Evaluation of two commercial molecular diagnostic assays: The Xpert Norovirus and the TRCReady NV.

INTRODUCTION: Norovirus is highly contagious, and a few particles of this virus are sufficient to make people sick. It is desirable to develop quick and accurate laboratory methods to detect norovirus. METHODS: We evaluated two commercial molecular diagnostic assays, the Xpert Norovirus and the TRCReady NV, using clinical fecal samples. A reference method was performed using in-house real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). RESULTS: The results of the real-time RT-PCR analysis of 60 suspected cases of norovirus infection showed 5 cases of Genogroup I (GI) positives and 21 cases of GII positives, among which was 1 GI and GII coinfection. The viral titers of the norovirus-positive samples ranged from 1.54 × 101 to 3.14 × 108 copies/µL. Norovirus GII.17 (12 cases, 48%) was the most frequently detected genotype in this study, followed by GII.4 (6 cases, 24%), GII.13 (2 cases, 8%), GI.2 (2 cases, 8%), GI.3 (2 cases, 8%), GI.1 (1 case, 4%), and GII.2 (1 case, 4%). The kappa coefficient was 1.000 (95% CI: 1.000-1.000) for Xpert Norovirus and 0.966 (95% CI: 0.896-1.000) for TRCReady NV, indicating a strong agreement. CONCLUSIONS: Norovirus detection using Xpert Norovirus and TRCReady NV is highly useful for diagnosis and infection control because these assays are easy to operate, quick, and exhibit almost the same performance as that of real-time RT-PCR.

Clonality investigation of clinical Escherichia coli isolates by polymerase chain reaction-based open-reading frame typing method.

Escherichia coli (E. coli) causes urinary tract infections, pneumonia, surgical site infections, and bloodstream infections and is the important pathogen for both community-acquired and healthcare-associated infections. To investigate the clonality of E. coli is important for infection control and prevention. We aimed to investigate the clonality of clinical E. coli isolates using Cica Geneus E. coli polymerase chain reaction (PCR)-based open-reading frame typing (POT) KIT and clarify the clinical usefulness of this kit. About 124 E. coli isolates obtained from inpatients at Sapporo Medical University Hospital were used. The POT method was used to classify 124 clinical isolates into 87 POT numbers. In addition to the clonality, it was possible to obtain additional information that 20 of the 124 isolates were extended-spectrum ß-lactamase (ESBL) producing E. coli (5 isolates of CTX-M-1 group and 15 isolates of CTX-M-9 group) and 13 were sequence type (ST) 131 clone. Furthermore, when these ESBL-producing 20 isolates were compared with pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing (MLST), Simpson's index of diversity was 0.968 in POT method, 0.979 in PFGE, and 0.584 in MLST. POT method had an analytical power similar to that of PFGE. In conclusion, attention should be paid to the difference in the interpretation of the results between the POT method and the PFGE, but POT method may be useful to timely monitor the spread of E. coli in medical facilities.

Evaluation of new algorithm using TPLA as an initial syphilis screening test.

We retrospectively compared the performance of an existing syphilis diagnostic algorithm with a new algorithm that analyzes the results of Treponema pallidum latex agglutination (TPLA) tests. Of the 100 clinical blood samples, 51 were classified as positive through both Mediace TPLA and ESPLINE TP; 2/51 were classified as negative by Architect Syphilis TP, whereas 1/51 was negative as per LUMIPULSE Presto TP. The false positive rate when the results of Mediace TPLA and ESPLINE TP were combined was 1.96% versus 0% for both Architect Syphilis TP and LUMIPULSE Presto TP. The sensitivity of Mediace TPLA (98%) was comparable to that of Architect Syphilis TP (98%) but lower than that of LUMIPULSE Presto TP (100%). The specificity of Mediace TPLA was 98.0% versus 100% for Architect Syphilis TP, and versus 100% for LUMIPULSE Presto TP. We conclude that the performance of Mediace TPLA in combination with a reverse algorithm is nearly equal to that of enzyme immunoassay (EIA) or chemiluminescence immunoassay (CIA). Because TPLA is low cost, highly sensitive method for IgM detection, and is easy to operate, we have recommended its adoption for initial syphilis screening tests.

