Clinical evaluation of the rapid nucleic acid amplification point-of-care test (Smart Gene SARS-CoV-2) in the analysis of nasopharyngeal and anterior nasal samples.

INTRODUCTION: Smart Gene is a point-of-care (POC)-type automated molecular testing platform that can be performed with 1 min of hands-on-time. Smart Gene SARS-CoV-2 is a newly developed Smart Gene molecular assay for the detection of SARS-CoV-2. The analytical and clinical performance of Smart Gene SARS-CoV-2 has not been evaluated. METHODS: Nasopharyngeal and anterior nasal samples were prospectively collected from subjects referred to the local PCR center from March 25 to July 5, 2021. Two swabs were simultaneously obtained for the Smart Gene SARS-CoV-2 assay and the reference real-time RT-PCR assay, and the results of Smart Gene SARS-CoV-2 were compared to the reference real-time RT-PCR assay. RESULTS: Among a total of 1150 samples, 68 of 791 nasopharyngeal samples and 51 of 359 anterior nasal samples were positive for SARS-CoV-2 in the reference real-time RT-PCR assay. In the testing of nasopharyngeal samples, Smart Gene SARS-CoV-2 showed the total, positive and negative concordance of 99.2% (95% confidence interval [CI]: 98.4-99.7%), 94.1% (95% CI: 85.6-98.4%) and 99.7% (95% CI: 99.0-100%), respectively. For anterior nasal samples, Smart Gene SARS-CoV-2 showed the total, positive and negative concordance of 98.9% (95% CI: 97.2-99.7%), 98.0% (95% CI: 89.6-100%) and 99.0% (95% CI: 97.2-99.8%), respectively. In total, 5 samples were positive in the reference real-time RT-PCR assay and negative in the Smart Gene SARS-CoV-2 assay, whereas 5 samples were negative in the reference real-time RT-PCR assay and positive in the Smart Gene SARS-CoV-2 assay. CONCLUSION: Smart Gene SARS-CoV-2 showed sufficient analytical performance for the detection of SARS-CoV-2 in nasopharyngeal and anterior nasal samples.

The evaluation of a novel digital immunochromatographic assay with silver amplification to detect SARS-CoV-2.

INTRODUCTION: Rapid antigen tests are convenient for diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, they have lower sensitivities than nucleic acid amplification tests. In this study, we evaluated the diagnostic performance of Quick Chaser® Auto SARS-CoV-2, a novel digital immunochromatographic assay that is expected to have higher sensitivity than conventional antigen tests. METHODS: A prospective observational study was conducted between February 8 and March 24, 2021. We simultaneously obtained two nasopharyngeal samples, one for evaluation with the QuickChaser® Auto SARS-CoV-2 antigen test and the other for assessment with reverse transcription PCR (RT-PCR), considered the gold-standard reference test. The limit of detection (LOD) of the new antigen test was compared with those of four other commercially available rapid antigen tests. RESULTS: A total of 1401 samples were analyzed. SARS-CoV-2 was detected by reference RT-PCR in 83 (5.9%) samples, of which 36 (43.4%) were collected from symptomatic patients. The sensitivity, specificity, positive predictive value, and negative predictive value were 74.7% (95% confidence interval (CI): 64.0-83.6%), 99.8% (95% CI: 99.5-100%), 96.9% (95% CI: 89.2-99.6%), and 98.4% (95% CI: 97.6-99.0%), respectively. When limited to samples with a cycle threshold (Ct) < 30 or those from symptomatic patients, the sensitivity increased to 98.3% and 88.9%, respectively. The QuickChaser® Auto SARS-CoV-2 detected 34-120 copies/test, which indicated greater sensitivity than the other rapid antigen tests. CONCLUSIONS: QuickChaser® Auto SARS-CoV-2 showed sufficient sensitivity and specificity in clinical samples of symptomatic patients. The sensitivity was comparable to RT-PCR in samples with Ct < 30.

