Identifying pathogenic microorganisms as early as possible is critical for selecting the appropriate antimicrobial therapy in infected patients. We previously reported the development of the Tm mapping method for identifying a broad range of pathogenic bacteria within 3 h of blood collection. However, the Tm mapping identification requires an analytical instrument with a tube-to-tube variation of no more than 0.1 °C, so we can only use a few instruments that have such high thermal accuracy. To address the problem, we developed the improved Tm mapping method using imperfect-match linear long quenching probes (IMLL Q-probes). Using IMLL Q-probes, almost all commercially available analytical instruments can be used for the Tm mapping method. Some bacterial species cannot be narrowed down to one species, but they can at least be narrowed down to the genus level. The Tm mapping method using IMLL Q-probes is useful for deciding on antimicrobial therapy in infected patients.

Sepsis is life-threatening organ dysfunction and is considered a major cause of health loss. However, since the current biomarkers of sepsis reflect the host's immune response to microorganisms, they would inevitably cause a time-lag. This means that there is still no truly reliable biomarker of sepsis. In the present study, we developed a novel method for identifying and quantifying unknown pathogenic bacteria within four hours of sample collection. The most important point of this study is that the novel method can be used to determine the number of bacteria in a sample as a novel biomarker of infectious diseases. Indeed, based on the number of bacteria, we were able to accurately estimate the severity of microbial infection. Furthermore, using the time-dependent changes in the number of bacteria, we were able to monitor the therapeutic effect accurately. The rapid identification and quantification of bacteria may change our approach to medical care.

Background Cell population data (CPD) parameters related to neutrophils, such as fluorescent light intensity (NE-SFL) and fluorescent light distribution width index (NE-WY), have emerged as potential biomarkers for sepsis. However, the diagnostic implication in acute bacterial infection remains unclear. This study assessed the diagnostic value of NE-WY and NE-SFL for bacteremia in patients with acute bacterial infections, and those associations with other sepsis biomarkers. Methods Patients with acute bacterial infections were enrolled in this prospective observational cohort study. For all patients, a blood sample, with at least two sets of blood cultures, were collected at the onset of infection. Microbiological evaluation included examination of the blood bacterial load using PCR. CPD was assessed using Automated Hematology analyzer Sysmex series XN-2000. Serum levels of procalcitonin (PCT), interleukin-6 (IL-6), presepsin, and CRP were also assessed. Results Of 93 patients with acute bacterial infection, 24 developed culture-proven bacteremia and 69 did not. NE-SFL and NE-WY were significantly higher in patients with bacteremia than in those without bacteremia (p < 0.005, respectively), and were significantly correlated with the bacterial load determined by PCR (r = 0.384 and r = 0.374, p < 0.005, respectively). To assess the diagnostic value for bacteremia, receiver operating characteristic curve analysis was used. NE-SFL and NE-WY showed an area under the curve of 0.685 and 0.708, respectively, while those of PCT, IL-6, presepsin, and CRP were 0.744, 0.778, 0.685, and 0.528, respectively. Correlation analysis showed that the levels of NE-WY and NE-SFL were strongly correlated with PCT and IL-6 levels. Conclusion This study demonstrated that NE-WY and NE-SFL could predict bacteremia in a manner that may be different from that of other indicators. These findings suggest there are potential benefits of NE-WY/NE-SFL in predicting severe bacterial infections.

Protein S Tokushima variant is a single nucleotide polymorphism in exon 6 of the PROS1 gene, which causes type II congenital Protein S deficiency. This deficiency is difficult to diagnose by conventional coagulation tests and therefore genetic testing of PROS1 is usually required. Direct sequencing tests are a gold standard genetic test; however, they are expensive and require time and labor to perform. In this study, we aimed to develop a rapid and easy gene-based detection method for Protein S Tokushima variant. We applied the quenching probe technique in combination with polymerase chain reaction. Two different probes were prepared; one for wild-type alleles and one for Protein S Tokushima variant alleles. To evaluate the specificity of these probes, we prepared DNA sample from two subjects without any variant and two subject with heterozygous variant of Protein S Tokushima. Moreover, synthetic oligonucleotides with one of known seven variants or Protein S Tokushima variant were also prepared.The quenching probes were highly specific for the Protein S Tokushima variant; thus, we could reliably detect the variant allele. Moreover, we could distinguish wild-type, Protein S Tokushima homozygotes, and Protein S Tokushima heterozygotes. In addition to peripheral blood samples, scraped mucosal cell samples could be also applied as specimens in our method. We developed a rapid and easy gene-based detection method for Protein S Tokushima variant using quenching probes. Because the scraped buccal mucosal cells can be also applied as specimens, our method is non-invasive and very convenient.Funding Information: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Competing interests.Conflict of Interests: The authors declare that they have no competing interests.Ethical Approval Written informed consent was obtained from all participants. All procedures were performed under a protocol approved by the University of Toyama Ethics Committee.