Analysis of major BCR-ABL1 mRNA by digital polymerase chain reaction is useful for prediction of international scale.

BACKGROUND: Major BCR-ABL1 mRNA in patients with chronic myeloid leukemia (CML) has generally been analysed by real-time polymerase chain reaction (PCR). Application of the international scale (IS) for the quantification of major BCR-ABL1 mRNA has been recommended in several sets of guidelines, including those of the European LeukemiaNet. The aim of this study was to clarify the efficacy of digital PCR technology for the IS of BCR-ABL1 mRNA in the patients with CML by comparing with real-time PCR. METHODS: The analysis of BCR-ABL1 mRNA was carried out by the Ipsogen® BCR-ABL1 Mbcr IS-MMR DX Kit (Qiagen), and the QuantStudio 3D Digital PCR System (Thermo Fisher Scientific ) using 20 peripheral blood samples obtained from the 9 patients with CML at Sapporo Medical University Hospital. RESULTS: The correlation between the data obtained by digital PCR and by real-time PCR was really high at R = 0.96. The detection limit of digital PCR was up to 0.003% and was equal to IS with 0.01% or less in comparison with real-time PCR. CONCLUSIONS: Digital PCR technology is promising for predicting the IS value with similar efficacy to real-time PCR and should be useful for simple monitoring of the effects of tyrosine kinase inhibitor (TKI) treatments.

A Case of Acquired Hemophilia A: Usefulness of Various Methods for Judging Mixing Test Results for Monitoring the Effect of Immunosuppressive Therapy.

BACKGROUND: Measurement of FVIII inhibitor (FVIII INH) levels is important for determining the effect of immunosuppressive therapy on acquired hemophilia A (AHA). However, FVIII INH can only be measured at a limited number of laboratories, which means that there are delays in obtaining the results at many sites. METHODS: A series of mixing tests were carried out in a case of AHA, followed by comparison of various methods for judging the obtained results in association with a change of FVIII INH. The mixing test results were judged using the visual waveform pattern method and the index of circulating anticoagulant (ICA), as well as the difference between the APTT values of delayed-type and immediate-type waveforms (APTT D-I) as a numerical index. RESULTS: All examined judgment methods reflected the change in FVIII INH, but ICA and APTT D-I were particularly sensitive for capturing this. CONCLUSIONS: Our results suggest that a series of mixing tests are useful for rapid monitoring of the effect of immunosuppressive therapy on AHA.

Inoculum effect of high concentrations of methicillin-susceptible Staphylococcus aureus on the efficacy of cefazolin and other beta-lactams.

The existence of a cefazolin inoculum effect (InE) of methicillin-susceptible Staphylococcus aureus (MSSA), which is speculated to be a reason for cefazolin treatment failure in MSSA infections, is controversial. In Japan, although cefazolin is one of the therapeutic choices for patients with MSSA infection, there are few reports of this effect. Additionally, the association between InE and blaZ type in beta-lactams other than cefazolin has not been well documented. In this study, we confirmed an MSSA InE in several beta-lactams, including cefazolin, and its relationship with blaZ, using 52 MSSA isolates from blood cultures. Three isolates (5.8%) that possessed type A blaZ showed a pronounced cefazolin InE. Five isolates (9.6%) showed pronounced InE with sulbactam/ampicillin; four isolates had type C blaZ and one had type A blaZ. However, we confirmed InE in MSSA isolates with blaZ not only type A and C but also B and D. For cefotaxime, ceftriaxone, imipenem, and meropenem, regardless of the presence of blaZ, we did not observe a significant increase in MICs at a high inoculum of MSSA. Hence, our results suggest that the above four beta-lactams are good alternatives to cefazolin if InE leads to treatment failure in a patient.

Identification of a bacteriolysis-associated virulence factor against lung epithelial cells in Pseudomonas aeruginosa PAO-1 cell lysate.

The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells.

