Diagnostic accuracy of direct reverse transcription-polymerase chain reaction using guanidine-based and guanidine-free inactivators for SARS-CoV-2 detection in saliva samples.

This study aimed to evaluate diagnostic accuracy of SARS-CoV-2 RNA detection in saliva samples treated with a guanidine-based or guanidine-free inactivator, using nasopharyngeal swab samples (NPS) as referents. Based on the NPS reverse transcription-polymerase chain reaction (RT-PCR) results, participants were classified as with or without COVID-19. Fifty sets of samples comprising NPS, self-collected raw saliva, and saliva with a guanidine-based, and guanidine-free inactivator were collected from each group. In patients with COVID-19, the sensitivity of direct RT-PCR using raw saliva and saliva treated with a guanidine-based and guanidine-free inactivator was 100.0%, 65.9%, and 82.9%, respectively, with corresponding concordance rates of 94.3% (κ=88.5), 82.8% (κ=64.8), and 92.0% (κ=83.7). Among patients with a PCR Ct value of <30 in the NPS sample, the positive predictive value for the three samples was 100.0%, 80.0%, and 96.0%, respectively. The sensitivity of SARS-CoV-2 RNA detection was lower in inactivated saliva than in raw saliva and lower in samples treated with a guanidine-based than with a guanidine-free inactivator. However, in individuals contributing to infection spread, inactivated saliva showed adequate accuracy regardless of the inactivator used. Inactivators can be added to saliva samples collected for RT-PCR to reduce viral transmission risk while maintaining adequate diagnostic accuracy.

A case of prosthetic joint infection caused by Mycobacterium tuberculosis complicated secondary bacterial infection after knee joint replacement surgery.

Mycobacterium tuberculosis (M. tuberculosis) is a rare cause of prosthetic joint infection (PJI). Previous studies have reported that many cases of PJI caused by M. tuberculosis have no medical history of active tuberculosis (TB) or other localization, which contributes to diagnostic difficulties. Furthermore, owing to the limited number of studies on treatment, appropriate treatment strategies, such as the duration of anti-tuberculosis (anti-TB) drugs and surgical indications, remain unclear. We report a case of PJI caused by M. tuberculosis and secondary pyogenic arthritis caused by Staphylococcus aureus and Streptococcus dysgalactiae in a 67-year-old man after knee joint replacement surgery in Japan, which was a moderately endemic country until 2020 and a low endemic country since 2021. Although he had no past medical history or close contact with TB, he was diagnosed with PJI caused by M. tuberculosis, following the culture of a synovectomy specimen. He underwent two-stage surgery and was treated with anti-TB drugs for a total of 12 months and recovered without recurrence. Based on our case and previous studies, there are three points of clinical significance for PJI caused by M. tuberculosis. First, about one year of anti-TB drugs with two staged joint revision resulted in a good course of treatment. Second, surgical treatment might be considered in cases complicated by secondary bacterial infection. Third, because the diagnosis of PJI caused by M. tuberculosis is difficult, TB should be considered in the differential diagnosis of routine bacterial culture-negative PJI, especially in endemic areas.

The development of SARS-CoV-2 PCR testing methods at a designated medical institution for specific infectious diseases in Japan.

Due to the coronavirus disease 2019 (COVID-19) pandemic, we have been conducting polymerase chain reaction (PCR) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at this facility since March 2020. In the early days, the PCR testing system had limited capabilities, so testing had to be conducted manually and only a few tests were conducted. Moreover, we lacked sufficient experience to conduct PCR testing manually, so we struggled with the manual work, which required intense concentration, and we felt pressured not to make mistakes such as allowing contamination. Since we introduced upgraded equipment, new methods, and additional staff for testing and we cooperated with the clinical technologist on the night shift in the Emergency Department, we are currently able to conduct urgent PCR testing on more than 2,000 specimens per month 24 hours a day. We will continue to meet new needs for COVID-19 treatment with the cooperation of other departments.

Negative Results of Nucleic Acid Amplification Tests for SARS-CoV-2 in Clinical Practice May Vary among Six Molecular Assays in Patients with COVID-19.

Several commercial nucleic acid amplification tests (NAATs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed. We used 6 kits available in Japan in 13 NAAT-positive specimens with crossing point values >36 and 7 NAAT-negative specimens from patients with coronavirus disease 2019 (COVID-19), and their results were compared. Specimens positive in ≥2 assays were considered true-positive and examined for concordance with the specimen results. The SARS-CoV-2 Detection Kit -Multi- (Toyobo M; Toyobo, Osaka, Japan) using extracted RNA had the highest concordance (κ = 1.00). This was followed by Cobas® SARS-CoV-2 (Roche, Basel, Switzerland) (κ = 0.79). There was a weak correlation between the number of negative results for each kit and the number of days between onset and testing (Spearman rank correlation: ρ = 0.44; P < 0.05). We believe that the variations in results among kits for specimens with low viral loads should not be problematic when these kits are used for screening infectious patients because these variations are more likely to be observed in specimens tested many days after onset (i.e., those that have lost their infectivity). However, it may be better to use a test for suspected late-stage COVID-19 with a low viral load, such as Toyobo M or Cobas.