Background: Ossification of the posterior longitudinal ligament (OPLL), characterized by ectopic new bone formation in the spinal ligament, causes neurological impairment due to narrowing of the spinal canal. However, the etiology has not been fully elucidated yet. Several biomarkers may be related to the pathogenesis of OPLL. The present study focused on the serum level of periostin, which is recognized as an important bone formation regulator. Methods: This study included 92 patients with OPLL and 54 control patients without OPLL. For the case-control analysis, 54 age and sex-matched patients were randomly included in the OPLL group. The serum fibroblast growth factor-23 (FGF-23), creatinine, inorganic phosphate, calcium, alkaline phosphatase, and periostin levels were assessed. Furthermore, the calcium, creatinine, and inorganic phosphate levels in urine and the percentage of tubular reabsorption of phosphate were also analyzed. Moreover, the relationship between the biomarkers and the extent of OPLL was analyzed. The data were compared between patients with OPLL progression (the progression group) and without OPLL progression (the non-progression group). Results: The mean serum FGF-23 and periostin levels in the OPLL group were higher than that in the control group. The serum inorganic phosphate level in the OPLL group was lower than that in the control group. No correlation was found between any of the biomarkers and the extent of ossification. The serum periostin level in the progression group was higher than that in the non-progression group. No significant difference in the serum FGF-23 level was noted between the progression and non-progression groups. Moreover, no correlation was found between serum periostin and FGF-23 levels. Conclusions: The serum periostin level is related to OPLL progression. Level of Evidence: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Abstract We developed a novel pretreatment procedure that destroys the plasma membrane of platelets but not the bacterial cell wall, allowing viable bacterial ATP in septic blood samples to be measured with minimal human-derived ATP background. After the pretreatment procedure with this approach, the plasma membranes of platelets are completely destroyed, and human-derived ATP background is dramatically decreased. To test the recovery rates of viable bacteria after pretreatment, we evaluated 19 bacterial genera. As a result, the mean recovery rates of viable bacteria were 93.6% using ATP bioluminescence and 71.9% using the colony-counting method. Finally, using the pretreatment procedure and ATP bioluminescence, rapid antimicrobial susceptibility testing of pathogenic bacteria in septic blood samples was performed, and LVFX resistance was determined within 6.5 h of whole blood collection. This novel pretreatment procedure will open up new possibilities concerning the utility of ATP bioluminescence in medicine.

Despite many clinical decision support systems (CDSSs) being rated as highly usable, CDSSs have not been widely adopted in clinical practice. We posit that there are factors aside from usability that impact adoption of CDSSs; in particular we are interested in the role played by MDs intrinsic motivation to use computer-based support. Our research aim is to investigate the relationship between usability and intrinsic motivation in order to learn about adoption of CDSS in clinical practice.Following the evaluation of a CDSS, 19 MDs completed a 2 part questionnaire about their intrinsic motivation to use computer-based support in general and the usability of the evaluated CDSS.The analysis of MDs motivation to use computer-based support demonstrated that MDs are comfortable using computer-based support and in general find using it quite easy (a motivation rating of 0.66 on a (0, 1) scale was computed). However MDs also reported a perceived lack of competence associated with a lack of prior experience using technology in practice, which results in pressure and tension. The considered CDSS scored highly on all usability dimensions and a usability rating of 0.74 was recorded. The examination of the relationship between motivation and usability suggested that users who were motivated to use computer-based support experienced better usability than those who reported low levels of motivation.Our small case study suggests that an important factor supplementing the usability of CDSSs is intrinsic motivation to use computer-based support in general. We posit that the lack of such a measure thus far in CDSS evaluation may to some extent explain seeming MD satisfaction with CDSSs on one hand, but their limited adoption on the other. We recommend that clinical managers responsible for deploying CDSS should invest in training MDs to use technology underlying computer-based support applications instead of focusing only on the features of the specific CDSS to be deployed.

