Detection of Anti-SARS-CoV-2 Nucleocapsid and Spike Antibodies in Patients with Coronavirus Disease 2019 in Japan.

OBJECTIVES: Coronavirus Disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Serological testing for anti-SARS-CoV-2 nucleocapsid (N) antibodies (Abs) and anti-SARS-CoV-2 spike (S) Abs is performed to detect prior COVID-19 infection. It is still controversial which antibodies are the most sensitive and specific, and which can be detected earliest after infection. Here, we evaluated the results of serological tests of anti-SARS-CoV-2 N and S Abs in Japan. METHODS: Symptomatic COVID-19 patients (n = 84) and control patients with rheumatoid arthritis (n = 93) were recruited at Tokyo National Hospital. Anti-SARS-CoV-2 N and S Abs were measured by commercial electrochemiluminescence immunoassays. RESULTS: The fraction of patients positive for anti-SARS-CoV-2 N and S Abs was highest >14 days after symptom onset. The frequency of anti-SARS-CoV-2 S Ab positivity at this time (80.4%) tended to be slightly but not significantly lower than anti-SARS-CoV-2 N Ab positivity (84.8%). Optimized cut-off levels for anti-SARS-CoV-2 N and S Ab positivity were lower than the manufacturer's recommended cut-off levels. Using multiple linear regression analyzes with anti-SARS-CoV-2 N and S Abs, we created an Ab-index with high sensitivity. CONCLUSION: To increase the sensitivity of serological diagnostic tests for COVID-19, it is suggested that both anti-SARS-CoV-2 N and S Abs should be measured and cut-off levels decreased.

High detection rate of azole-resistant Aspergillus fumigatus after treatment with azole antifungal drugs among patients with chronic pulmonary aspergillosis in a single hospital setting with low azole resistance.

The prevalence of azole-resistant Aspergillus fumigatus (ARAF) among chronic pulmonary aspergillosis (CPA) patients treated with azoles in Japan is unknown. The aim of this study was to determine the detection rate of ARAF in isolates from CPA patients who were treated with azoles for varying durations. The potential mechanism of acquiring resistance was examined by sequencing cyp51A and hmg1, two genes associated with ARAF. A. fumigatus isolates (n = 120) were collected from CPA patients (n = 104) between February 2012 and February 2019, at National Hospital Organization Tokyo National Hospital. The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF among all isolates was 8.3% (n = 10). Of the 10 resistant isolates, eight were ITCZ-resistant and five were VRCZ-resistant. Among 47 isolates obtained from 36 CPA patients who were treated with ITCZ (for an average of 256 days) and/or VRCZ (for an average of 29 days), the resistance rates were 17.0% and 10.6%, respectively. In addition, 46.2% of 13 isolates obtained from CPA patients with ongoing azole treatment at the time of antifungal therapy failure were resistant to azoles. Among the 10 ARAF isolates, a point mutation was detected in cyp51A in seven isolates and in hmg1 in two isolates. ARAF was detected at a high rate in CPA patients, particularly in those with ongoing long-term azole treatment, at the time of azole antifungal therapy failure.

Aspergillus fumigatus can acquire azole resistance during long-term treatment with azole drugs in patients with chronic pulmonary aspergillosis (CPA). The aim of this study was to determine the detection rate of azole-resistant A. fumigatus (ARAF) in isolates from CPA patients who had been treated with azoles. In addition, a potential mechanism of acquiring resistance was examined by sequencing cyp51A and hmg1, two genes associated with ARAF. A. fumigatus isolates (n = 120) were collected from CPA patients (n = 104). The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF from all isolates was 8.3% (n = 10). Greater than 10% of the 47 isolates obtained from 36 CPA patients who had been treated with azoles exhibited resistance. Furthermore, 46.2% of 13 isolates obtained from CPA patients with ongoing azole treatment at the time of antifungal therapy failure were resistant to azoles. Among the 10 ARAF isolates, a point mutation was detected in cyp51A in seven isolates and in hmg1 in two isolates. ARAF was detected at a high rate in CPA patients undergoing long-term azole treatment at the time of antifungal therapy failure.

Validation of a Method for Quantification of Lutein in Spinach Using High-Performance Liquid Chromatography: Interlaboratory Study.