Dissemination in Japan of multidrug-resistant Pseudomonas aeruginosa isolates producing IMP-type metallo-ß-lactamases and AAC(6')-Iae/AAC(6')-Ib.

The spread throughout Japan of antibiotic-resistance factors in multidrug-resistant (MDR) Pseudomonas aeruginosa isolates was investigated epidemiologically, using immunochromatographic assays specific for IMP-type metallo-ß-lactamases (IMPs) and aminoglycoside 6'-N-acetyltransferase [AAC(6')]-Iae and -Ib. Three hundred MDR P. aeruginosa isolates were obtained during each of two years, 2011 and 2012, from 190 hospitals in 39 prefectures in Japan. The percentage of P. aeruginosa isolates producing IMPs, AAC(6')-Iae or AAC(6')-Ib increased significantly from 170/300 (56.7%) in 2011 to 230/300 (76.7%) in 2012, with 134/170 (78.8%) in 2011 and 179/230 (77.8%) in 2012 producing both IMP and either AAC(6')-Iae or AAC(6')-Ib. The MICs of antibiotics, including cephalosporins and carbapenems, were markedly higher for isolates that did than did not produce these resistance factors. These results indicated that MDR P. aeruginosa producing IMPs, AAC(6')-Iae or AAC(6')-Ib have spread throughout Japan and that these antibiotic-resistance factors are useful markers for monitoring MDR P. aeruginosa in Japan.

Production of monoclonal antibodies specific for the recombinant viral coat protein of Apple stem grooving virus-citrus isolate and their application for a simple, rapid diagnosis by an immunochromatographic assay.

A simple and rapid immunochromatographic assay (ICA) for the diagnosis of Apple stem grooving virus (ASGV) in citrus was developed. Nine lines of monoclonal antibodies (mAbs) were produced by immunizing with a recombinant viral coat protein of ASGV as the antigen. According to the competitive-binding ELISA results, the 9 mAbs comprised 2 paratope groups, A and B. After screening for the most effective combination of mAbs, the two lines from different paratope groups (4A12 from group A and 6N31 from group B) were used to create a colloidal gold conjugate and for the test line, respectively, in ICA test plate preparation. The ICA detection using this test plate was accurate for positive and negative samples, and ASGV was detectable to a dilution of 1:2430 for the infected citrus sample. Furthermore, ICA was more sensitive than ELISA for the detection of ASGV isolates in citrus. The simple and sensitive ICA for ASGV provides a straightforward method for diagnosis by non-experts, including nursery workers and growers.

Development of an immunochromatographic assay for rapid detection of AAC(6')-Ib-producing Pseudomonas aeruginosa.

To detect aminoglycoside 6'-N-acetyltransferase-Ib [AAC(6')-Ib]-producing, Pseudomonas aeruginosa isolates which are a frequent cause of nosocomial infections in Japan, an immunochromatographic assay was developed using two kinds of monoclonal antibodies (mAbs) recognizing AAC(6')-Ib. The results of the assessment were fully consistent with those of aac(6')-Ib PCR analyses.

Emergence of a novel multidrug-resistant Pseudomonas aeruginosa strain producing IMP-type metallo-ß-lactamases and AAC(6')-Iae in Japan.

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates producing IMP-type metallo-ß-lactamases (MBLs) and aminoglycoside 6'-N-acetyltransferase [AAC(6')-Iae] has become a serious problem in medical settings in Japan. A total of 217 MDR P. aeruginosa isolates were obtained from August 2009 to April 2010 from patients at 144 hospitals in Japan, of which 145 (66.8%) were positive for IMP-type MBLs and AAC(6')-Iae when tested with an immunochromatographic assay. Polymerase chain reaction (PCR) showed that these isolates were also positive for blaIMP and aac(6')-Iae genes. When these IMP-type MBL- and AAC(6')-Iae-producing isolates were analysed by pulsed-field gel electrophoresis (PFGE), two clusters (I and II) were detected. Most of the isolates (88.3%; 128/145) were grouped under cluster I and had multilocus sequence type ST235 and serotype O11, except for one isolate that was ST991 and serotype O3. The isolates were mainly isolated from the urinary tract (82/145; 56.6%) and respiratory tract (58/145; 40.0%). The epidemiological properties of the isolates belonging to cluster I were similar to those of MDR P. aeruginosa isolates that have been previously reported in Japan. The remaining 16 isolates belonged to cluster II, had identical PFGE patterns and were multilocus sequence type ST991 and serotype O18; all of these isolates were isolated from the respiratory tract. The properties of isolates belonging to cluster II have not been previously described, indicating that a novel IMP-type MBL- and AAC(6')-Iae producing P. aeruginosa strain is emerging in Japan. Isolates belonging to both clusters were isolated from different parts of the country.

