Multidrug-resistant Klebsiella pneumoniae clinical isolates producing NDM- and OXA-type carbapenemase in Nepal.

OBJECTIVES: The emergence of multidrug-resistant Klebsiella pneumoniae has become a serious problem in medical settings worldwide. METHODS: A total of 46 isolates of multidrug-resistant K. pneumoniae were obtained from 2 hospitals in Nepal from October 2018 to April 2019. RESULTS: Most of these isolates were highly resistant to carbapenems, aminoglycosides, and fluoroquinolones with the minimum inhibitory concentrations (MICs) of more than 64 µg/mL. These isolates harboured carbapenemase-encoding genes, including blaNDM-1, blaNDM-5, blaOXA-181 and blaOXA-232, and 16S rRNA methyltransferase-encoding genes, including armA, rmtB, rmtC, and rmtF. Multilocus sequence typing revealed that 44 of 46 isolates were high-risk clones such as ST11 (2%), ST14 (4%), ST15 (11%), ST37 (2%), ST101 (2%), ST147 (28%), ST231 (13%), ST340 (4%), and ST395 (28%). In particular, ST395 isolates, which spread across medical settings in Nepal, co-harboured blaNDM-5 and rmtB on IncFII plasmids and co-harboured blaOXA-181/-232 and rmtF on ColKP3 plasmids. Several isolates harboured blaOXA-181 or blaNDM-5 on their chromosomes and multi-copies of blaNDM-1 or genes encoding 16S rRNA methyltransferases on their plasmids. CONCLUSIONS: The presented study demonstrates that the high-risk clones of multidrug-resistant K. pneumoniae spread in a clonal manner across hospitals in Nepal.

Identification of Mycobacterium abscessus using the peaks of ribosomal protein L29, L30 and hemophore-related protein by MALDI-MS proteotyping.

Mycobacteroides (Mycobacterium) abscessus, which causes a variety of infectious diseases in humans, is becoming detected more frequently in clinical specimens as cases are spreading worldwide. Taxonomically, M. abscessus is composed of three subspecies of M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, with different susceptibilities to macrolides. In order to identify rapidly these three subspecies, we determined useful biomarker proteins, including ribosomal protein L29, L30, and hemophore-related protein, for distinguishing the subspecies of M. abscessus using the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiles. Thirty-three clinical strains of M. abscessus were correctly identified at the subspecies-level by the three biomarker protein peaks. This study ultimately demonstrates the potential of routine MALDI-MS-based laboratory methods for early identification and treatment for M. abscessus infections.

Assessment of antibody dynamics and neutralizing activity using serological assay after SARS-CoV-2 infection and vaccination.

The COVID-19 antibody test was developed to investigate the humoral immune response to SARS-CoV-2 infection. In this study, we examined whether S antibody titers measured using the anti-SARS-CoV-2 IgG II Quant assay (S-IgG), a high-throughput test method, reflects the neutralizing capacity acquired after SARS-CoV-2 infection or vaccination. To assess the antibody dynamics and neutralizing potency, we utilized a total of 457 serum samples from 253 individuals: 325 samples from 128 COVID-19 patients including 136 samples from 29 severe/critical cases (Group S), 155 samples from 71 mild/moderate cases (Group M), and 132 samples from 132 health care workers (HCWs) who have received 2 doses of the BNT162b2 vaccinations. The authentic virus neutralization assay, the surrogate virus neutralizing antibody test (sVNT), and the Anti-N SARS-CoV-2 IgG assay (N-IgG) have been performed along with the S-IgG. The S-IgG correlated well with the neutralizing activity detected by the authentic virus neutralization assay (0.8904. of Spearman's rho value, p < 0.0001) and sVNT (0.9206. of Spearman's rho value, p < 0.0001). However, 4 samples (2.3%) of S-IgG and 8 samples (4.5%) of sVNT were inconsistent with negative results for neutralizing activity of the authentic virus neutralization assay. The kinetics of the SARS-CoV-2 neutralizing antibodies and anti-S IgG in severe cases were faster than the mild cases. All the HCWs elicited anti-S IgG titer after the second vaccination. However, the HCWs with history of COVID-19 or positive N-IgG elicited higher anti-S IgG titers than those who did not have it previously. Furthermore, it is difficult to predict the risk of breakthrough infection from anti-S IgG or sVNT antibody titers in HCWs after the second vaccination. Our data shows that the use of anti-S IgG titers as direct quantitative markers of neutralizing capacity is limited. Thus, antibody tests should be carefully interpreted when used as serological markers for diagnosis, treatment, and prophylaxis of COVID-19.

