Development of a Novel Loop-Mediated Isothermal Amplification Method to Detect Guiana Extended-Spectrum (GES) ß-Lactamase Genes in Pseudomonas aeruginosa.

Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) ß-lactamase-producing strains are of growing concern. Several genotypes of the GES ß-lactamase gene (bla GES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of bla GES and another LAMP method to discriminate carbapenemase genotypes of bla GES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting bla GES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting bla GES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected bla GES with high sensitivity in all DNA-spiked samples; PCR did not detect bla GES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing bla GES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two bla GES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES ß-lactamase gene detection assay using the LAMP method. Our new assays effectively detect bla GES and critical unique mutations.

Functional mannose-binding lectin levels in patients with end-stage renal disease on maintenance hemodialysis.

BACKGROUND: Innate immunity is generally impaired in chronic renal failure (CRF). Mannose-binding lectin (MBL) has an important role in first-line host defense against pathogens via the lectin pathway. We recently reported that functional MBL was significantly lower in CRF patients than in healthy subjects. In this study, we aimed to determine whether functional MBL would be improved following hemodialysis (HD) therapy. METHODS: This study included 22 patients with end-stage renal disease (ESRD) on maintenance HD. Functional MBL was measured every 6 months for 1 year after HD using an enzyme-linked immunosorbent assay. RESULTS: Median serum functional MBL levels of ESRD patients were significantly higher after 6 and 12 months than at the start of HD therapy (p < 0.05 and p < 0.01, respectively). Furthermore, median functional MBL levels at 12 months were significantly higher than those at 6 months (p < 0.05). CONCLUSIONS: We found significant increases in serum functional MBL levels in patients on HD. Our results indicated that HD tailored to remove uremic toxins could improve functional MBL levels in these patients.

Liver abscess of Actinomyces israelii in a hemodialysis patient: case report and review of the literature.

We had encountered a 74-year-old woman on hemodialysis therapy suffering from liver abscess of Actinomyces israelii. Percutaneous drainage of the abscess before starting antimicrobial therapy followed by correct microbiological identification and susceptibility test led us to determine long treatment with ampicillin and to a successful outcome. Periodontitis was thought to be a possible entry of actinomyces. Hepatic actinomycosis should be recognized as one of the important infectious diseases among patients of end-stage renal disease.

Relationship between oligomer and functional serum mannose-binding lectin in chronic renal failure.

INTRODUCTION: Mannose-binding lectin (MBL) plays an important role in first-line host defence against pathogens via the lectin pathway. The binding affinity for ligands is greatly increased by oligomerization, although the basic triplet does not bind solid phase mannan and cannot activate complement. Besides, MBL is a positive acute-phase protein. In this study, we examined the relationship between oligomer and functional serum MBL in chronic renal failure patients who were either uraemic [Pre-haemodialysis (pre-HD) patients], or who were receiving maintenance haemodialysis treatment (HD patients). MATERIALS AND METHODS: This study included a total of 20 Pre-HD patients, 130 HD patients and 28 healthy subjects. The oligomer and functional serum MBL levels were measured using enzyme-linked immunosorbent assays established previously. RESULTS: The median serum functional MBL levels were significantly reduced in both Pre-HD and HD patients compared with healthy subjects (P<0·05 for both). Furthermore, the median functional MBL level in Pre-HD patients was significantly lower than that in HD patients (P<0·05). The median serum oligomer MBL levels in both Pre-HD and HD patients were significantly higher compared with healthy subjects (P<0·05 for both). Furthermore, the median oligomer MBL level in HD patients was significantly (P<0·05) higher than that in Pre-HD patients. The ratios of median serum functional MBL levels to oligomer MBL levels were significantly reduced in both Pre-HD and HD patients compared with healthy subjects (P<0·05 for both). CONCLUSIONS: We found significant reductions in the ratios of serum functional MBL levels to oligomer MBL levels in HD and Pre-HD patients compared with healthy subjects.

Peritonitis associated with Pasteurella multocida: molecular evidence of zoonotic etiology.

