Yasuko Aoki

Simultaneous Detection of Pathogens in Clinical Samples from Patients with Community-Acquired Pneumonia by Real-Time PCR with Pathogen-Specific Molecular Beacon Probes

ABSTRACT In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Streptococcus pyogenes, simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children (n = 389) and from adults (n = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae, the mip gene for L. pneumophila, and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, 100% and 100% for S. pyogenes, and 100% and 95.4% for M. pneumoniae, respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae, the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.

Nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy in 2007: general view of the pathogens' antibacterial susceptibility.

For the purpose of nationwide surveillance of the antimicrobial susceptibility of bacterial respiratory pathogens collected from patients in Japan, the Japanese Society of Chemotherapy conducted a third year of nationwide surveillance during the period from January to April 2008. A total of 1,097 strains were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections. Susceptibility testing was evaluable with 987 strains (189 Staphylococcus aureus, 211 Streptococcus pneumoniae, 6 Streptococcus pyogenes, 187 Haemophilus influenzae, 106 Moraxella catarrhalis, 126 Klebsiella pneumoniae, and 162 Pseudomonas aeruginosa). A total of 44 antibacterial agents, including 26 β-lactams (four penicillins, three penicillins in combination with β-lactamase inhibitors, four oral cephems, eight parenteral cephems, one monobactam, five carbapenems, and one penem), three aminoglycosides, four macrolides (including a ketolide), one lincosamide, one tetracycline, two glycopeptides, six fluoroquinolones, and one oxazolidinone were used for the study. Analysis was conducted at the central reference laboratory according to the method recommended by the Clinical and Laboratory Standard Institute (CLSI). The incidence of methicillin-resistant S. aureus (MRSA) was as high as 59.8%, and those of penicillin-intermediate and penicillin-resistant S. pneumoniae (PISP and PRSP) were 35.5 and 11.8%, respectively. Among H. influenzae, 13.9% of them were found to be β-lactamase-non-producing ampicillin (ABPC)-intermediately resistant (BLNAI), 26.7% to be β-lactamase-non-producing ABPC-resistant (BLNAR), and 5.3% to be β-lactamase-producing ABPC-resistant (BLPAR) strains. A high frequency (76.5%) of β-lactamase-producing strains was suspected in Moraxella catarrhalis isolates. Four (3.2%) extended-spectrum β-lactamase-producing K. pneumoniae were found among 126 strains. Four isolates (2.5%) of P.aeruginosa were found to be metallo β-lactamase-producing strains, including three (1.9%) suspected multidrug-resistant strains showing resistance to imipenem, amikacin, and ciprofloxacin. Continual national surveillance of the antimicrobial susceptibility of respiratory pathogens is crucial in order to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis.

The first nationwide surveillance of bacterial respiratory pathogens conducted by the Japanese Society of Chemotherapy. Part 1: a general view of antibacterial susceptibility

The Japanese Society of Chemotherapy (JSC) conducted the first nationwide surveillance of bacterial respiratory pathogens during the period from January to August 2006. With the cooperation of 32 medical institutions throughout Japan, a total of 924 strains belonging to seven clinically relevant bacterial species were collected from adult patients with well-diagnosed respiratory tract infections (RTIs). Antimicrobial susceptibility testing of the 887 evaluable strains (205 Staphylococcus aureus, 200 Streptococcus pneumoniae, 9 Streptococcus pyogenes, 165 Haemophilus influenzae, 91 Moraxella catarrhalis, 74 Klebsiella pneumoniae, and 143 Pseudomonas aeruginosa) to 42 antibacterial agents was conducted at the Central Laboratory of the Research Center for Anti-infective Drugs of the Kitasato Institute, according to recommendations issued by the Clinical and Laboratory Standards Institute (CLSI). The antibacterial agents employed were 25 β-lactams, three aminoglycosides, four macrolides (including one azalide and one ketolide), one lincosamide, one tetracycline, two glycopeptides, five fluoroquinolones, and one oxazolidinone. The incidence of methicillin-resistant S. aureus (MRSA) was 63.4%, and the incidences of penicillin-intermediately resistant S. pneumoniae (PISP) and penicillin-resistant S. pneumoniae (PRSP) were 35.0% and 4.0%, respectively. Among H. influenzae, 21.2% of the strains were found to be β-lactamase-nonproducing ampicillin (ABPC)-intermediately resistant (BLNAI), 29.1% to be β-lactamase-nonproducing ABPC-resistant (BLNAR), and 4.8% to be β-lactamaseproducing ABPC-resistant (BLPAR) strains. The incidence of extended-spectrum β-lactamase-producing K. pneumoniae was 2.7% (2 of 74 strains). Three (2.1%) of the 143 P. aeruginosa strains were found to be metallo-β-lactamaseproducing, including 1 (0.7%) multidrug-resistant strain. Through the nationwide surveillance, we obtained fundamental antimicrobial susceptibility data of clinically relevant bacterial pathogens in adult RTI to various antibacterial agents. These data will be a useful reference for future periodic surveillance studies, as well as for investigations to control antimicrobial-resistant pathogens.