Evaluation of calculation processes of apparent diffusion coefficient subtraction method (ASM) imaging.

A number of restricted diffusion (RD) imaging techniques, such as diffusion kurtosis (DK) imaging and Q space imaging, have been developed and proven to be useful for the diagnosis of diseases, including cerebral gliomas and cerebrovascular infarction. In particular, apparent diffusion coefficient (ADC) subtraction method (ASM) imaging has become available recently as a novel RD imaging technique. ASM is based on the difference between the ADC values in an image pair of two ADC maps, ADC basic (ADCb) and ADC modify (ADCm), which are created from diffusion-weighted images taken using short and long effective diffusion times, respectively. The present study aimed to assess the potential of different types of ASM imaging by comparing them with DK imaging which is the gold-standard RD imaging technique. In the present basic study using both polyethylene glycol phantom and cell-containing bio-phantom, three different types of ASM images were created using different calculation processes. ASM/A is an image calculated by dividing the absolute difference between ADCb and ADCm by ADCb several times. By contrast, ASM/S is an image created by dividing the absolute difference between ADCb and ADCm by the standard deviation of ADCb several times. As for positive ASM/A image (PASM/A), the positive image, which was resultant after subtracting ADCb from ADCm, was divided by ADCb several times. A comparison was made between the types of ASM and DK images. The results showed the same tendency between ASM/A in addition to both ASM/S and PASM/A. By increasing the number of divisions by ADCb from three to five times, ASM/A images transformed from DK-mimicking to more RD-sensitive images compared with DK images. These observations suggest that ASM/A images may prove useful for future clinical applications in RD imaging protocols for the diagnosis of diseases.

Performance evaluation of the Ortho VITROS SARS-CoV-2 Spike-Specific Quantitative IgG test by comparison with the surrogate virus neutralizing antibody test and clinical assessment.

BACKGROUND: Despite the worldwide campaigns of COVID-19 vaccinations, the pandemic is still a major medical and social problem. The Ortho VITROS SARS-CoV-2 spike-specific quantitative IgG (VITROS S-IgG) assay has been developed to assess neutralizing antibody (NT antibody) against SARS-CoV-2 spike (S) antibodies. However, it has not been evaluated in Japan, where the total cases and death toll are lower than the rest of the world. METHODS: The clinical performance of VITROS S-IgG was evaluated by comparing with the NT antibody levels measured by the surrogate virus neutralizing antibody test (sVNT). A total of 332 serum samples from 188 individuals were used. Of these, 219 samples were from 75 COVID-19 patients: 96 samples from 20 severe/critical cases (Group S), and 123 samples from 55 mild/moderate cases (Group M). The remaining 113 samples were from 113 healthcare workers who had received 2 doses of the BNT162b2 vaccine. RESULTS: VITROS S-IgG showed good correlation with the cPass sVNT assay (Spearman rho = 0.91). Both VITROS S-IgG and cPass sVNT showed significantly higher plateau levels of antibodies in Group S compared to Group M. Regarding the humoral immune responses after BNT162b2 vaccination, individuals who were negative for SARS-CoV-2 nucleocapsid (N)-specific antibodies had statistically lower titers of both S-IgG and sVNT compared to individuals with a history of COVID-19 and individuals who were positive for N-specific antibodies without history of COVID-19. In individuals who were positive for N-specific antibodies, S-IgG and sVNT titers were similar to individuals with a history of COVID-19. CONCLUSIONS: Although the automated quantitative immunoassay VITROS S-IgG showed a reasonable correlation with sVNT antibodies, there is some discrepancy between Vitros S-IgG and cPass sVNT in milder cases. Thus, VITROS S-IgG can be a useful diagnostic tool in assessing the immune responses to vaccination and herd immunity. However, careful analysis is necessary to interpret the results.