BACKGROUND: Lutein is gaining attention as a strong antioxidant contained in foods. It accumulates in the human blood and retina, and is considered to play an important role in the body, especially in the eyes. OBJECTIVE: A method to determine the lutein content of raw spinach (Spinacia oleracea L.) was developed with the aim of its enactment as a Japanese agricultural standard (JAS) measurement method for components beneficial to human health. METHODS: An interlaboratory study was conducted to evaluate an analytical method for the determination of lutein in spinach. The detection limit and quantification limit of lutein for this method were 0.2 and 0.7 mg/kg, respectively. Twelve participating laboratories independently analyzed test samples (five pairs of blind duplicates) using high-performance liquid chromatography (HPLC). RESULTS: After removal of a few outliers, the repeatability relative standard deviation (RSDr), reproducibility (RSDR), and predicted RSDR of the evaluated method were 3.4-7.5, 4.6-13, and 7.5-8.5%, respectively, in a concentration range from 64.9-150 mg/kg. CONCLUSIONS: The HorRat values (RSDR/predicted RSDR) of the lutein concentration were calculated to be 0.61-1.6. HIGHLIGHTS: The study results indicate the acceptable precision of this method.

A case of pembrolizumab-induced hemophagocytic lymphohistiocytosis successfully treated with pulse glucocorticoid therapy.

Treatments using immune checkpoint inhibitors such as pembrolizumab lead to immune mediated adverse effects including hemophagocytic lymphohistiocytosis (HLH). Herein, we present a case where HLH developed after pembrolizumab administration, which was treated using high dose prednisolone. He developed high-grade fever complicated with liver dysfunction and diarrhea 7 days after pembrolizumab administration. Although treatment with oral prednisolone alleviated the symptoms, other adverse effects arose owing to a tapered prednisolone dose. Hyperferritinemia suggested the diagnosis of HLH and met the criteria for HLH diagnosis. He was thus administered intravenous pulses of methylprednisolone followed by high-dose oral prednisolone, which resolved these symptoms.

Interlaboratory study of qualitative PCR methods for genetically modified maize events MON810, bt11, GA21, and CaMV P35S.

Qualitative PCR methods for the genetically modified (GM) maize events MON810, Bt11, and GA21, and the 35S promoter (P35S) region of the cauliflower mosaic virus (CaMV) were evaluated in an interlaboratory study. Real-time PCR-based quantitative methods for these GM events using the same primer pairs had already been validated in previous studies. Fifteen laboratories in Japan participated in this interlaboratory study. Each participant extracted DNA from blind samples, performed qualitative PCR assays, and then detected the PCR products with agarose gel electrophoresis. The specificity, sensitivity, and false-negative and false-positive rates of these methods were determined with different concentrations of GM mixing samples. LODs of these methods for MON810, Bt11, GA21, and the P35S segment calculated as the amount of MON810 were 0.2, 0.2, 0.1, and 0.2% or less, respectively, indicating that the LODs of MON810, Bt11, and P35S were lower than 10 copies, and the LOD of GA21 was lower than 25 copies of maize haploid genome. The current study demonstrated that the qualitative methods would be fit for the detection and identification of these GM maize events and the P35S segment.

[Development and validation of event-specific quantitative PCR method for genetically modified maize LY038].

In this article, we report a novel real-time PCR-based analytical method for quantitation of the GM maize event LY038. We designed LY038-specific and maize endogenous reference DNA-specific PCR amplifications. After confirming the specificity and linearity of the LY038-specific PCR amplification, we determined the conversion factor required to calculate the weight-based content of GM organism (GMO) in a multilaboratory evaluation. Finally, in order to validate the developed method, an interlaboratory collaborative trial according to the internationally harmonized guidelines was performed with blind DNA samples containing LY038 at the mixing levels of 0, 0.5, 1.0, 5.0 and 10.0%. The precision of the method was evaluated as the RSD of reproducibility (RSDR), and the values obtained were all less than 25%. The limit of quantitation of the method was judged to be 0.5% based on the definition of ISO 24276 guideline. The results from the collaborative trial suggested that the developed quantitative method would be suitable for practical testing of LY038 maize.