A patient on continuous cyclic peritoneal dialysis for chronic kidney disease due to type 2 diabetes mellitus developed peritoneal dialysis-associated peritonitis induced by Pasteurella multocida that was isolated from a sample of dialysis effluent. The route of infection was unknown for this case; however, P. multocida was also isolated from a culture of a pharyngeal swab obtained from the patient's cat. There was no evidence that the cat had bitten and ruptured the peritoneal dialysis tubing or bags. Pulsed-field gel electrophoresis (PFGE) showed that the P. multocida isolated from the patient was completely identical to the strain isolated from the domestic cat. As there is a rise in the pet-keeping population, an increase in zoonoses is to be expected. It is necessary to be carefully informed of hygiene rules in keeping pets because a pet may transmit zoonoses, even on casual contact.

Trial of influenza HA vaccination for healthcare workers in consecutive years.

We investigated the trial of the influenza HA vaccination for healthcare workers in 2 consecutive years at Nihon University Itabashi Hospital. The vaccination rate increased significantly (P<0.01) from the first season to the second season. The number of missed working days in the vaccinated group was significantly lower (P<0.01) than that in the unvaccinated group in a mild-pandemic year. Furthermore, the relative risk of infection was 0.53 and the effectiveness rate of the vaccine was 46.8%. Absenteeism and influenza infection rates were also significantly lower in the vaccinated group. This study may support the possibility of influenza vaccination for healthcare workers to prevent the outbreak of influenza in hospitals.

Impact of reporting gram stain results from blood culture bottles on the selection of antimicrobial agents.

We assessed the usefulness of reporting direct blood Gram stain results compared with the results of positive blood cultures in 482 episodes and monitored impact on selection of antimicrobial treatment. We found that the reporting groups "Staphylococcus spp," "Pseudomonas spp and related organisms," and "yeasts" identified in this way matched perfectly with later culture identification. When the report indicated Staphylococcus spp or Pseudomonas spp and related organisms, physicians started or changed antimicrobials suitable for these bacteria more frequently than when "other streptococci" and "family Enterobacteriaceae" were reported (P < .05). Incorrect recognition of Acinetobacter spp as Enterobacteriaceae family is still the most challenging problem in this context. Gram stain results that definitively identify Staphylococcus spp, Pseudomonas spp and related organisms, and yeasts reliably can be rapidly provided by clinical laboratories; this information has a significant impact on early selection of effective antimicrobials. Further investigation is needed to assess the clinical impact of reporting Gram stain results in bacteremia.

Development and performance of an enzyme immunoassay to detect creatine kinase isoenzyme MB activity using anti-mitochondrial creatine kinase monoclonal antibodies.

OBJECTIVE: The MB fraction of creatine kinase (CK-MB) has long been used as a cardiac marker. It is known that the CK-MB immunoinhibition method lacks selectivity and accuracy, because the appearance of macro CK type 2, corresponding to mitochondrial creatine kinase (MtCK) in some patient serum may render CK-MB activity measured by conventional method abnormally high. Thus, to improve the specificity and accuracy of the CK-MB assay, we developed two types of monoclonal anti-MtCK antibodies against sarcomeric MtCK and ubiquitous MtCK, and present herein the performance of a new method using these antibodies. MATERIAL AND METHODS: The performance of our test for detecting CK-MB activity was compared with other methods, and the range of CK-MB activities in normal human serum was investigated. RESULTS: The two types of monoclonal antibodies developed by us were isoenzyme-specific to sMtCK or uMtCK. The correlation coefficients of our method and conventional method to electrophoresis were 0.973 and 0.873, respectively. The mean CK-MB activity in normal human serum by our method and the conventional method was 2.4 and 11.7 U/L, respectively. Thus, our data indicated that about 80% of CK-MB activity, determined using the conventional method, seems to correspond to the MtCK activity. CONCLUSION: Our method is novel in offering higher accuracy of measuring true CK-MB contents in human serum as compared to the conventional method. The possibility of accurately estimating CK-MB activity by our method which can inhibit MtCKs in healthy person and patient serum is likely to bring a break-through in clinical diagnostics.

[Medical reimbursement for the management of laboratory testing and a 24-hour system for microbiological testing].

In recent years, annual revenues of hospitals in Japan for their health care services have been declining because of the frequent downward revisions of the reimbursement rates to curb growth in national medical spending, exerting a marked influence on the area of laboratory testing. Particularly, unprofitable microbiological testing has been neglected. However, microbiological laboratory testing is not only essential for the diagnosis and treatment of infections, but also plays an important role in the prevention of hospital-acquired infections. The Japanese Society of Laboratory Medicine has been involved in various activities to help clinical laboratories in hospitals ensure stable health care revenues from their practice, as well as improve the status of clinical laboratory physicians. In response to recent changes in clinical laboratory settings, we will hold a symposium to develop and improve a 24-hour system for microbiological testing.