Th1 cytokine endotype discriminates and predicts severe complications in COVID-19

Treatment of severe and critical cases of coronavirus disease 2019 (COVID-19) is still a top priority in public health. Previously, we reported distinct Th1 cytokines related to the pathophysiology of severe COVID-19 condition. In the present study, we investigated the association of Th1 and Th2 cytokine/chemokine endotypes with cell-mediated immunity via multiplex immunophenotyping, single-cell RNA-Seq analysis of peripheral blood mononuclear cells, and analysis of the clinical features of COVID-19 patients. Based on serum cytokine and systemic inflammatory markers, COVID-19 cases were classified into four clusters of increasing (I-IV) severity. Two prominent clusters were of interest and could be used as prognostic reference for a targeted treatment of severe COVID-19 cases. Cluster III reflected severe/critical pathology and was characterized by decreased in CCL17 levels and increase in IL-6, C-reactive protein CXCL9, IL-18, and IL-10 levels. The second cluster (Cluster II) showed mild to moderate pathology and was characterized by predominated CXCL9 and IL-18 levels, levels of IL-6 and CRP were relatively low. Cluster II patients received anti-inflammatory treatment in early-stage, which may have led prevent disease prognosis which is accompanied to IL-6 and CRP induction. In Cluster III, a decrease in the proportion of effector T cells with signs of T cell exhaustion was observed. This study highlights the mechanisms of endotype clustering based on specific inflammatory markers in related the clinical outcome of COVID-19.

Activation of SARS-CoV-2 neutralizing antibody is slower than elevation of spike-specific IgG, IgM, and nucleocapsid-specific IgG antibodies.

COVID-19 antibody testing has been developed to investigate humoral immune response in SARS-CoV-2 infection. To assess the serological dynamics and neutralizing potency following SARS-CoV-2 infection, we investigated the neutralizing (NT) antibody, anti-spike, and anti-nucleocapsid antibodies responses using a total of 168 samples obtained from 68 SARS-CoV-2 infected patients. Antibodies were measured using an authentic virus neutralization assay, the high-throughput laboratory measurements of the Abbott Alinity quantitative anti-spike receptor-binding domain IgG (S-IgG), semiquantitative anti-spike IgM (S-IgM), and anti-nucleocapsid IgG (N-IgG) assays. The quantitative measurement of S-IgG antibodies was well correlated with the neutralizing activity detected by the neutralization assay (r = 0.8943, p < 0.0001). However, the kinetics of the SARS-CoV-2 NT antibody in severe cases were slower than that of anti-S and anti-N specific antibodies. These findings indicate a limitation of using the S-IgG antibody titer, detected by the chemiluminescent immunoassay, as a direct quantitative marker of neutralizing activity capacity. Antibody testing should be carefully interpreted when utilized as a marker for serological responses to facilitate diagnostic, therapeutic, and prophylactic interventions.

Performance evaluation of the Roche Elecsys® Anti-SARS-CoV-2 immunoassays by comparison with neutralizing antibodies and clinical assessment.