Prophylactic and therapeutic effects of Acanthopanax senticosus Harms extract on murine collagen-induced arthritis.

Evidences are accumulating that extract of Acanthopanax senticosus Harms (ASH; syn Eleutherococcus senticosus [Rupr. & Maxim.] Maxim), a shrub native to Northeastern Asia, has antiinflammatory effects. In this study, we examined prophylactic and therapeutic effects of ASH extract (ASHE) on rheumatoid arthritis using collagen-induced arthritis (CIA) mouse model. Acanthopanax senticosus Harms extract was administered before the onset of arthritis in the prophylaxis model. In the therapeutic model, ASHE was administered after the onset of arthritis with or without anti-TNF-α antibody. The ASHE treatment showed efficacy before onset of CIA but there was no effect after CIA was established. The ASHE treatment delayed the onset and decreased severity of CIA. In vitro examinations showed that ASHE is an antioxidant and that ASHE suppresses TNF-α and interleukin-6 production in human peripheral blood mononuclear cells. The combination therapy with ASHE and anti-TNF-α antibody reduced the severity of arthritis compared with anti-TNF-α antibody alone. The present study shows that ASHE has prophylactic effect against CIA and support therapeutic effect of anti-TNF-α antibody.

Prognostic value of 6-min walk stress echocardiography in patients with interstitial lung disease.

BACKGROUND: The 6-min walk test (6MWT) provides prognostic information for patients with interstitial lung disease (ILD). Parameter determined by Doppler echocardiography after the 6MWT (6 MW stress echocardiography) is shown to be a predictor of future development of pulmonary hypertension in patients with connective tissue disease. However, the clinical utility of 6 MW stress echocardiography in predicting cardiopulmonary events in patients with ILD remains unknown. We examined whether parameters determined by 6 MW stress echocardiography independent predictors of adverse events in patients with ILD. METHODS: Echocardiographic examinations were performed in 68 consecutively enrolled patients with ILD (age, 65 ± 10 years, 65% men). A pressure gradient of tricuspid regurgitation (TRPG) and pulmonary vascular resistance (PVRecho) calculated using the following formula [PVRecho = (peak velocity of TR × 10/time-velocity integral of right ventricular outflow (RVOT-VTI)) + 0.16] were measured at baseline and at post 6MWT. Data for parameters of pulmonary functional tests and for 6MWT were collected. RESULTS: During a mean follow-up period of 22 ± 12 months, 22 patients experienced cardiopulmonary events. In univariate analysis, %VC, TRPG, PVRecho, TRPG post 6MWT, and PVRecho post 6MWT were significantly associated with cardiopulmonary events. Multivariate analysis using the Cox proportional hazards model indicated that %VC [hazard ratio (HR): 0.97, p = 0.009] and PVRecho post 6MWT (HR: 1.77, p = 0.004) were independent predictors of cardiopulmonary events in patients with ILD. CONCLUSIONS: In addition to parameters of pulmonary function tests, increased PVRecho post 6MWT is a significant predictor of cardiopulmonary events in patients with ILD. A 6 MW stress echocardiography is useful in assessing the risk of adverse events in patients with ILD.

Quantification of Wilms' tumor 1 mRNA by digital polymerase chain reaction.

Wilms' tumor 1 (WT1) is overexpressed in various hematopoietic tumors and widely used as a marker of minimal residual disease. WT1 mRNA has been analyzed using quantitative real-time polymerase chain reaction (real-time PCR). In the present study, we analyzed 40 peripheral blood and bone marrow samples obtained from cases of acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome at Sapporo Medical University Hospital from April 2012 to January 2015. We performed quantification of WT1 was performed using QuantStudio 3D Digital PCR System (Thermo Fisher Scientific ) and compared the results between digital PCR and real-time PCR technology. The correlation between digital PCR and real-time PCR was very strong (R = 0.99), and the detection limits of the two methods were equivalent. Digital PCR was able to accurately detect lower WT levels compared with real-time PCR. Digital PCR technology can thus be utilized to predict WT1/ABL1 expression level accurately and should thus be useful for diagnosis or the evaluation of drug efficiency in patients with leukemia.

