Prompt identification of causative pathogenic bacteria is imperative for the treatment of patients suffering from infectious diseases, including sepsis and pneumonia. However, current culture-based methodologies have several drawbacks including their limitation of use to culturable bacterial species. To circumvent these problems, we attempted to detect bacterial DNA in blood using next-generation DNA sequencing (NGS) technology. We conducted metagenomic and 16S ribosomal RNA (rRNA) gene amplicon sequencing of DNA extracted from bacteria-spiked blood using an Ion Personal Genome Machine. NGS data was analyzed using our in-house pipeline Genome Search Toolkit and database GenomeSync. The metagenomic sequencing analysis successfully detected three gram-positive and three gram-negative bacteria spiked in the blood, which was associated with a significant portion of non-bacterial reads, even though human blood cells were separated by low-speed centrifugation prior to DNA extraction. Sequencing analysis of seven variable regions of the 16S rRNA gene amplicon also successfully detected all six bacteria spiked in the blood. The methodology using 16S rRNA gene amplicon analysis was verified using DNA from the blood of six patients with sepsis and four healthy volunteers with potential pathogenic bacteria in the blood being identified at the species level. These findings suggest that our system will be a potential platform for practical diagnosis in the future.

Acute urinary tract infection (UTI) is a highly common clinical condition. Although bacterial culture is the gold standard diagnostic test, false negative results may be possible, leading to the pathogen being unidentified. In recent years, bacterial DNA sequencing analysis has garnered much attention, but clinical studies are rare in Japan. In this study, we assessed the usefulness of next-generation DNA sequencing (NGS) analysis for acute UTI patients. We thus performed an observational, retrospective case series study. Urine and blood samples were collected from ten acute UTI patients, of whom four had also been diagnosed with urosepsis. Seven variable regions of bacterial 16S rRNA genes were amplified by PCR and then sequenced by IonPGM. The identified bacterial species were compared with those identified using the culture tests and the clinical parameters were analyzed. As a result, the NGS method effectively identified predominant culture-positive bacteria in urine samples. The urine NGS also detected several culture-negative species, which have been reported to be potentially pathogenic. Out of four urosepsis cases, three were pathogen-positive in blood NGS results, while two were pathogen-negative in blood culture. In one sepsis case, although blood culture was negative for Escherichia coli, this species was detected by blood NGS. For nonsepsis cases, however, blood NGS, as well as blood culture, was less effective in detecting bacterial signals. In conclusion, NGS is potentially useful for identifying pathogenic bacteria in urine from acute UTI patients but is less applicable in patients who do not meet clinical criteria for sepsis.

Although not common, herpes simplex virus (HSV) pneumonia can occur in immunocompromised patients. However, HSV pneumonia in immunocompetent hosts is very rare. The authors encountered a very rare case of severe HSV pneumonia in an immunocompetent host. The patient was an 85-year-old Japanese woman who presented with severe intractable pneumonia refractory to empirical antimicrobial therapy. Furthermore, the causative microorganisms remained unknown. Therefore, cytological examination of bronchoalveolar lavage fluid and protected brush biopsy of the lower respiratory tract were performed, which indicated herpes virus-infected cells with nuclear inclusions; PCR assay was positive for HSV DNA. Accordingly, the patient was diagnosed with HSV pneumonia. Her respiratory condition improved immediately after initiation of acyclovir monotherapy. In selected cases of intractable pneumonia refractory to standard antimicrobial therapy, the possibility of HSV pneumonia should be pursued.

Highlights F. necrophorum pelvic peritonitis and bacteremia were seen without Lemierre's syndrome. Hepatoportal venous gas and pneumatosis intestinalis mimicked intestinal necrosis. Unprotected receptive oral sex may have transmitted the F. necrophorum to the vagina.

A 60-year-old Japanese woman was admitted to hospital for impaired consciousness and convulsive status epilepticus. She had received supportive care for bloody diarrhea at a nearby hospital 9 days earlier. Blood test results at admission were as follows: white blood cell count, 43.5 (normal 4.