RATIONALE:Factor X (FX) deficiency is a rare autosomal recessive bleeding disorder. The majority of patients carry a missense mutation in F10, and patients with bleeding disorders are either homozygous or compound heterozygous for F10. Nonsense mutations are exceptionally rare, and a heterozygous nonsense mutation is not considered to cause bleeding disorders.PATIENT CONCERNS:A 35-year-old Japanese female with an incidental hemorrhage after gynecologic polypectomy was referred to our hospital.DIAGNOSES:Following differential diagnostic workup, including cross-mixing test, congenital FX deficiency was strongly suspected.INTERVENTION:Coagulation tests and mutation analyses were conducted for the patient and her parents.OUTCOMES:Mutation analysis revealed that she carried a heterozygous nonsense mutation in F10. Pedigree analysis revealed that the mutation was inherited from her mother although there was no familial history of bleeding or hemostatic disturbance.LESSONS:Hemostatic disturbance may occur even in a patient with heterozygous F10. Because heterozygous nonsense mutation in F10 is expected to be hidden in an apparently healthy population, as observed in our patient, unexpected hemostatic disturbance may occur, particularly during the use of direct oral anticoagulant (DOAC)-targeting factor Xa for thrombotic diseases. FX activity should be evaluated before prescribing DOACs to patients.

STUDY DESIGN:Case-control study. OBJECTIVES:To determine the possible pathogenesis of ossification of the posterior longitudinal ligament (OPLL) in regard to the serum concentration of fibroblast growth factor 23 (FGF-23). METHODS:The study included 95 patients with OPLL and a control group of 73 age- and sex-matched volunteers. The serum concentrations of FGF-23, creatinine (Cre), alkaline phosphatase, calcium (Ca), inorganic phosphate (Pi), and hypersensitive C-reactive protein (hs-CRP) were analyzed from blood samples, and Cre, Ca, Pi, and tubular reabsorption of phosphate were measured using urine samples. We evaluated the severity of ossified spinal lesions in patients with OPLL according to the ossification index (the OP index and the OS index). Data was compared between the OPLL and control group and between the OPLL progression and no progression group. RESULTS:Serum FGF-23 and hs-CRP were higher, and serum Pi was lower in patients with OPLL than in the controls. There was a positive correlation between FGF-23 and hs-CRP and a negative correlation between serum Pi and the OS index; however, the correlations were very weak. Overall, 31.7% of patients had progression of OPLL during follow-up. FGF-23 and hs-CRP were higher in the progression group than in the no progression group. CONCLUSIONS:These results might indicate that FGF-23 and hs-CRP are positive markers for OPLL. Phosphate metabolism via FGF-23 might be a target for future study on the pathogenesis of OPLL.

Problem It is not known whether 17‐alpha‐hydroxyprogesterone caproate (17 OHP ‐C) is effective for preventing preterm delivery with an episode of preterm labor ( PTL ) with or without intra‐amniotic inflammation/infection. Methods of study This was a retrospective cohort study. One hundred and seven PTL patients were selected and divided into a 17 OHP ‐C group (use of 17 OHP ‐C: n = 53) and a no‐treatment group (no use of 17 OHP ‐C: n = 54). Moreover, the patients were divided into three subgroups (subgroup A: without intra‐amniotic inflammation, B: with mild intra‐amniotic inflammation, and C: with severe intra‐amniotic inflammation) according to their level of amniotic interleukin ( IL )‐8, and perinatal prognosis was analyzed. Results Interval from admission to delivery (days) in the 17 OHP ‐C group (76 [13‐126], n = 34) was significantly longer than that in the no‐treatment group (50 [8‐104], n = 33; P = .012) in subgroup B. In cases without intra‐amniotic microbes in subgroup B, a significant prolongation of gestational days was associated with the 17 OHP ‐C group (79 [13‐126], n = 25) compared with the no‐treatment group (50 [8‐104], n = 29; P = .029). However, there were no significant differences in subgroups A or C. Conclusion 17 OHP ‐C could prolong gestational period in limited PTL cases with sterile mild intra‐amniotic inflammation.

ProblemTo investigate whether amniotic fluid (AF) “sludge” in patients with preterm labor (PTL) with intact membranes is related to intra‐amniotic infection or inflammation.Method of study105 PTL patients before 29 weeks’ gestation were enrolled. AF “sludge” was evaluated by transvaginal sonography. Microorganisms were identified in AF by our newly established PCR method using a eukaryote‐made thermostable DNA polymerase.ResultsAF “sludge” was present in 18.1% (19/105) of patients. The results obtained in the AF “sludge” group vs the no “sludge” group were as follows: (i) a similar positive rate of microorganisms in AF by PCR, 31.6% (6/19) vs 38.4% (33/86); (ii) a higher level of AF interleukin‐8, 15.2 (0.2‐381.5) ng/mL vs 5.8 (0.1‐413.7) ng/mL; P = .005); and (3) a higher frequency of histological chorioamnionitis, 52.6% (10/19) vs 23.3% (20/86); P = .010.ConclusionThe presence of AF “sludge” is related to intra‐amniotic inflammation with or without microorganisms.