Significance of SALL4 as a drug­resistant factor in lung cancer.

We examined first evidence for the significance of SALL4, a transcription factor essential for embryonic development and the self-renewal of embryonic stem (ES) cells, as a natural resistance factor against anticancer drugs in lung cancer. To determine the significance of SALL4 expression in lung cancer cells, small inhibitory RNA (siRNA) against SALL4 was transduced into A549 and SBC-3 cells, resulting in increased sensitivity towards anticancer drugs [cisplatin (CDDP), carboplatin (CBDCA), and paclitaxel (PTX)]. SALL4 cancer tissues from 31 lung cancer patients were used to assess clinical significance. The analysis showed differences in SALL4 expression corresponding to the therapeutic outcomes. SALL4 expression measured before adjuvant chemotherapy was significantly higher in the patients showing recurrence of cancer than in the disease-free patients. In addition, the period until recurrence was shorter in the patients showing high SALL4 expression. These results indicate that SALL4 overexpression acts as a natural resistance factor and may be involved in the recurrence of lung cancer after adjuvant chemotherapy.

Significance of serine threonine tyrosine kinase 1 as a drug resistance factor and therapeutic predictor in acute leukemia.

Alterations in the mRNA expression or the mutation of previously reported tyrosine kinases have been detected only in a limited number of patients with acute leukemia. In this study, we examined whether the widely expressed serine threonine tyrosine kinase 1 (STYK1)/novel oncogene with kinase domain (NOK) acts as a drug resistance factor in acute leukemia. The transfection of leukemic HL-60 cells with an STYK1 expression vector resulted in the resistance to doxorubicin and etoposide and decreased drug-induced caspase-3/7 activity and sub-G1 population. To investigate the mechanism of STYK1-induced drug resistance, microarray analysis was performed using HL-60 cells transfected with control or STYK1 expression vectors. Three tyrosine kinases (EphA4, FLT4 and STK31), two NF-κB inducers (MAPK4 and TNF-RSF11A), and two genes essential for stem cell replication (SALL4 and NOV) were identified as novel STYK1-induced genes. In addition to the data using cell line, a comparison of the leukemic patients who did and did not respond to therapy revealed that STYK1 expression before therapy was significantly higher in the non-responder group compared with the group that responded completely. These results suggest that STYK1 is a novel drug resistance factor and could be a predictor of the therapeutic response in acute leukemia.

CD7 promotes extramedullary involvement of the B-cell acute lymphoblastic leukemia line Tanoue by enhancing integrin ß2-dependent cell adhesiveness.

Extramedullary involvement (EMI) is a factor that defines prognosis of acute lymphoblastic leukemia; however, the molecular mechanism(s) remain elusive. Here, we show that CD7 promotes EMI of the human B-cell acute lymphoblastic leukemia cell line Tanoue. The Tanoue cell line expressing firefly luciferase, Luc-Tanoue, was transplanted into non-obese diabetic/severe combined immunodeficient mice, and cells infiltrated into the brain were cultured ex vivo. This process was repeated 4 times to obtain the highly invasive line Luc-Tanoue-F4. Comparison of the global gene expression signatures of Luc-Tanoue-F4 and Luc-Tanoue indicated that the CD7 gene showed the largest increase in expression among EMI-related genes in Luc-Tanoue-F4 cells. Overexpression of CD7 in Tanoue enhanced cell invasiveness. Among cell migration, proliferation, adhesion and protease activity, only cell adhesiveness showed enhancement in Luc-Tanoue-F4. Expression of the intracellular domain, but not the extracellular domain, of CD7 enhanced cell adhesiveness. Luc-Tanoue-F4 showed a higher level of integrin ß2 expression; overexpression of CD7 induced the expression of integrin ß2 in Luc-Tanoue. These results show that CD7 induces integrin ß2 and enhances cell adhesiveness and invasiveness in Tanoue cells. This study highlights the role of the CD7/integrin ß2 axis as a critical pathway in the process of EMI of human B-cell acute lymphoblastic leukemia.