduced into various solid tumors, including stomach cancer, to predict lymph node status. Ideally, if the SN has no metastasis, extensive lymphadenectomy can be avoided, the extent of resection of the stomach reduced, function preserved, complications lessened, recovery accelerated, and the quality of life for the patient improved. SN navigation surgery has been widely performed for the treatment of breast cancer and malignant melanoma. Regarding gastric cancer, it seemed that SN concept is difficult to apply because the lymphatic flow is multidirectional. However, many studies reported that SN mapping is feasible in gastric cancer especially in early gastric cancer even though many issues still remain to be solved. Sensitivity of SN biopsy is highly important in gastric cancer compared with breast cancer and malignant melanoma. When false-negative cases develop in patients with malignant melanoma or breast cancer, other treatment options, such as chemotherapy and radiotherapy exist. Because gastric cancer has no other treatment option except surgery, a gastric cancer patient with false-negative SN loses an opportunity for cure. The detection rate is not as important as sensitivity in gastric cancer SN biopsy. Surgeons should perform just conventional lymphadenectomy for detection failure cases. Sensitivities of SN mapping in gastric cancer reported until now are not adequate for clinical application. Until now, the sensitivities were generally low and even the studies with high sensitivity had small number of cases with lymph node metastasis. There are several methods to increase the sensitivity of SN biopsy in gastric cancer. As pathologic methods, multiple sections of the SN may raise the rate of detecting lymph node metastases. Immunohistochemistry (IHC) staining with anti-cytokeratin antibody helps to detect micrometastases in SNs in addition to conventional hematoxylin and eosin (HE) staining. Reverse transcription-polymerase chain reaction (RT-PCR) using tumor markers, such as carcinoembryonic antigen, cytokeratin, or CD44, also is good tool for molecular detection of SN micrometastases. Rapid IHC or real-time RT-PCR is needed so that IHC or RT-PCR is applicable to SN biopsy for gastric cancer. Takeuchi et al. 1 introduced a novel realtime multiplex RT-PCR assay that is suitable for intraoperative detection of micrometastasis in SNs for the patients with clinical N0 gastric carcinoma. In the present study, 31% of patients with no metastasis over histological diagnosis were RT-PCR positive and the diagnostic accuracy is significantly higher in their multiplex RT-PCR assay than in intraoperative histopathology. Because the time taken for the assay is just 80 minutes, real-time multiplex RT-PCR is thought to be applicable for use in the intraoperative diagnosis of SN. Society of Surgical Oncology 2011

Rapid detection for primary screening of influenza A virus: microfluidic RT-PCR chip and electrochemical DNA sensor

Rapid and definitive diagnosis is critical to the prevention of the spread of endemic human pathogenic viruses. Detection of variant specific genes by reverse transcription polymerase chain reaction (RT-PCR) has become a routine diagnostic test for accurate subtyping of RNA viruses, such as influenza. In this paper, we demonstrate the use of a continuous-flow polydimethylsiloxane (PDMS) microfluidic RT-PCR chip and disposable electrical printed (DEP) chips for rapid amplification and sensing of new influenza (AH1pdm) virus of swine-origin. The RT-PCR chip consisted of four zones: RT reaction zone, initial denaturation zone, thermal cycle zone for PCR (2-step PCR) and pressurizing-channel zone for preventing air-bubble formation. In order to measure electrochemical signals, methylene blue (MB), an electro-active DNA intercalator, was added to the RT-PCR mixture. The RT-PCR was completed within 15 min which was the total flow-through time from the inlet to the outlet, and the reduction signals from amplifications could be detected quickly on the DEP chip. The MB reduction current on the DEP chip with the amplicon significantly reduced compared to non-amplified controls. This microfluidic platform for rapid RT-PCR and the DEP chip for quick electrochemical sensing are suitable for integration, and have the potential to be a portable system for diagnostic tests.

High-speed RNA microextraction technology using magnetic oligo-dT beads and lateral magnetophoresis

This paper presents a high-speed RNA microextractor for the direct isolation of RNA from peripheral blood lysate using magnetic oligo-dT beads. The extraction is achieved through lateral magnetophoresis, generated by a ferromagnetic wire array inlaid on a glass substrate. This RNA microextractor separated more than 80% of magnetic beads with a flow rate up to 20 ml h(-1), and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein (A(260)/A(280)) was >1.7, indicating that the extraction technology yielded nearly pure RNA. The feasibility of this technique was evaluated further for its applicability to reverse transcription polymerase chain reaction (RT-PCR) procedures by performing cDNA synthesis and PCR. The analysis verified that the RNA microextractor is a practical method for easy, rapid, and high-precision RT-PCR using minimal reagent volumes without requiring highly trained personnel. In addition, it can be readily incorporated into genetic analysis procedures for realizing automated on-chip genetic platforms in a micro format.

A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at −80 °C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.

A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at −80 °C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.