Evaluation of SARS-CoV-2 RNA quantification by RT-LAMP compared to RT-qPCR.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is a global pandemic caused by a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral load of SARS-CoV-2 is associated with mortality in COVID-19 patients. Measurement of viral load requires the use of reverse transcription quantitative PCR (RT-qPCR), which in turn requires advanced equipment and techniques. In this study, we aimed to evaluate the viral load measurement using reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is a simpler procedure compared to RT-qPCR. MATERIALS AND METHODS: RNA was extracted by using the QIAamp Viral RNA Mini Kit. The RT-LAMP assay was performed by using the Loopamp® 2019-SARS-CoV-2 detection reagent kit and 10-fold serial dilutions of known viral load RT-LAMP were used to measure Tt, which is the time until the turbidity exceeds the threshold. Based on the relationship between viral load and Tt, the linearity and detection sensitivity of the calibration curve were evaluated. In addition, 117 clinical specimens were measured, and RT-qPCR and RT-LAMP assay results were compared. RESULTS: The dilution linearity of the calibration curve was maintained at five orders of magnitude 1.0× 106 to 1.0 × 101 copies/µL, and was confirmed to be detectable down to 1.0 × 100 copies/µL. The limit of quantification of RNA extracted from clinical specimens using RT-LAMP correlated well with that obtained using RT-qPCR (r2 = 0.930). CONCLUSION: The findings indicate that RT-LAMP is an effective method to determine the viral load of SARS-CoV-2.

[Emergency medical care and POCT].

Emergency testing is intended to elucidate the patient's condition; therefore, the ideal emergency testing should be able to be implemented quickly and easily at the patient bedside for 24 hours a day, and the time from test order to result should be as short as possible. In other words, a shorter turn around time (TAT) is better. Needless to say, the most effective way to reduce TAT is to omit all other processes except testing from the test procedure, and bring it as close to the measuring time = TAT as possible. Because we think of emergency testing in this way, the concept of point of care testing (POCT) is the essence of emergency testing and does not threaten the emergency testing domain of laboratory technicians but is of medical origin and aims at the rapid appropriate treatment of patients.

[Development of analysis of drugs and toxic substances that can play an immediate role in emergency medical service: what is to be analyzed?].

Our laboratory was capable of analyzing less than 20 drugs and toxic substances at the time of the establishment of the Center in 1994. Since the poisoning crimes in 1998, such as the curry poisoning with arsenic in Wakayama, the sodium azide poisoning in Niigata, and the potassium cyanide poisoning in Nagano, we have introduced methods for rapid qualitative analysis of arsenic compounds, cyanides and azides, and developed methods for qualitative analysis of three types of surfactants (cationic, anionic, and nonionic) on the basis of the statistics for intoxication patients transferred to the Center. In 1999, the Analysis Method Investigation Committee of the Japanese Society for Clinical Toxicology requested individual medical institutions to analyze 15 selected intoxicating substances, focusing on the following three aspects. 1. Intoxication with a high degree of fatality. 2. Intoxication where analysis plays an immediate role in treatment. 3. Intoxication with a high frequency of requests by clinical physicians for analysis. The selected substances included methanol, barbital drugs, benzodiazepines, tricyclic or tetracyclic antidepressants, methamphetamine, acetaminophen, salicylic acid, bromovalerylurea, organophosphorus pesticides, carbamate pesticides, paraquat, glufosinate, cyanides, arsenic, and theophylline. Responding to the Committee's request, out laboratory has been making efforts so that analysis of drugs and intoxicating substances can play an immediate role in emergency medical service, giving the highest priority to the aforementioned 15 substances. As a result, anyone of us can now rapidly analyze about 35 substances, including those listed by the Society, day and night.

Impact of mobile clinical analyzers on disaster medicine: a lesson from crush syndrome in the 1995 Hanshin-Awaji earthquake.

The Great Hanshin-Awaji earthquake caused many people to develop crush syndrome. Analysis of these patients revealed that the severity is not related to their hemodynamics but to hematocrit, base deficits, and potassium concentrations soon after their extrications. In the disaster site, these parameters can only be measured using whole-blood samples by mobile instruments. The present study was made to evaluate the possibility of uses of a mobile measuring device, ABL77, for the triage of crush syndrome patients in disaster sites. Heparin-added blood samples and serum samples were collected from patients admitted to Senshu Critical Care Medical Center. Blood gases, electrolytes, and hematocrit of the heparin-added blood samples were measured using ABL77 and compared with those measured using the stationary device ABL725. Potassium concentrations of the heparin-added blood samples measured by the ABL77 were compared with those of the serum samples measured by the stationary EA06T. Significant correlations were observed between the measurements. We conclude that the ABL77 was satisfactorily compatible with stationary devices in the hospital. Medical institutions should have simplified, mobile measuring devices as a precaution against disasters, so that they can get ready to take appropriate action promptly.

[Examination of drugs and poisons in institutions not equipped with an analyzer of them and poisons--focusing on the simple qualitative test of surfactants].

We examined the simple qualitative test of surfactants, and discussed its possible usefulness in clinical emergency medicine and the analysis system in institutions not equipped with an analyzer of drugs and poisons. The procedures for the cobalt thiocyanate ammonium test, methylene blue chloroform test, and bromphenol blue test are easy, if reagents are prepared beforehand. If concomitantly used, these tests enable analysis of ion properties of surfactants. These tests thus seem useful for the screening of drugs and poisons in emergency medicine. We think that it is possible for institutions not equipped with an analyzer of drugs and poisons to establish a system to support clinical practice by giving priority to the establishment of drugs and poisons that frequently cause intoxication as well as of items for the simple qualitative test that are specified in "proposals for the guidelines for the analysis of drugs and poisons" advanced by the Japanese Society for Clinical Toxicology.