Circannual rhythm of laboratory test parameters among chronic haemodialysis patients.

BACKGROUND/AIMS: Seasonal variations in laboratory test results have been pointed out in dialysis patients. Although the mechanism for this phenomenon is not clear, this could result in changes in dialysis and medication prescriptions. We investigated the effect of the circannual rhythm on laboratory test parameters in chronic haemodialysis patients. METHODS: Data of 38 laboratory test parameters were collected every month and analyzed for 150 stable haemodialysis patients, with non-linear sine wave regression and paired t test between data of peak and trough months. RESULTS: Serum urea nitrogen, unsaturated iron binding capacity, lactate dehydrogenase, alkaline phosphatase, amylase, and neutrophil count showed significant circannual rhythms with high amplitudes. Additionally, serum creatinine, uric acid, chloride, calcium, phosphate, magnesium, total cholesterol, total protein, leucocyte count, mean corpuscular haemoglobin level, mean corpuscular haemoglobin concentration, and platelet count showed significant circannual rhythms with little amplitudes. CONCLUSIONS: The circannual rhythm of laboratory test parameters could be attributed to seasonal variations in food intake. Awareness of these variations should be taken into account in the interpretation of laboratory results.

Supplemental utility of nested PCR for the pathological diagnosis of disseminated trichosporonosis.

Disseminated trichosporonosis is known to be a severe opportunistic mycosis and has a high mortality rate. In autopsy cases, it is often difficult to diagnose as trichosporonosis because the causative Trichosporon species are pathologically similar to other fungi, especially the Candida species. Immunohistochemical analysis is essential for the differential diagnosis, but an antibody to Trichosporon is not available commercially. In the present study, we investigated the supplemental utility of nested polymerase chain reaction (PCR) for the pathological diagnosis of trichosporonosis from formalin-fixed and paraffin-embedded tissues. Total DNA was purified from 30 major organs in three autopsy cases, and Trichosporon DNA was specifically amplified by nested PCR using three sets of primers. Of 22 organs in which Grocott's stain was positive for fungal infection, 170- and 259-bp PCR products were detected in 20 (91%) and 12 (55%) organs, respectively. In short-term fixation (about 1 day), these bands were highly detected in ten (100%) and nine (90%) organs, whereas the detection efficiency tended to decrease after long-term fixation and decalcification. No PCR product of 412 bp was detected in any organs. These findings suggest that nested PCR from short-term-fixed tissues is useful for supportive pathological diagnosis of disseminated trichosporonosis.

[The impact of the amendment to the Infectious Diseases Control Law on the management of clinical laboratories].

In December 2006, the Japanese parliament promulgated an amendment of the Law Concerning the Prevention of Infectious Diseases and Medical Care for Patients of Infections (the Infectious Diseases Control Law). The main elements of the amendment provide a pathogen control system that will prevent biological terrorism, global and accidental spread of infectious diseases and allow for comprehensive control of infectious diseases, including tuberculosis. State intervention was substantially curtailed in the original Infectious Diseases Control Law because of the demand at the time for decentralization. However, the rising danger of bio-terrorism has established an urgent need for direct control by the state. Developing the necessary regulations for laboratory safety and pathogen collection to prevent bioterrorism is an onerous task. The rules must take account of real conditions on the ground and to be seen to work. However, whereas the new rules may be efficient in the prevention of terrorism, there is a real suspicion that they will impede and obstruct the day-to-day routine of the clinical laboratories. We report a questionnaire concerning the influence of this law on the clinical laboratory management in 84 university hospital laboratories. The imposition of well-intentioned but implausible regulations that impair or disrupt routine laboratory work encourages employees to selectively ignore regulations that they feel are impractical or irrelevant.

[Interpretation of the results of blood culture and interventional role by clinical laboratory physicians].