Background: It has been suggested that more than 100 bacterial species can be identified using only seven universal bacterial primer sets in the melting temperature (Tm) mapping method and that these findings can be obtained within 3 h of sterile site collection.Case presentation: A 67-year-old Japanese man with type 2 diabetes visited our hospital complaining of progressive lower back pain for 2 months. The patient was suspected to have spondylodiscitis on magnetic resonance imaging of the spine. Blood culture and transcutaneous vertebral biopsy were subsequently performed. Using the Tm mapping method, Parvimonas micra was detected from a transcutaneous vertebral biopsy specimen in 3 h. Gram-positive cocci were also detected by Gram staining and P. micra was identified directly from the anaerobic blood culture by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Four days after admission, the biopsy specimen culture isolate was identified as P. micra.Conclusions: The Tm mapping method may be useful for the diagnosis of bacterial infections where diagnosis is challenging because of the difficulty of culturing.

OBJECTIVES:To examine the efficacy of the use of antibiotics in preterm labor (PTL) with intact membranes, after evaluating intra-amniotic microbes by our rapid and bacteria-free polymerase chain reaction (PCR) system.MATERIALS AND METHODS:One hundred and four PTL patients before 32 weeks of gestation were recruited. Until 2012, antibiotics were empirically prescribed based on the clinical severity of PTL. Intra-amniotic microbes in stored samples were evaluated later by our newly established PCR system, and the efficacy of the use of antibiotics in PTL was evaluated.RESULTS:In the amniotic fluid (AF) microbe-negative patients (n = 67), antibiotic therapy significantly shortened the gestation period (P < 0.0001), whereas in the microbe-positive patients (n = 37), appropriate antibiotic therapy (proper antibiotic selection against identified AF microbes) was significantly associated with an increase in gestation period (P < 0.0001).CONCLUSION:Appropriate antibiotic therapy in PTL with intact membranes prolonged the gestation period.

PROBLEM:To study the relationship between perinatal prognosis in cases of preterm labor (PTL) and polymicrobial infection in amniotic fluid (AF) and intra-amniotic (IA) inflammation using a highly sensitive and reliable PCR-based method.METHOD OF STUDY:To detect prokaryotes using a nested PCR-based method, eukaryote-made thermostable DNA polymerase without bacterial DNA contamination was used in combination with bacterial universal primers. We collected AF aseptically from 118 PTL cases and 50 term subjects.RESULTS:The prevalence of microorganisms was 33% (39/118) by PCR and only 7.6% (9/118) by culture. PTL caused by a combination of positive Mycoplasma/Ureaplasma and other bacteria had significantly higher AF IL-8 levels and a significantly shorter amniocentesis-to-delivery interval.CONCLUSIONS:Our newly established PCR method is useful for detecting IA microorganisms. Polymicrobial infection with Mycoplasma/Ureaplasma and other bacteria induces severe IA inflammation associated with poor perinatal prognosis in PTL.

Acquiring the earliest possible identification of pathogenic microorganisms is critical for selecting the appropriate antimicrobial therapy in infected patients. We herein report the novel "melting temperature (Tm) mapping method" for rapidly identifying the dominant bacteria in a clinical sample from sterile sites. Employing only seven primer sets, more than 100 bacterial species can be identified. In particular, using the Difference Value, it is possible to identify samples suitable for Tm mapping identification. Moreover, this method can be used to rapidly diagnose the absence of bacteria in clinical samples. We tested the Tm mapping method using 200 whole blood samples obtained from patients with suspected sepsis, 85% (171/200) of which matched the culture results based on the detection level. A total of 130 samples were negative according to the Tm mapping method, 98% (128/130) of which were also negative based on the culture method. Meanwhile, 70 samples were positive according to the Tm mapping method, and of the 59 suitable for identification, 100% (59/59) exhibited a "match" or "broad match" with the culture or sequencing results. These findings were obtained within three hours of whole blood collection. The Tm mapping method is therefore useful for identifying infectious diseases requiring prompt treatment.

Background Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. Methods We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Results Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. Conclusions We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.

To achieve the production of a thermostable DNA polymerase free from bacterial DNA contamination, we developed eukaryote-made thermostable DNA (Taq) polymerase. The novel eukaryote-made thermostable DNA polymerase resolves the problem of contaminating bacterial DNA in conventional bacterially made thermostable DNA polymerase as a result of its manufacture and incomplete purification. Using eukaryote-made thermostable DNA polymerase, the sensitive and reliable detection of bacteria becomes feasible for large fields, thereby making the development of a wide range of powerful applications possible.