Quantitative measurement of SARS-CoV-2 neutralizing antibodies is highly expected to evaluate immune status, vaccine response, and antiviral therapy. The Elecsys® Anti-SARS-CoV-2 S (Elecsys® anti-S) was developed to measure anti-SARS-CoV-2 S proteins. We sought to investigate whether Elecsys® anti-S can be used to predict neutralizing activities in patients' serums using an authentic virus neutralization assay. One hundred forty-six serum samples were obtained from 59 patients with COVID-19 at multiple time points. Of the 59 patients, 44 cases were included in Group M (mild 23, moderate 21) and produced 84 samples (mild 35, moderate 49), while 15 cases were included in Group S (severe 11, critical 4) and produced 62 samples (severe 43, critical 19). The neutralization assay detected 73% positive cases, and Elecsys® anti-S and Elecsys® Anti-SARS-CoV-2 (Elecsys® anti-N) showed 72% and 66% positive cases, respectively. A linear correlation between the Elecsys® anti-S assay and the neutralization assay were highly correlated (r = 0.7253, r2 = 0.5261) than a linear correlation between the Elecsys® anti-N and neutralization assay (r = 0.5824, r2 = 0.3392). The levels of Elecsys® anti-S antibody and neutralizing activities were significantly higher in Group S than in Group M after 6 weeks from onset of symptoms (p < 0.05). Conversely, the levels of Elecsys® anti-N were comparable in both groups. Three immunosuppressed patients, including cancer patients, showed low levels of anti-S and anti-N antibodies and neutralizing activities throughout the measurement period, indicating the need for careful follow-up. Our data indicate that Elecsys® anti-S can predict the neutralization antibodies in COVID-19.

Influence of mesiodens on adjacent teeth and the timing of its safe removal.

Purpose: To focus on the effects of the presence of mesiodens on adjacent teeth and to investigate the timing of its safe removal. Materials and Methods: Cone-beam computed tomography examinations, obtained at Okayama University Hospital over a three-year period, were inspected. Data were recorded including the number of mesiodens; associated abnormalities; and the relationship with neighboring structures. Depending on multiple factors, the risk of developing complications due to early extraction of a mesiodens was divided into three categories: high, medium, and low risk. Results: A total of 5,958 cone-beam computed tomography exams were obtained, 460 patients aged 3-85 years were diagnosed with a total of 568 mesiodens, 382 (67.3%) of which were discovered in young patients (age <10 years), and 333 (87.2%) of these were associated with abnormalities. Regarding the risk categories, 11 (1.9%) were considered to be in the high-risk, five (0.9%) in the medium-risk and 552 (97.2%) in the low-risk categories. Moreover, eight out of 11 high-risk mesiodens were extracted and no post-operative complications have been seen. Conclusion: As the results showed that no postoperative complications were seen in all the extracted cases of high-risk mesiodens, this indicates the possibility of safe extraction at an early age which could reduce related future complications.

Development and Evaluation of a Short-time Imaging Method for the Clinical Study of the Apparent Diffusion Coefficient Subtraction Method.

The apparent diffusion coefficient subtraction method (ASM) was developed as a new restricted diffusionweighted imaging technique for magnetic resonance imaging (MRI). The usefulness of the ASM has been established by in vitro basic research using a bio-phantom, and clinical research on the application of the ASM for the human body is needed. Herein, we developed a short-time sequence for ASM imaging of the heads of healthy volunteers (n=2), and we investigated the similarity between the obtained ASM images and diffusion kurtosis (DK) images to determine the utility of the ASM for clinical uses. This study appears to be the first to report ASM images of the human head. We observed that the short-time sequence for the ASM imaging of the head can be scanned in approx. 3 min at 1.5T MRI. The noise reduction effect of median filter processing was confirmed on the ASM images scanned by this sequence. The obtained ASM images showed a weak correlation with the DK images, indicating that the ASM images are restricted diffusion-weighted images. The new shorttime imaging sequence could thus be used in clinical studies applying the ASM.

New field-in-field with two reference points method for whole breast radiotherapy: Dosimetric analysis and radiation-induced skin toxicities assessment.