Development and interlaboratory validation of quantitative polymerase chain reaction method for screening analysis of genetically modified soybeans.

A novel real-time polymerase chain reaction (PCR)-based quantitative screening method was developed for three genetically modified soybeans: RRS, A2704-12, and MON89788. The 35S promoter (P35S) of cauliflower mosaic virus is introduced into RRS and A2704-12 but not MON89788. We then designed a screening method comprised of the combination of the quantification of P35S and the event-specific quantification of MON89788. The conversion factor (Cf) required to convert the amount of a genetically modified organism (GMO) from a copy number ratio to a weight ratio was determined experimentally. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDR), respectively. The determined RSDR values for the method were less than 25% for both targets. We consider that the developed method would be suitable for the simple detection and approximate quantification of GMO.

Development and validation of event-specific quantitative PCR method for genetically modified maize MIR604.

A GM maize event, MIR604, has been widely distributed and an analytical method to quantify its content is required to monitor the validity of food labeling. Here we report a novel real-time PCR-based quantitation method for MIR604 maize. We developed real-time PCR assays specific for MIR604 using event-specific primers designed by the trait developer, and for maize endogenous starch synthase IIb gene (SSIIb). Then, we determined the conversion factor, which is required to calculate the weight-based GM maize content from the copy number ratio of MIR604-specific DNA to the endogenous reference DNA. Finally, to validate the developed method, an interlaboratory collaborative trial according to the internationally harmonized guidelines was performed with blind samples containing MIR604 at the mixing levels of 0, 0.5, 1.0, 5.0 and 10.0%. The reproducibility (RSDr) of the developed method was evaluated to be less than 25%. The limit of quantitation of the method was estimated to be 0.5% based on the ISO 24276 guideline. These results suggested that the developed method would be suitable for practical quantitative analyses of MIR604 maize.

Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

Practicable group testing method to evaluate weight/weight GMO content in maize grains.

Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the method's user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.

Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12.

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

Establishment and evaluation of event-specific quantitative PCR method for genetically modified soybean MON89788.

A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C(f)) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON89788.

Synthesis, characterization, and detailed electrochemistry of binuclear ruthenium(III) complexes bridged by bisacetylacetonate. Crystal and molecular structures of [[Ru(acac)2]2(tae)] (acac = 2,4-pentanedionate ion, tae = 1,1,2,2-tetraacetylethanate dianion).

Binuclear beta-diketonatoruthenium(III) complexes [[Ru(acac)(2)](2)(tae)], [[Ru(phpa)(2)](2)(tae)], and [(acac)(2)Ru(tae)Ru(phpa)(2)] and binuclear and mononuclear bipyridine complexes [[Ru(bpy)(2)](2)(tae)](PF(6))(2) and [Ru(bpy)(2)(Htae)]PF(6) (acac = 2,4-pentanedionate ion, phpa = 2,2,6,6-tetramethyl-3,5-heptanedionate ion, tae = 1,1,2,2-tetraacetylethanate dianion, and bpy = 2,2'-bipyridine) were synthesized. The new complexes have been characterized by (1)H NMR, MS, and electronic spectral data. Crystal and molecular structures of [[Ru(acac)(2)](2)(tae)] have been solved by single-crystal X-ray diffraction studies. Crystal data for the meso isomer of [[Ru(acac)(2)](2)(tae)] have been confirmed by the dihedral angle result that two acetylacetone units of the bridging tae ligand are almost perpendicular to one another. A detailed investigation on the electrochemistry of the binuclear complexes has been carried out. The electrochemical behavior details of the binuclear complexes have been compared with those of the mononuclear complexes obtained from the half-structures of the corresponding binuclear complexes. Studies on the effects of solvents on the mixed-valence states of Ru(II)-Ru(III) and Ru(III)-Ru(IV) complexes have been carried out by various voltammetric and electrospectroscopic techniques. A correlation between the comproportionation constant (K(c)) and the donor number of the solvent has been obtained. The K(c) values for the binuclear complexes have been found to be low because of the fact that two acetylacetone units of the bridging tae ligand are not in the same plane, as revealed by the crystal structure of [[Ru(acac)(2)](2)(tae)].