Pure red cell aplasia: relationship between inhibitory activity of T cells to CFU-E and erythropoiesis

T cell‐mediated inhibition of autologous late erythroid colony formation was found in two patients with PRCA. Each patient was treated separately with immunosuppressive agents, ALG, bolus methylprednisolone or cyclophosphamide. Cyclophosphamide was the most effective among these immunosuppressive therapies. Peripheral blood T cells, which were taken serially from the patients during the course of the disease, were cryopreserved until use. The inhibitory activity of T cells was assayed after remission using autologous bone marrow. It was found that the decrease of inhibitory activity was closely correlated with clinical improvement and that the effectiveness of the immunosuppressive therapy on inhibitory activities of T cells differed between therapies. These findings suggest that T cell‐mediated inhibition of erythropoiesis may be pathogenetic for PRCA in some patients.

Clinical characteristics and risk factors for community-acquired Clostridium difficile infection: A retrospective, case-control study in a tertiary care hospital in Japan

The epidemiology of Clostridium difficile infection (CDI) has changed in the past decade. The incidence, prevalence, and severity of community-acquired CDI (CA-CDI) have increased. However, the epidemiology of CA-CDI in Japan has not been investigated. To evaluate the clinical characteristics and risk factors for CA-CDI in Japan, we conducted a retrospective, case-control study of CA-CDI at the National Hospital Organization Tokyo Medical Center between January 2010 and December 2014. Two age- and sex-matched C. difficile toxin- and culture-negative controls were assigned for each case. A total of 26 patients were identified with CA-CDI were identified. The incidence rate for CA-CDI was 1.4 per 100,000 patient-years. Of the CA-CDI patients, 6 (23.1%) had no underlying comorbidity, 22 (84.6%) had prior exposure to antimicrobials, and 5 (19.2%) had prior exposure to antacids. Although 5 patients (19.2%) required hospitalization, none required intensive care or died. Recurrence was observed in 1 patient (3.8%). Patients with CA-CDI cases were more likely to have been recently exposed to antimicrobials compared to controls (odds ratio [OR]: 8.12, 95% confidence interval [CI]: 2.43-26.98). However, exposure to antacids was not associated with CA-CDI (OR: 0.59, 95% CI: 0.19-1.85). Our findings indicate that the incidence rate for CA-CDI in Japan is relatively low compared to the US and Europe, and that CA-CDI is usually not severe. Previous antimicrobial exposure was the main risk factor for CA-CDI, suggesting that clinicians should consider CDI in patients presenting with diarrhea who have recently received antimicrobials.

T cell-mediated inhibition of haematopoiesis in aplastic anaemia: serial assay of inhibitory activities of T cells to autologous CFU-E during immunosuppressive therapy.

T cell‐mediated inhibition of autologous erythroid colony formation was found in two patients with aplastic anaemia. Each patient was treated separately with ALG and methylprednisolone. Peripheral blood T cells were cryopreserved serially during the course of the disease. The inhibitory activity of T cells was assayed after remission using autologous bone marrow. The inhibitory activity of T cells was lost following the treatment and preceded haematopoietic recovery.

Characterization of Small Colony Variants of Methicillin-Resistant Staphylococcus aureus Regrown in the Presence of Arbekacin

We examined the biologic and pathogenic characteristics of small colony variants of methicillin-resistantStaphylococcus aureus regrown in the presence of arbekacin at concentrations above the MIC. The cells possessed both coagulase and catalase activities, but not DNase activity. Ultrastructural observations revealed that the cell structure was polymorphic and fragile. The growth rate of these cells was slower, but they were more resistant to arbekacin than those of the parent strain. They were also more easily phagocytized by human polymorphonuclear leukocytes than the parent strain. The 50% lethal dose in mice of small colony variants was higher than that of the parent strain and they were rapidly cleared from the peripheral blood of challenged mice. These differences were more pronounced in normal mice than in leukopenic mice. These findings suggest that the pathogenicity of methicillin-resistantStaphylococcus aureus small colony variants regrown in the presence of arbekacin is low. However, this organism may produce a latent infection by reducing the efficacy of arbekacin in compromised hosts, or in infected foci with deficient neutrophil activity.