Discrimination of influenza A subtype by antibodies recognizing host-specific amino acids in the viral nucleoprotein.

BACKGROUND: Nucleoprotein (NP) of influenza viruses is utilized to differentiate between the A, B, and C viral serotypes. The availability of influenza genome sequence data has allowed us to identify specific amino acids at particular positions in viral proteins, including NP, known as "signature residues," which can be used to discriminate human influenza A viruses from H5N1 highly pathogenic avian influenza in human cases (HPAI) and pandemic H1N1(2009) (H1N1/2009) viruses. METHODS: Screening and epitope mapping of monoclonal antibodies (mAb) against NP of influenza A, which reacted differently with NP from human influenza A virus from HPAI and H1N1/2009 A virus. To identify the epitope(s) responsible for the discrimination of viral NP by mAbs, we prepared mutant NP proteins in the 293 cell expression system because some of the mAbs reacted with non-linear epitopes. RESULTS AND CONCLUSIONS: In the present study, we identified 3 mAbs. The results of epitope mapping showed that the epitopes were located at the signature residues. These results indicated that signature residues of NP could discriminate influenza A viruses from different origin.

Development of an immunochromatographic assay for diagnosing the production of IMP-type metallo-ß-lactamases that mediate carbapenem resistance in Pseudomonas.

Rapid and reliable detection of carbapenem-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. IMP-type metallo-ß-lactamase (MBL) is an emzyme that mediate carbapenem resistance in bacteria. Here, an immunochromatographic assay was newly developed using novel monoclonal antibodies (mAbs) recognizing IMP-type MBL. Epitope mapping of mAbs and mutational analysis of the epitope region in IMP antigen suggested that the mAbs could react to all known subtypes of IMP-type MBL. Evaluation of the assay using Pseudomonas aeruginosa strains (n=248) showed that the results of the immunochromatographic detection of the IMP-type MBLs were fully consistent with those of the PCR analysis for bla(IMP) genes, showing false positives and negatives. All positive strains were resistant to carbapenem (MIC ≥ 16 µg/ml). The assay also accurately distinguished the production of IMP-type MBLs in Pseudomonas putida, Acinetobacter baumannii, and Alcaligenes xylosoxidans. The detection limit of the assay was 5.7×10(4)cfu per test. Taken together, these data suggest that the developed assay can be used for rapid and reliable diagnosis of the production of IMP-type MBLs in Gram-negative bacteria.

Multicenter prospective evaluation of a novel rapid immunochromatographic diagnostic kit specifically detecting influenza A H1N1 2009 virus.

BACKGROUND: Definitive diagnosis is crucial in reducing morbidity and mortality from pandemic influenza A H1N1 2009 (A/H1N1/2009), especially in high-risk populations. We recently developed a rapid diagnosis kit (RDK) capable of specifically detecting A/H1N1/2009. OBJECTIVES: To evaluate the diagnostic capability of the RDK in a multicenter, prospective trial. STUDY DESIGN: Samples were obtained by nasal swab from patients with suspected influenza. The diagnostic capability of the RDK was compared with that of the standard, real-time reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Of 266 patients who met the criteria, 122 and 92 were positive for A/H1N1/2009 influenza by PCR and by the newly developed RDK, respectively. The sensitivity, specificity and positive and negative predictive values of the RDK were 73.0%, 97.9%, 96.7% and 81.0%, respectively. A/H1N1/2009 detection rates by the RDK were significantly lower in samples obtained from patients more than 3 days after onset than in samples obtained between 1 and 2 days. CONCLUSIONS: The A/H1N1/2009-specific RDK is a reliable test that can be used easily at a patient's bedside for rapid diagnosis of A/H1N1/2009. This test will be of key importance in the control of A/H1N1/2009.

