Hideki Niimi

A novel “eukaryote-made” thermostable DNA polymerase which is free from bacterial DNA contamination

ABSTRACT 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.

Polymicrobial Amniotic Fluid Infection with Mycoplasma/Ureaplasma and Other Bacteria Induces Severe Intra-Amniotic Inflammation Associated with Poor Perinatal Prognosis in Preterm Labor.

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.

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases

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.

A novel Y231del mutation of HFE in hereditary haemochromatosis provides in vivo evidence that the Huh-7 is a human haemochromatotic cell line.

Hereditary haemochromatosis (HH), which is mainly associated with a C282Y polymorphism in HFE, is common among Caucasians of north European descent, but is very rare among Asians. Herein, we report a 43‐year‐old Japanese man who was diagnosed as having HH. A laboratory examination revealed an elevated serum iron level (280 μg/dl), hyperferritinemia (1698 ng/ml) and a low serum level of hepcidin‐25 (4.0 ng/ml). Abdominal magnetic resonance imaging revealed findings suggestive of iron accumulation in the liver and pancreas. HFE gene sequencing in the patient revealed a novel homozygous TAC nucleotide deletion (c. 691_693del) responsible for the loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This homozygous Y231del mutation was recently found in the Huh‐7 hepatoma cell line and was shown to prevent the translocation of HFE to the cell surface. This clinical case provides in vivo evidence suggesting that Huh‐7 is undoubtedly a human haemochromatotic cell line and, as such, is a valuable tool for investigating the pathogenesis of HFE‐related HH in humans.

Antibiotic Therapy Increases the Risk of Preterm Birth in Preterm Labor without Intra-Amniotic Microbes, but may Prolong the Gestation Period in Preterm Labor with Microbes, Evaluated by Rapid and High-Sensitive PCR System

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.

Melting Temperature Mapping Method: A Novel Method for Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Sample Collection

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.

Open-heart surgery in an infant with heterozygous factor VII deficiency.

A four-month-old male with Taussig-Bing anomaly and multiple ventricular septal defects underwent an open-heart palliative procedure. He suffered from massive postoperative gastrointestinal bleeding. Heterozygous Arg402Stop-related factor VII deficiency was detected by genomic examinations. When he was 14 months old, a subsequent open-heart surgery with replacement therapy of recombinant factor VIIa was performed without any bleeding or thromboembolic complications. Although heterozygous factor VII deficiency is generally recognized as clinically asymptomatic, this latent bleeding disorder can appear perioperatively or postoperatively in patients who undergo cardiopulmonary bypass procedures. Consequently, the prophylactic replacement therapy with recombinant factor VII is recommended during cardiac operations.

Sludge reflects intra-amniotic inflammation with or without microorganisms

To investigate whether amniotic fluid (AF) “sludge” in patients with preterm labor (PTL) with intact membranes is related to intra‐amniotic infection or inflammation.

Pyogenic liver abscess due to hypervirulent Klebsiella pneumoniae in a 14-year-old boy

A 14-year-old otherwise healthy boy presented with right-sided back pain following high fever. Abdominal computed tomography scan showed a large liver abscess. Klebsiella pneumoniae (KP) was rapidly identified from peripheral blood using the melting temperature mapping (Tm) method, which enables identification of pathogenic microorganisms within four hours after patient sample collection. He was diagnosed with pyogenic liver abscess (PLA) caused by KP on the day of admission. The KP was the hypervirulent (hv) clinical variant (string test positive, serotype K1, sequence type 23, rmpA and magA positive). After intravenous antibiotic therapy and drainage of the abscess, his condition resolved. The highlights of this case report are a healthy child with hypervirulent Klebsiella pneumoniae liver abscess in Japan and the new Tm mapping method for rapid and accurate identification of the pathogenic microorganism.

17OHP-C in patients with spontaneous preterm labor and intact membranes: is there an effect according to the presence of intra-amniotic inflammation?

It is not known whether 17‐alpha‐hydroxyprogesterone caproate (17OHP‐C) is effective for preventing preterm delivery with an episode of preterm labor (PTL) with or without intra‐amniotic inflammation/infection.