Peripherally inserted central catheter-related bloodstream infection caused by Kocuria marina in an elderly man.

BackgroundKocuria species are Gram-positive, aerobic cocci, and members of the Micrcoccaceae family that are known to be opportunistic pathogens. Although there have been sporadic reports of infections caused by Kocuria species, little is known regarding their human pathogenicity and clinical characteristics.Case reportWe herein report a case of a peripherally inserted central catheter (PICC)-related bloodstream infection caused by Kocuria marina in a 90-year-old Japanese with multiple cancer. The patient, who was admitted due to adhesive intestinal obstruction, suddenly developed sepsis on day 29 following admission. Three sets of blood cultures and a culture of the PICC tip revealed the growth of Gram-positive cocci arranged in clusters. The patient improved quickly after treatment with an antimicrobial agent and catheter removal. The organism was identified as Kocuria varians using the MicroScan Walkaway system and K. varians/Kocuria rosea with a 99.7% probability using an API Staph system. However, 16S rRNA gene sequencing analysis identified the pathogen as K. marina.Conclusion Although K. marina is a rare pathogen, physicians should consider it in case of catheter-related infections in patients with serious underlying conditions. As commercial identification systems can misidentify species within the Kocuria genus, the use of genomic methods such as 16S rRNA sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry may be useful for the precise identification of Kocuria to the species level.

Pelvic abscess due to Mycoplasma hominis following caesarean section

Introduction: Mycoplasma hominis is associated with genito-urinary tract infection and adverse pregnancy outcomes. However, whether the species is a true pathogen or part of the genito-urinary tracts natural flora remains unclear. Case presentation: A 41-year-old pregnant woman was admitted to our hospital at 38 weeks and 5 days of gestation owing to premature rupture of the membranes. The patient delivered by caesarean section. Subsequently, the patient complained of lower abdominal pain and had persistent fever. Enhanced computed tomography revealed pelvic abscesses. Gram staining of pus from the abscess and vaginal secretions indicated presence of polymorphonuclear leucocytes but no pathogens. Cultures on blood agar showed growth of pinpoint-sized colonies in an anaerobic environment within 48 h. Although administration of carbapenem and metronidazole was ineffective and we could not fully drain the abscess, administration of clindamycin led to clinical improvement. The isolates 16S rRNA gene and yidC gene sequences exhibited identity with those of M. hominis. Conclusion: Physicians should consider M. hominis in cases of pelvic abscesses where Gram staining yields negative results, small colonies are isolated from the abscess and treatment with β-lactam antibiotics is ineffective.

Characteristics of mucoid Streptococcus pyogenes isolated from two patients with pneumonia in a local community

Streptococcus pyogenes (Group A Streptococcus, GAS) infections can develop into life-threatening disorders. However, the occurrence of some GAS pneumonia cases is relatively rare in a local community. We report here characteristics of mucoid GAS isolates obtained from the sputum of two patients with pneumonia in a local community. Although case-patients did not have contact with each other, case-patient 1′s child and case-patient 2′s grandchild attended the same kindergarten where a GAS pharyngitis epidemic had occurred. We conducted phenotypic and genotypic analyses with the GAS isolates from sputum of both patients, to examine (1) colony appearance between the isolates, (2) numerical profile based on API-20 Strep system, (3) similarity to the type strain using 16S rRNA sequencing, (4) emm type (subtype) and emm full-length sequence, (5) sequence type, (6) sic allele, (7) antimicrobial susceptibility result and the resistance determinant, (8) genome profile following a random amplified polymorphic DNA fragments, and (9) pattern of digested DNA fragments by pulse-field gel electrophoresis. These phenotypic and genotypic analyses revealed similar matching between the isolates from both cases. Our findings suggest that when clinicians examine adult patients having infection with the mucoid GAS, they should confirm whether anyone within the same household also developed the infection and need to investigate epidemic situations in local communities, including kindergartens and elementary schools.