Development of an immunochromatographic assay for the rapid detection of AAC(6')-Iae-producing multidrug-resistant Pseudomonas aeruginosa.

OBJECTIVES: To develop an easy-to-use method for the rapid detection of antibiotic-resistant bacteria. Here, a new immunochromatographic assay specific for aminoglycoside 6'-N-acetyltransferase AAC(6')-Iae was designed. AAC(6')-Iae is a significant marker molecule for multidrug-resistant (MDR) Pseudomonas aeruginosa isolates in Japan. METHODS: Monoclonal antibodies specific for AAC(6')-Iae were used to construct the assay. The assessment of the assay was performed using 116 P. aeruginosa clinical isolates obtained from hospitals in the Kanto area of Japan where little was known about AAC(6')-Iae producers. PCR analyses of the aac(6')-Iae and class 1 integron, antimicrobial susceptibility testing and PFGE analysis were performed to characterize positive strains. RESULTS: The detection limit of the assay was 1.0 x 10(5) cfu. Of 116 clinical isolates, 60 were positive for AAC(6')-Iae using the assay. The results of assessment with clinical isolates were fully consistent with those of aac(6')-Iae PCR analyses, showing no false positives or negatives. All positive strains detected by the assay showed MDR phenotypes that were resistant to several classes of antibiotic. PFGE analysis showed that 59 of 60 positive strains tightly clustered, and these included clonal expansions. CONCLUSIONS: The developed assay is an easy-to-use and reliable detection method for AAC(6')-Iae-producing MDR P. aeruginosa. This approach may be applicable for screening and investigation of antibiotic-resistant bacteria as an alternative to PCR analysis.

Development of an immunochromatographic assay specifically detecting pandemic H1N1 (2009) influenza virus.

The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. Since hemagglutinin (HA) genes are among the most specific genes in the influenza virus genome, AH1pdm can be definitively diagnosed by viral gene analysis targeting the HA genes. This type of analysis, however, cannot be easily performed in clinical settings. While commercially available rapid diagnosis kits (RDKs) based on immunochromatography can be used to detect nucleoproteins (NPs) of influenza A and B viruses in clinical samples, there are no such kits that are specific for AH1pdm. We show here that an RDK using a combination of monoclonal antibodies against NP can be used to specifically detect AH1pdm. The RDK recognized AH1pdm virus isolates but did not recognize seasonal H1N1 and H3N2 and influenza B viruses, indicating that the specificity of the RDK is 100%. A parallel comparison of RDK with a commercial influenza A/B virus kit revealed that both types of kits had equal sensitivities in detecting their respective viruses. Preliminary evaluation of clinical samples from 5 individuals with PCR-confirmed human AH1pdm infection showed that the RDK was positive for all samples, with the same detection intensity as that of a commercial influenza A/B virus kit. This RDK, together with a new vaccine and the stockpiling of anti-influenza drugs, will make aggressive measures to contain AH1pdm infections possible.

Polar coordinate representation of Hb(rc) versus (h2/8m)nabla2rhob(rc) at BCP in AIM analysis: classification and evaluation of weak to strong interactions.