Blood culture has long been recognized as the gold standard for the definitive diagnosis of bacterial and fungal infections. However, fewer blood cultures have been tested and their results have not been fully used in Japan. Clinical laboratory physicians should play an interventional role, such as recommending blood culture tests in patients with infectious disease or fever of unknown origin. In our hospital, clinical laboratory physicians act as on-call consultants. The yearly number of consultations is between 500 and 700, and consultations concerning infectious disease have increased up to 40% in the past 5 years. As a result, the number of blood cultures and the percentage of 2-set blood collections have increased in order to increase the positivity rate and determine whether the results obtained were contaminated. However, physicians sometimes misunderstand the results of blood culture, and they assume that the identified organism was causative, or that sepsis did not exist if the culture is negative. Clinical laboratory physicians should act as consultants more frequently, concerning the interpretation of blood culture results, and the choice of antimicrobial agents, because the inappropriate use of antimicrobial agents leads to higher mortality and higher medical costs. Finally, collaboration between clinical laboratory physicians and co-medical staff such as the infection control team, nurses and pharmacists is necessary.

Evaluation of serological diagnosis tests for tuberculosis in hemodialysis patients.

Patients receiving hemodialysis are generally considered to be at increased risk of developing tuberculosis. In the current study, in order to evaluate the usefulness of serological tests in dialysis patients, serum antibodies for tuberculous glycolipids antigen (TBGL) and for lipoarabinomannan (LAM) were measured in hemodialysis patients. The present study included 243 hemodialysis patients. Serum antibodies for TBGL and LAM were measured. Tuberculin skin tests were carried out and chest X-rays evaluated at the same time. There were no patients with active tuberculosis at the time of blood sampling. Thirty-six patients (14.8%) and 25 patients (10.3%) were positive for anti-TBGL antibody and anti-LAM antibody, respectively. One hundred and fifty-five patients (63.8%) were positive for tuberculin skin testing and 123 patients (50.6%) had old pulmonary tuberculosis on their chest X-ray. There was no significant correlation between the results of anti-TBGL antibody and anti-LAM antibody. There were no relationships among the results of tuberculin skin test and the two serological tests. However, positivity of anti-TBGL antibody and anti-LAM antibody was significantly higher in patients with findings of old tuberculosis on the chest X-ray than those without findings. The current results show that these serological tests are positive more frequently in hemodialysis patients without any proof of active tuberculosis than in healthy subjects (2%) and careful interpretation is necessary for relevant results.

[In-vitro susceptibilites to levofloxacin and various antibacterial agents of 18,639 clinical isolates obtained from 77 centers in 2004].

A total of 18,639 clinical isolates in 19 species collected from 77 centers during 2004 in Japan were tested for their susceptibility to fluoroquinolones (FQs) and other selected antibiotics. The common respiratory pathogens, Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae showed a high susceptible rate against FQs. The isolation rate of beta lactamase non-producing ampicillin-resistant H. influenzae was approximately three times as large as those of western countries. Most strains of Enterobacteriaceae were also susceptible to FQs. The resistance rate of Escherichia coli against FQs has however been rapidly increasing so far as we surveyed since 1994. The FQs-resistant rate in methicillin-resistant Staphylococcus aureus (MRSA) showed approximately 90% except for 36%. of sitafloxacin while FQs-resistant rate in methicillin-susceptible S. aureus (MSSA) was around 5%. The FQs-resistant rate of methicillin-resistant coagulase negative Staphylococci (MRCNS) was also higher than that of methicillin-susceptible coagulase negative Staphylococci (MSCNS), however, it was lower than that of MRSA. In Pseudomonas aeruginosa clinical isolates, 32-34% from UTI and 15-19% of from RTI was resistant to FQs. Acinetobacter spp. showed a high susceptibility to FQs. Although FQs-resistant Neisseria gonorrhoeae have not been increased in western countries, it is remarkably high in Japan. In this survey, isolates of approximately 85% was resistant to FQs.

[The clinical usefulness of a newly rapid diagnosis kit, detection of respiratory syncytial virus].