The usefulness of the field-in-field with two reference points (FIF w/ 2RP) method, in which the dose reference points are set simultaneously at two positions in the irradiation field and the high-dose range is completely eliminated, was examined in the present study with the aim of decreasing acute skin toxicity in adjuvant breast radiotherapy (RT). A total of 573 patients with breast cancer who underwent postoperative whole breast RT were classified into 178 cases with wedge (W) method, 142 cases with field-in-field without 2 reference points (FIF w/o 2RP) method and 253 cases with FIF w/ 2RP method. Using the FIF w/ 2RP method, the high-dose range was the lowest among the three irradiation methods. The planning target volume (PTV) V105% and the breast PTV for evaluation (BPe) V105% decreased to 0.09 and 0.10%, respectively. The FIF w/ 2RP method vs. the FIF w/o 2RP method had a strong association (η) with PTV V105% (η=0.79; P<0.001) and BPe V105% (η=0.76; P<0.001). The FIF w/ 2RP method had a significant impact on lowering the skin toxicity grade in weeks 3 and 4, and increasing the occurrence of skin toxicity grade 0. The FIF w/ 2RP method vs. the W method had a moderate association with skin toxicity grade at week 3 (η=0.49; P<0.001). Using the FIF w/ 2RP method, the high-dose range V105% of the target decreased to 0%, and skin adverse events were decreased in conjunction. For patients with early-stage breast cancer, particularly patients with relatively small-sized breasts, the FIF w/ 2RP method may be an optimal irradiation method.

Investigation into the Effect of Breast Volume on Irradiation Dose Distribution in Asian Women with Breast Cancer.

Reports on irradiation dose distribution in breast cancer radiotherapy with sufficient sample size are limited in Asian patients. Elucidating dose distribution in Asian patients is particularly important as their breast volume differs compared to patients in Europe and North America. Here, we examined dose distribution in the irradiation field relative to breast volume for three irradiation methods historically used in our facility. We investigated the influence of breast volume on each irradiation method for Asian women. A total of 573 women with early-stage breast cancer were treated with breast-conserving surgery and adjuvant radiotherapy. Three methods were compared: wedge (W), field-in-field (FIF), and wedge-field-in-field (W-FIF). In patients with small breast volume, FIF decreased low- and high-dose areas within the planning target volume, and increased optimal dose area more than W. In patients with medium and large breast volumes, FIF decreased high-dose area more than W. The absolute values of correlation coefficients of breast volume to low-, optimal-, and high-dose areas and mean dose were significantly lower in FIF than in W. The correlation coefficients of V107% were 0.00 and 0.28 for FIF and W, respectively. FIF is an excellent irradiation method that is less affected by breast volume than W in Asian breast cancer patients.

Evaluation of the Imaging Process for a Novel Subtraction Method Using Apparent Diffusion Coefficient Values.

Diffusion-weighted imaging may be used to obtain the apparent diffusion coefficient (ADC), which aids the diagnosis of cerebral infarction and tumors. An ADC reflects elements of free diffusion. Diffusion kurtosis imaging (DKI) has attracted attention as a restricted diffusion imaging technique. The ADC subtraction method (ASM) was developed to visualize restricted diffusion with high resolution by using two ADC maps taken with different diffusion times. We conducted the present study to provide a bridge between the reported basic ASM research and clinical research. We developed new imaging software for clinical use and evaluated its performance herein. This software performs the imaging process automatically and continuously at the pixel level, using ImageJ software. The new software uses a macro or a plugin which is compatible with various operating systems via a Java Virtual Machine. We tested the new imaging software's performance by using a Jurkat cell bio-phantom, and the statistical evaluation of the performance clarified that the ASM values of 99.98% of the pixels in the bio-phantom and physiological saline were calculated accurately (p<0.001). The new software may serve as a useful tool for future clinical applications and restricted diffusion imaging research.