Polar coordinate (R, theta) representation is proposed for the plot of Hb(rc) versus (h2/8m)nabla2rhob(rc) in AIM analysis to classify, evaluate, and understand weak to strong interactions in a unified way and in more detail; Hb(rc) and nabla2rhob(rc) are total electron energy densities and the Laplacian of rhob(rc) at bond critical points (BCPs: rc), respectively, where rhob(rc) are electron densities at rc. Both the x- and y-axes of the plot are expressed in the common unit of energy since Hb(rc) = Gb(rc) + Vb(rc) and (h2/8m)nabla2rhob(rc) = Hb(rc) - Vb(rc)/2 (= Gb(rc) + Vb(rc)/2), where Gb(rc) and Vb(rc) are kinetic energy densities and potential energy densities, respectively. Data employed for the plot are calculated at BCPs for full-optimized structures and optimized structures with the fixed distances (r) of r = r(o) + wa(o), where r(o) are the full-optimized distances, a(o) is the Bohr radius, and w = +/-0.1 and +/-0.2. The plot draws a helical stream starting from near origin (Hb(rc) = (h2/8m)nabla2rhob(rc) = 0) for very weak interactions and turns to the right as interactions become stronger. The helical stream is well described by the polar coordinate representation with (R, theta); R is given in the energy unit, and theta in degrees is measured from the y-axis. The ratio of Vb(rc)/Gb(rc) (= k) controls theta, of which an acceptable range in the plot is 45.0 < theta < 206.6 degrees. Each plot for an interaction gives a curve, which supplies important information. It is expressed by theta(p) and kappa(p); theta(p) corresponds to the tangent line measured from the y-direction, and kappa(p) is the curvature of the plot at w = 0. The polar coordinate (R, theta) representation with (theta(p), kappa(p)) helps us to classify, evaluate, and understand the nature of weak to strong interactions in a unified way.

Atoms-in-molecules dual parameter analysis of weak to strong interactions: behaviors of electronic energy densities versus Laplacian of electron densities at bond critical points.

AIM dual parameter analysis is proposed for the better understanding of weak to strong interactions: Total electron energy densities (H(b)(r(c))) are plotted versus Laplacian of electron densities (Delta rho(b)(r(c))) at bond critical points (BCPs). Interactions examined in this work are those in van der Waals adducts, hydrogen bonded complexes, molecular complexes and hypervalent adducts through charge transfer (CT) interactions, and some classical covalent bonds. Data calculated at BCPs for the optimized distances (r(o)), together with r(o) - 0.1 A, r(o) + 0.1 A, and r(o) + 0.2 A, are employed for the plots. The plots of H(b)(r(c)) versus Delta rho(b)(r(c)) start from near origin (H(b)(r(c)) = Delta rho(b)(r(c)) = 0) and turn to the right drawing a helical stream as a whole. The helical nature is demonstrated to be controlled by the relative magnitudes of kinetic energy densities (G(b)(r(c))) and potential energy densities (V(b)(r(c))), where G(b)(r(c)) + V(b)(r(c)) = H(b)(r(c)). Requirements for the data to appear in the specified quadrant are clarified. Points corresponding to the data will appear in the first quadrant (Delta rho(b)(r(c)) > 0 and H(b)(r(c)) > 0) when -V(b)(r(c)) < G(b)(r(c)), they drop in the forth one (Delta rho(b)(r(c)) > 0 and H(b)(r(c)) < 0) if -(1/2)V(b)(r(c)) < G(b)(r(c)) < -V(b)(r(c)), and they appear in the third quadrant (Delta rho(b)(r(c)) < 0 and H(b)(r(c)) < 0) when G(b)(r(c)) < -(1/2)V(b)(r(c)). No points will appear in the second quadrant (Delta rho(b)(r(c)) < 0 and H(b)(r(c)) > 0). The physical meanings of the plots proposed in this work are also considered. The helical nature of the interactions in the plots helps us to understand the interactions in a unified way.

Contributions from atomic p(Se), d(Se), and f(Se) orbitals to absolute paramagnetic shielding tensors in neutral and charged SeHn and some oxides including the effect of methyl and halogen substitutions on sigmap(Se).

