Two cases of Streptococcus dysgalactiae subspecies equisimilis infection transmitted through transfusion of platelet concentrate derived from separate blood donations by the same donor.

BACKGROUND: Transfusion-transmitted bacterial infections (TTBIs) in Japan have been largely prevented due to a short shelf life of 3.5 days after blood collection for platelet concentrate (PC) and washed PCs (WPCs; PC in which 95% plasma is replaced by platelet additive solution). CASE PRESENTATION: Case 1: In January 2018, a woman in her 50s with aplastic anaemia who received WPC transfusion and developed a fever the next day and Streptococcus dysgalactiae subspecies equisimilis (SDSE) was detected in the residual WPC. Case 2: In May 2018, a man in his 60s with a haematologic malignancy who received PC transfusion and developed chills during the transfusion. SDSE was detected in the patient's blood and residual PC. The contaminated platelet products were both manufactured from blood donated by the same donor. The multi-locus sequencing typing revealed that SDSE detected in case 1 was identical to that from case 2; however, whole blood subsequently obtained from the donor was culture negative. CONCLUSION: WPC and PC produced from two blood donated 106 days apart by the same donor were contaminated with SDSE of the same strain and both caused TTBIs. Safety measures should be considered regarding blood collection from a donor with a history of bacterial contamination.

High throughput method of 16S rRNA gene sequencing library preparation for plant root microbial community profiling.

Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have increased sample handling efficiency in a two-step PCR amplification protocol for 16S rRNA gene sequencing of plant root microbiota, improving DNA extraction using AMPure XP magnetic beads and PCR purification using exonuclease. These modifications reduce sample handling and capture microbial diversity comparable to that obtained by the manual method. We found a buffer with AMPure XP magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. We also demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. In addition, our method generated comparable microbiome profile data in plant roots and soils to that of using common commercially available DNA extraction kits, such as DNeasy PowerSoil Pro Kit and FastDNA SPIN Kit for Soil. Our method offers a simple and high-throughput option for maintaining the quality of plant root microbial community profiling.

Different growth kinetics in blood components and genetic analysis of Lactococcus garvieae isolated from platelet concentrates.

BACKGROUND: In 2014, we experienced the first isolation of Lactococcus garvieae from a platelet concentrate (PC). Thereafter, L. garvieae contamination of PCs occurred in two more cases in Japan. It is rare that bacterial contamination with uncommon strains like this species occurs frequently within a short period. Therefore, we performed a detailed analysis of the characteristics of these strains. STUDY DESIGN AND METHODS: Three bacterial strains were identified by biochemical testing and molecular analysis. Genomic diversity was characterized by multilocus sequence typing (MLST). To observe growth kinetics in blood components, PCs were inoculated with the three different strains. RESULTS: All three strains were identified as L. garvieae by molecular analysis. Each strain belonged to a different phylogenetic group according to MLST analysis. In the spiking trial, the three strains demonstrated differences in their final concentrations and changes in appearance of PCs. CONCLUSION: In this study, all three L. garvieae strains were correctly identified by molecular analysis. Since the three strains were collected in different regions of Japan and belonged to different phylogenetic groups according to MLST analysis, it is suggested that L. garvieae have a wide distribution with diversity in Japan. In PCs, the three L. garvieae strains showed clear differences in growth kinetics and changes in appearance of PCs. These differences may have been the primary determinant of whether PC contamination was detected before transfusion. Moreover, L. garvieae represents an emerging foodborne bacterium that can cause transfusion-transmitted bacteremia. Understanding our cases may help prevent bacterial contamination of blood products.

Seroprevalence of Trypanosoma cruzi infection among at-risk blood donors in Japan.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi and is endemic in Latin America. In nonendemic countries, including Japan, Chagas disease is primarily a problem in the context of transfusion transmission. Approximately 250,000 immigrants from Latin America reside in Japan, and many of those individuals serve as active blood donors. This study surveyed the seroprevalence of T. cruzi infection among at-risk blood donors in Japan, defined as those who themselves (or whose mothers) were born (or raised) in Latin America, or those with a travel history to Latin America. STUDY DESIGN AND METHODS: Blood samples were obtained from at-risk donors in two periods, 2004-2012 and 2013-2016. Collected samples were tested for T. cruzi antibodies using both an enzyme-linked immunosorbent assay and a chemiluminescent immunoassay. Samples that tested positive in both assays were additionally tested by polymerase chain reaction, and look-back investigation was conducted when necessary. RESULTS: Of 18,484 samples obtained from 18,076 at-risk donors, 3 (1:6,025, 0.017%) donors showed seroreactivity by enzyme-linked immunosorbent assay and chemiluminescent immunoassay. All antibody-positive donors were born in Latin America. One of them also was positive for T. cruzi DNA. Eleven previous donations from this donor were subjected to look-back investigation, and five recipients were tested. All five recipients tested negative for T. cruzi antibodies. CONCLUSION: Seroprevalence of T. cruzi was 0.017% among at-risk donors in Japan. Transfusion-transmitted infection of Chagas disease has not been confirmed to date. Screening for T. cruzi antibodies by targeting at-risk donors is an appropriate strategy for ensuring blood safety in Japan.

Novel DNA sequence isolated from blood donors with high transaminase levels.

AIM: In Japan, the etiology of 10-20% of cases of acute hepatitis remains unclarified. This study was conducted to verify the agent causing non-A-E hepatitis. METHODS: Serum samples from 500 blood donors with elevated alanine aminotransferase (ALT) levels were screened by polymerase chain reaction using primers constructed from conserved areas of RNA virus helicase. The sequence obtained was investigated for viral properties. RESULTS: Four blood samples were found to contain a novel DNA sequence of 9496 bp, which was designated KIs-V. KIs-V was sensitive to the restriction enzyme SalI and BstXI. Rolling-circle amplification produced an excessive amount of KIs-V DNA. In sucrose density gradient ultracentrifugation, KIs-V banded at a 1.158-g/cm(3) density. Detergent treatment increased the density of KIs-V. There was no KIs-V DNA amplification from human leukocyte DNA. Serial filtration suggested that KIs-V was included in a 30-50-nm size particle. In silico analysis revealed that KIs-V contained 13 potential genes, none of which showed homology to any viral proteins reported. One gene showed similarity to a DNA polymerase domain. Strong signals for transcription initiation and a CpG island were identified. The nucleotide composition of KIs-V showed a characteristic feature of circular DNA genomes that contain a replication origin and a terminus. In a preliminary study, KIs-V was frequently identified among hepatitis E virus antibody positive individuals with elevated ALT levels. CONCLUSION: A new sequence KIs-V was isolated from blood donors with elevated ALT levels. It was suggested that KIs-V is a double-stranded circular DNA genome derived from a novel category of enveloped viruses.