ImmunoCard STAT! RSV (Meridian Bioscience, Inc, USA) is a rapid immunoassay method newly developed for detection of respiratory syncytial virus (RSV) by immunochromatography. We carried out an evaluation of the ImmunoCard STAT! RSV. One hundred fifty-nine nasal wash samples and nasopharyngeal aspirates from patients were used to evaluate three different kits, which are ImmunoCard STAT! RSV, RSV testpack (Abbott JAPAN) and Directigen EZ RSV (Nippon Becton, Dickinson and Company) . One hundred twenty-eight samples showed equivalent results. When nested reversed transcription-PCR (nested RT-PCR) results for 31 samples showing discrepancies among three kits, 10 samples were positive, and 21 samples were negative by nested RT-PCR. Compared to Nested RT-PCR results, ImmunoCard STAT! RSV showed a sensitivity of 90.5% (19/21) and a specificity of 80.0% (8/10), as well as RSV testpack showed a sensitivity of 10.0% (2/21) and a specificity of 100% (10/10), Directigen EZ RSV showed 95.2% (20/21) and 0.0% (0/ 10), respectively. Furthermore, the detection limits were also evaluated by using ACTT No. VR1540 for RSV A-2 strain, and ACTT No. VR1401 for Wash strain. The detection limit of ImmunoCard STAT! RSV was 5.15 x 10(6) TCID50/mL in subgroup A strain and was 7.58 x 10(5) TCID50/mL in subgroup B strain. This result was similar to RSV testpack, and was better than the detection limit of Directigen EZ RSV. It is concluded that ImmunoCard STAT! RSV is useful in detecting RSV in a clinical setting with equivalent performance to conventional other detecting kits.

[The role of regional research laboratories of the local government in administering regional EQA programs].

In 1981, the Japanese Ministry of Health and Welfare revised the enforcement of regulations of the Medical Technologists' Act. The amendments stipulate that all independent laboratories are legally obliged to introduce laboratory quality assurance programs and are responsible for the quality of all test results. To ensure adherence to these regulations, regional research laboratories of local governments such as the Tokyo Metropolitan Research Laboratory of Public Health should conduct regional external quality assessment (EQA) programs. We did a survey, in the form of a questionnaire, of the regional research laboratories of public health across the country. We found that commitment to the regional EQA in almost all of these public laboratories is insufficient. The main problem is that restructuring of local governments has resulted in lower budgets and so they are short of human resources. Nationwide EQA programs are only able to detect gross errors and use invalid methods for evaluating routine performance. We conclude that the regional EQA should be further developed.

[Evaluation of renal damage using urinary ATP analysis].

It is reported that urinary ATP concentration analysis is useful for determining urinary tract infection and renal damage caused by drugs. By means of the firefly luciferin-luciferase method, we determined the reference value of urinary free ATP and evaluated the effects of urine sediments and conditions of storage. The reference value was established as 1.77 x 10(-10) to approximately 7.70 x 10(-9)M using urine samples obtained from 63 outpatients who seemed to have no renal disease. There was no significant difference in ATP concentration between 33 males and 30 females. No significant changes were observed in 11 healthy volunteers during a 1-year period. Within-run reproducibility of ATP was satisfying (8.28% and 11.4% of coefficient value in low and high concentration samples, respectively). ATP concentration was significantly decreased after centrifugation (p < 0.05) and after filtration (p < 0.01). The amounts of the red blood cells (RBC) and white blood cells (WBC) in samples whose ATP concentration was decreased after centrifugation or filtration were significantly higher than those in samples whose concentration did not decrease (p < 0.05). Urine containing many RBCs and/or WBCs might show an artificially higher ATP concentration if no preparations has been performed. There were significant positive correlations between the ATP concentrations before and after refrigeration, but no correlations before and after freezing. It is concluded that the reference value of urinary free ATP concentration was 1.77 x 10(-10) to approximately 7.70 x 10(-9) M and that care is required in the estimation of urinary ATP concentrations in samples containing many sediments, especially with WBC and RBC.

[An attempt for standardization of serum CA19-9 levels, in order to dissolve the gap between three different methods].

It is well known that serious method-related differences exist in results of serum CA19-9, and the necessity of standardization has been pointed out. In this study, differences of serum tumor marker CA19-9 levels obtained by various immunoassay kits (CLEIA, FEIA, LPIA and RIA) were evaluated in sixty-seven clinical samples and five calibrators and the possibility to improve the inter-methodological differences were observed not only for clinical samples but also for calibrators. We supposed an assumed standard material using by a calibrator. We calculated the serum levels of CA19-9 when using the assumed standard material for three different measurement methods. We approximate the CA19-9 values using by this method. It is suggested that the obtained CA19-9 values could be approximated by recalculation with the assumed standard material would be able to correct between-method and between-laboratory discrepancies in particular systematic errors.