Contributions from atomic p(Se), d(Se), and f(Se) orbitals to sigmap(Se) are evaluated for neutral and charged Se*Hn (*=null, +, or -) and some oxides to build the image of the contributions. The effect of methyl and halogen substitutions is also examined employing RrSe*XxOo (*=null, +, or -) where R=H or Me; X=F, Cl, or Br. The p(Se) contributions are larger than 96 % for SeH- (Cinfinityv), SeH2 (C2v), SeH3 + (C3v), SeH3 + (D3h), and SeH4 (Td). Therefore, sigmap(Se) of these compounds can be analyzed based on p(Se). The p(Se) contributions are 79-75 % for SeH4 (TBP), SeH5 + (TBP), SeH5 + (SP), and SeH5 - (SP). Methyl and halogen substitutions increase the contributions by 1-2 % (per Me) and 4-7 % (per X), respectively. The contributions are 92-79 % for H2SeO (Cs), H2SeO2 (C2v), and H4SeO (C2v). The values are similarly increased by the substitutions. Consequently, sigmap(Se) of these compounds can be analyzed based on p(Se) with some corrections by d(Se). The p(Se) contribution of SeH6 (Oh) is 52 %: sigmap(Se: SeH6 (Oh)) must be analyzed based on both p(Se) and d(Se). The contributions for the Me and X derivatives of SeH(6) amount to 86-77 %. Therefore, sigmap(Se) of the derivatives can also be analyzed mainly based on p(Se) with some corrections by d(Se). Contributions from f(Se) are negligible. Contributions from 4p(Se) in vacant orbitals are also considered. A utility program derived from the Gaussian 03 (NMRANAL-NH03G) is applied to evaluate the contributions.

Evaluation of electron population terms for 4p, 3p, and (2p): how do HOMO and LUMO shrink or expand depending on nuclear charges?

Electron population terms are evaluated for N=Se, S, and O. Calculations are performed on HOMO and LUMO constructed by pure atomic 4p(Se), 3p(S), and 2p(O) orbitals, employing the 6-311+G(3d) and/or 6-311(++)G(3df,3pd) basis sets at the HF, MP2, and DFT (B3 LYP) levels. Se(4+), Se(2+), Se(0), and Se(2-) with the O(h) symmetry are called G(A: Se) and HSe(+), H(2)Se, and HSe(-) with the C(infinityh) or C(2v) symmetry are named G(B: Se), here [G(A+B: Se) in all]. HOMO and LUMO in G(A+B: N) (N=Se, S, and O) satisfy the conditions of the calculations for . The (4p), (3p), and (2p) values correlate well with the corresponding MO energies (epsilon(N)) for all calculation levels employed. Plots of (HOMO) and (LUMO) versus Q(N) (N=Se, S, and O) at the HF and MP2 levels are analyzed as two correlations. However, the plots at the DFT level can be analyzed as single correlation. A regression curve is assumed for the analysis. Behaviors of clarify how valence orbitals shrink or expand depending on Q(N). The applicability of is examined to establish a new method that enables us to analyze chemical shifts with the charge effect separately from others. A utility program derived from the Gaussian 03 (NMRANAL-NH03G) is applied to evaluate and examine the applicability to the NMR analysis.

Immunochromatographic assay for simple and rapid detection of Satsuma dwarf virus and related viruses using monoclonal antibodies.

A simple and rapid immunochromatographic assay (ICA) to detect Satsuma dwarf virus (SDV) was developed using colloidal gold conjugates of anti-SDV monoclonal antibodies. Of six homogenization buffers tested, 0.1 M citrate buffer (pH 7.0) gave the best results for the ICA. In the ICA, addition of 0.1% thioglycolic acid in the homogenization buffers that have been widely used in enzyme-linked immunosorbent assays (ELISA) was deleterious to the reaction because of undesirable coagulation of the colloidal gold. ICA using the anti-SDV monoclonal antibodies was 8 times and 16 times more sensitive than double antibody sandwich-ELISA and ICA using the anti-SDV polyclonal antibody, respectively. The analysis is complete in only 15 min. Furthermore, ICA using the anti-SDV monoclonal antibodies could also detect SDV-related viruses.