Rapid Detection of the Macrolide Sensitivity of Pneumonia-Causing Mycoplasma pneumoniae Using Quenching Probe Polymerase Chain Reaction (GENECUBE®).

BACKGROUND AND OBJECTIVES: Macrolide-resistant Mycoplasma pneumoniae (MR-MP) have been reported worldwide. Strategies for the treatment of MR-MP are a key focus of research. The GENECUBE® is a novel, fully automated rapid genetic analyzer. The goals of this study were to assess the macrolide sensitivity of M. pneumoniae (MP) isolates by analyzing 23S ribosomal RNA (rRNA) gene sequences using a GENECUBE®-based system and to determine the validity of this system in determining clinical treatment options for MP pneumonia. METHODS: This was an observational retrospective study including 150 children with MP pneumonia. We used quenching probe polymerase chain reaction (Q-probe PCR) as implemented in the GENECUBE® system to detect macrolide resistance-causing mutations in the MP 23S rRNA gene. We compared the duration of fever between patients receiving initial empirical antibiotic treatment (Empirical T group) and those receiving treatment after Q-probe PCR (PCR First group) diagnosis. RESULTS: Selecting antibiotic treatment after Q-probe PCR significantly shortened the duration of fever compared to empirical antibiotic treatment (PCR First group, median: 6.0 days [n = 32]; Empirical T group, median: 7.5 days [n = 66]; p = 0.002). Comparison of macrolide sensitivity using Q-probe PCR and clinical diagnosis showed that the reliability of Q-probe PCR was nearly validated for macrolide sensitivity. CONCLUSION: Q-probe PCR as implemented by GENECUBE® is a useful tool for the diagnosis of MP pneumonia and enables optimization of the selection of antibiotics in order to rapidly improve the clinical course of disease.

Development of rhabdomyolysis in a child after norovirus gastroenteritis.

BACKGROUND: In children, the most significant cause of rhabdomyolysis or muscle breakdown is viral infection. However, there are no reports that norovirus, a gastroenteric virus that commonly infects children, specifically causes rhabdomyolysis. Here, we report the first pediatric case of norovirus-associated rhabdomyolysis. CASE PRESENTATION: The patient, a 2-year-old boy with fever, diarrhea, and vomiting, was referred to our hospital with dysstasia and transaminitis. He was diagnosed with rhabdomyolysis. Additionally, norovirus genogroup GII was detected from stool samples by real-time quantitative reverse transcription Polymerase Chain Reaction, and thereafter, the norovirus GII.4 variant was identified. CONCLUSION: However, the association between rhabdomyolysis and the isolated norovirus variant was not clarified. After treatment the patient recovered without renal failure or disseminated intravascular coagulation. Rhabdomyolysis is a disease for which there is a need for early detection and treatment. If abnormal posture or muscle weakness is observed during the course of gastroenteritis, blood and urinary tests should be performed to rule out rhabdomyolysis.

[Molecular Detection Methods for Vibrio parahaemolyticus in Seafood].

To detect Vibrio parahaemolyticus in seafood, we evaluated efficient combinations of molecular methods with DNA extraction methods using heat extraction and alkaline heat extraction, and PCR, real-time PCR and loop-mediated isothermal amplification (LAMP) assays were performed targeting V parahaemolyticus species-specific genes (tlh and rpoD) and pathogenic factors genes (tdh and trh). The species-specific genes were detected in all combinations of two strains (a tdh * trh1-positive strain and a trh2-positive strain), two kinds of shellfish (oyster and bloody clams) and molecular methods with tlh-real time PCR or rpoD-LAMP assays with DNA of alkaline heat extraction at 85-145cfu/test level. tdh was detected in both seafoods with real time PCR assay with DNA of heat extraction at 85cfu/test level, and detected with the LAMP and real time PCR assays with DNA of alkaline heat extraction at 85cfu/test level. Detection of both trh1 and trh2 with the PCR assay with DNA of alkaline heat extraction was comparatively high though trh2 was detected with the LAMP assay with DNA of alkaline heat extraction at 145cfu/test level. It, however, is necessary to investigate more sensitive trh-detection methods. In this study, the results indicated that tlh-real time PCR or rpoD-LAMP, tdh-real time PCR and tdh-LAMP assays with DNA of alkaline heat extraction are relatively-sensitive methods to detect V. parahaemolyticus in seafood.

Epidemiological evidence of lesser role of thermostable direct hemolysin (TDH)-related hemolysin (TRH) than TDH on Vibrio parahaemolyticus pathogenicity.

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

Characteristics of a sharp decrease in Vibrio parahaemolyticus infections and seafood contamination in Japan.

Vibrio parahaemolyticus has been one of the most important foodborne pathogens in Japan since the 1960s, and a large epidemic was caused by the pandemic serotype O3:K6 from 1997 to 2001. V. parahaemolyticus infections, however, have sharply declined since that time. Data on serotypes isolated from 977 outbreaks were collected and analysed. Total and pathogenic, thermostable direct hemolysin (TDH) gene-positive V. parahaemolyticus were qualitatively and quantitatively detected in 842 seafood samples from wholesale markets in 2007-2009. Strains isolated from patients and seafood were analysed by serotyping, tdh-PCR, group-specific PCR for pandemic strains, and pulsed-field gel electrophoresis (PFGE). The sharp decrease in the infections from 1999 onwards was noted not only for O3:K6 infections but also for other serotypes. The change in the seafood contamination situation from 2001 to 2007-2009 was characterised by a decrease to three-fourths in the frequency of tdh-positive samples, although that decrease was small compared to the 18-fold decrease in the cases of V. parahaemolyticus outbreaks. PFGE detected the pandemic O3:K6 serotype in the same profile in seafood and patients from 1998 to the present. Because of no large decrease in seafood contamination by V. parahaemolyticus from the production to distribution stages and the presence of pandemic O3:K6 serotype in seafood to the present, it was suggested that the change of seafood contamination was unrelated to the sharp decrease in V. parahaemolyticus infections. V. parahaemolyticus infections might be prevented at the stages after the distribution stage.

Prevalence of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in food-producing animals.

To evaluate the diversity of extended-spectrum ß-lactamases (ESBL) genes among food-producing animals, 48 isolates of ESBL-producing Escherichia coli isolates were obtained from rectal samples of broilers, layers, beef cattle and pigs, at the slaughterhouse level. ESBL-carrying E. coli were isolated from 60.0% of individual broiler rectal samples, 5.9% of layers, 12.5% of beef cattle and 3% of pigs. One ESBL-producing Klebsiella pneumoniae was isolated from a broiler. The ESBL-positive E. coli isolates from broilers harbored various ESBL genes: bla (SHV-12), bla(CTX-M-2), bla(CTX-M-14), bla(CTX-M-15) and bla(CTX-M-44). The plasmid DNAs were analyzed by restriction patterns. Homogeneous band patterns were yielded in those of K. pneumoniae and E. coli isolates harboring the bla(CTX-M-2) gene from different farms. No genetic relation between the 2 CTX-M-14 ESBL-producing strains was found by pulsed-field gel electrophoresis, although 2 plasmids in these strains, obtained from different broiler farms, were similar to each other. This study provides evidence that the proliferation of CTX-M-producing E. coli is due to the growth of indigenous CTX-M-producing strains and the possible emergence of strains that acquired CTX-M genes by horizontal transfer in different broiler farms. CTX-M-producing coliforms in broilers should be controlled due to the critical importance of cephalosporins and the zoonotic potential of ESBL-producing bacteria.

Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood.

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.