Development of a novel phantom using polyethylene glycol for the visualization of restricted diffusion in diffusion kurtosis imaging and apparent diffusion coefficient subtraction method.

The present study aimed to investigate whether polyethylene glycol (PEG) phantoms have the potential to be used as standard phantoms for magnetic resonance imaging (MRI) in order to visualize restricted diffusion in diffusion kurtosis imaging (DKI), the ADC subtraction method (ASM) and the apparent diffusion coefficient (ADC). Diffusion-weighted images of 0-120 mM PEG phantoms were captured to create ADC, DKI and ASM images with post-processing. ASM is a recently developed method for restricted diffusion imaging using the readout segmentation of long variable echo-train sequences. As the PEG concentration increases, the ADC value decreases. Conversely, an increase in DKI and ASM values is associated with increasing PEG concentration. Formulae were constructed to represent the association between PEG concentrations and ADC, DKI and ASM values. These formulae can be used to determine the required PEG concentrations to mimic arbitrary ADC, DKI and ASM values of certain diseases, including tumors and infarctions. Validation experiments were conducted using bio-phantoms and clarified that the PEG phantoms cover the range of ADC and DKI values reported in previous clinical research using 3T MRI. PEG phantoms may be useful for future MRI research involving restricted diffusion.

Incidental findings in the thyroid gland on computed tomography images of the oral and maxillofacial region.

The numbers of abnormal findings incidentally detected in adjacent regions are increasing with advances in imaging modalities. The present study aimed to examine the prevalence and characteristics of incidental findings in the thyroid gland on computed tomography (CT) images of the oral and maxillofacial region. CT scans of the oral and maxillofacial region in patients obtained between January 2012 and December 2016 were retrospectively reviewed. Images that revealed incidental findings in the thyroid gland, including nodules, were recorded, together with the sizes and characteristics of the findings. The Japan Association of Breast and Thyroid Sonology (JABTS) guidelines were used for classification. The rate of descriptions of these findings in the radiographic interpretation reports were also examined. Of the 1,135 patients examined, 326 (28.7%) had several types of incidental findings. In particular, 169 (14.9%) of the 1,135 patients had nodules >5 mm in diameter, for which further careful examination is recommended in the JABTS guideline. The description rate for nodules >5 mm in diameter in the radiographic interpretation reports was 30.8% (52/169 patients), of whom 17.3% (9/52 patients) were referred to the endocrinology department for further careful examination. Incidental findings in the thyroid gland were relatively common on CT images of the oral and maxillofacial region. Oral radiologists tend to focus specifically on the oral and maxillofacial region during diagnosis on oral and maxillofacial CT images, but should pay the same careful attention to observe adjacent regions, such as the thyroid gland.

Development of a novel method for visualizing restricted diffusion using subtraction of apparent diffusion coefficient values.

In order to visualize restricted diffusion, the present study developed a novel method called 'apparent diffusion coefficient (ADC) subtraction method (ASM)' and compared it with diffusion kurtosis imaging (DKI). The diffusion-weighted images of physiological saline, in addtion to bio-phatoms of low cell density and the highest cell density were obtained using two sequences with different effective diffusion times. Then, the calculated ADC values were subtracted. The mean values and standard deviations (SD) of the ADC values of physiological saline, low cell density and the highest cell density phantoms were 2.95±0.08x10­3, 1.90±0.35x10­3 and 0.79±0.05x10­3 mm2/sec, respectively. The mean kurtosis values and SD of DKI were 0.04±0.01, 0.44±0.13 and 1.27±0.03, respectively. The ASM and SD values were 0.25±0.20x104, 0.51±0.41x104 and 4.80±4.51x104 (sec/mm2)2, respectively. Using bio­phantoms, the present study demonstrated that DKI exhibits restricted diffusion in the extracellular space. Similarly, ASM may reflect the extent of restricted diffusion in the extracellular space.