Effective screening strategies for detection of asymptomatic COVID-19 travelers at airport quarantine stations: Exploratory findings in Japan.

The quantitative reverse transcription polymerase chain reaction method using nasopharyngeal swabs (NPS RT-qPCR) is regarded as the reference standard for diagnosing coronavirus disease 2019 (COVID-19). However, when using NPS RT-qPCR at busy airport quarantine stations, there are constraints on testing capacity, time, travelerstolerance, and availability of personal protective equipment for quarantine officers. A feasible alternative is therefore needed to test incoming travelers, especially when passenger numbers increase with the resumption of business, tourism, and economic activities. To explore alternatives to NPS RT-qPCR, we collected nasopharyngeal, anterior nasal, and saliva samples chronologically over days 1-7 from asymptomatic COVID-19 air travelers who were under quarantine at a designated facility, and we then compared test results for 9 different methods, comprising RT-qPCR (including the reference method), loop-mediated isothermal amplification (LAMP), and qualitative and quantitative antigen testing. We evaluated sensitivity for 97 person-day samples independently to evaluate asymptomatic travelers regardless of their testing date and period of asymptomatic status upon entry. Sensitivity of the different tests varied from 46.6% to 81.0%, but this was improved from 72.7% to 100.0% when the viral load was > 10 4 copies/sample on NPS RT-qPCR. Thus, most high-risk asymptomatic travelers with higher viral load would be detected by the tests evaluated. Quantitative antigen testing using saliva samples showed 90.9% sensitivity and provided quicker results, and should be an acceptable alternative to NPS RT-qPCR at busy airport quarantine stations. We discuss the implications of our exploratory findings for establishing a comprehensive and feasible testing strategy for COVID-19 among air passengers.

Diagnostic accuracy of nasopharyngeal swab, nasal swab and saliva swab samples for the detection of SARS-CoV-2 using RT-PCR.

BACKGROUND: The current gold standard in coronavirus disease (COVID-19) diagnostics is the real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal swab (NPS) samples. Alternatively, nasal swab (NS) or saliva swab (SS) specimens are used, although available data on test accuracy are limited. We examined the diagnostic accuracy of NPS/NS/SS samples for this purpose. METHODS: Ten patients were included after being tested positive for SARS-CoV-2 RT-PCR in NPS samples according to the National Institute of Infectious Disease guidelines. In comparison with this conventional diagnostic method, NPS/NS/SS samples were tested using the cobas 6800 systems RT-PCR device. To investigate the usefulness of the cobas method and the difference among sample types, the agreement and sensitivity were calculated. Five to six samples were collected over a total period of 5-6 d from each patient. RESULTS: Fifty-seven sets of NPS/NS/SS samples were collected, of which 40 tested positive for COVID-19 by the conventional method. Overall, the concordance rates using the conventional method were 86.0%/70.2%/54.4% for NPS/NS/SS samples (cobas); however, for samples collected up to and including on Day 9 after disease onset (22 negative and one positive specimens), the corresponding rates were 95.7%/87.0%/65.2%. The overall sensitivity estimates were 100.0%/67.5%/37.5% for NPS/NS/SS samples (cobas). For samples up to 9 d after onset, the corresponding values were 100.0%/86.4%/63.6%. CONCLUSIONS: NS samples are more reliable than SS samples and can be an alternative to NPS samples. They can be a useful diagnostic method in the future.

Molecular epidemiology of ß-lactamase production in penicillin-susceptible Staphylococcus aureus under high-susceptibility conditions.

Little is known about the prevalence of ß-lactamase production in penicillin-susceptible Staphylococcus aureus isolates under high-susceptibility conditions. We analyzed S. aureus isolates with penicillin G minimum inhibitory concentration (MIC) ≤ 0.12 µg/ml that were recovered from in-/outpatients (n = 108) between 2016 and 2017 in Japan. ß-Lactamase production was detected by nitrocefin-based and Clinical and Laboratory Standards Institute penicillin zone edge testing and blaZ PCR. All isolates were categorized as having penicillin G MIC ≤0.03 µg/ml using an automated system; MICs determined based on the microdilution method were 0.016 µg/ml (2%), 0.03 µg/ml (44%), and 0.06 µg/ml (54%). Notably, no isolates harbored the blaZ gene. The results from the nitrocefin-based and zone edge tests were consistent with those obtained by PCR. S. aureus isolates with penicillin G MIC ≤0.03 µg/ml exhibited a low frequency of ß-lactamase production. Thus, screening for ß-lactamase production may be unnecessary for isolates showing such high susceptibility.