Isothermal microcalorimetry minimal inhibitory concentration testing in extensively drug resistant Gram-negative bacilli: a multicentre study

ObjectivesTo evaluate the performance of an isothermal microcalorimetry (IMC) method for determining the MICs among extensively drug-resistant Gram-negative bacilli.MethodsA collection of 320 clinical isolates (n = 80 of each) of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii from Sweden, Spain, Italy and the Netherlands were tested. The MICs were determined using the IMC device calScreener (Symcel, Stockholm, Sweden) and ISO-broth microdilution as the reference method. Essential agreement, categorical agreement, very major errors (VME), major errors (ME) and minor (mE) errors for each antibiotic were determined.ResultsData from 316 isolates were evaluated. Four errors (two ME, one VME, one mE) among 80 K. pneumoniae, six errors (four ME, one VME, one mE) among 79 E. coli, 15 errors (seven VME, three ME, five mE) among 77 P. aeruginosa and 18 errors (12 VME, two ME, four mE) among 80 A. baumannii were observed. Average essential agreement and categorical agreement of the IMC method were 96.6% (95% confidence interval, 94.2–99) and 97.1% (95% confidence interval, 95.4–98.5) respectively when the MICs were determined at the end of 18 hours. Categorical agreement of the IMC method for prediction of MIC by the end of 8 hours for colistin, meropenem, amikacin, ciprofloxacin and piperacillin/tazobactam were 95%, 91.4%, 94%, 95.2% and 93.7% respectively.ConclusionsThe IMC method could accurately determine the MICs among extensively drug-resistant clinical isolates of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii isolates.     

Sensitivity to cefotiam of 494 hospital bacterial strains. Determination of minimal inhibitory concentration and comparison with other beta-lactams

Susceptibility to cefotiam of 494 bacterial strains was studied by MIC determination using an agar dilution assay. Activity of cefotiam was also compared with that of six other beta-lactams (ampicillin, mezlocillin, ticarcillin, cefalotin, cefotaxime, and moxalactam) using an agar diffusion method. Cefotiam showed a wide antibacterial spectrum including more than 70% of Enterobacteriaceae (except Serratia), beta-lactamase-producing and non-beta-lactamase-producing Haemophilus influenzae, non-enterococcus streptococci, and penicillinase-producing and non-penicillinase-producing staphylococci susceptible to methicillin. Conversely, cefotiam was inactive against Pseudomonas aeruginosa, Acinetobacter anitratum, Streptococcus faecalis, and methicillin-resistant Staphylococcus aureus.     

Influence of technical factors in the determination of minimal inhibitory concentration by microdilution

Minimal inhibitory concentrations (MICs) of a new quinolone, norfloxacin, as determined by agar dilution are greater than those found using a liquid dilution micromethod. We report herein an analysis of the various parameters possibly involved in this discrepancy: Mueller-Hinton and BioMérieux, glass or plastic, small or large inoculum, and comparative volumes in which the inoculum (IN) and antibiotic (AB) are presented (1 ml IN + 1 ml AB or 1.5 microliter IN + 50 microliter AB or 50 microliter IN + 50 microliter AB). Volume of the inoculum suspension had a bearing on the results obtained with all three reference strains tested. Norfloxacin MICs for S. aureus 7625 and P. aeruginosa 76110 increased commensurately with the ratio of inoculum volume to antibiotic volume, and vice versa. In contrast, no significant variation was found for E. coli 7624. To evaluate the frequency of this effect, we tested 40 antibiotics on the reference strains, and several antibiotics on savage strains (16 Enterobacteriaceae, 40 Staphylococcus, 12 Pseudomonas and 17 P. aeruginosa). The significance of inoculum and antibiotic volumes was corroborated for some antibiotics. Results most consistent with the reference method were obtained with 50 microliter inoculum and 50 microliter antibiotic solution.     

Effect of Prompt Inoculator on results of Sceptor system minimal inhibitory concentration determinations for Listeria monocytogenes

The minimal inhibitory concentrations (MICs) of eight antimicrobial agents were determined for each of 36 isolates of Listeria monocytogenes with the Sceptor system. Two different inoculation procedures, the standard Sceptor log-phase broth (LB) culture and the Prompt Inoculator (PI) (Bauer-Kirby type), were used. The PI MIC/LB MIC ratio (PI/LB ratio) was determined for each antimicrobial agent for each of the isolates. Of the 217 on-scale PI/LB ratio results, all were within the expected range of 0.5 to 2.0. The PI was found to be a convenient, rapid, and suitable method of preparing an inoculum of L monocytogenes for use with the Sceptor system and should function equally well when testing L monocytogenes isolates with other commercially available MIC systems.     

Gentamicin and amikacin disk susceptibility tests with Pseudomonas aeruginosa: definition of minimal inhibitory concentration correlates for susceptible and resistant categories

With currently recommended disk susceptibility tests, minimal inhibitory concentration correlates for amikacin were greater than 16 micrograms/ml for resistance and less than or equal to 12 micrograms/ml (not less than or equal to 8 micrograms/ml) for susceptibility. For gentamicin, they were greater than 8 micrograms/ml for resistance and less than or equal to 6 micrograms/ml (not less than or equal to 4 micrograms/ml) for susceptibility. Minor discrepancies between disk tests and minimal inhibitory concentration determinations will occur if only doubling dilutions of drug are used for measuring minimal inhibitory concentration values.     

Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.     

Rapid colormetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.     

Comparative study of minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) values for tetracycline, monocycline, erythromycin and rokitamycin against eleven strains of Chlamydia trachomatis

We evaluated the efficacy of tetracycline, minocycline, erythromycin and rokitamycin (rikamycine, TMS-19Q) in controlling in vitro propagation of Chlamydia trachomatis in HeLa 229 cells. Ten recent clinical isolates of Chlamydia trachomatis and one fast-growing strain were tested with inocula of 100-1,000 inclusion forming units per well of a 96-wheel microculture plate. Chlamydia trachomatis inclusions were detected by an immunoperoxidase-antiperoxidase procedure (PAP), including a genus-specific monoclonal antibody. Minimal inhibitory concentration (MIC) geometric means and ranges were respectively 0.128, 0.015-0.25 mg/l tetracycline, 0.001, less than or equal to 0.001-0.004 mg/l minocycline, 0.187, 0.031-0.5 mg/l erythromycin, and 0.005, less than or equal to 0.001-0.062 mg/l rokitamycin; minimal lethal concentration (MLC) geometric means and ranges were 6.79, 0.125-32 mg/l tetracycline, 0.225, 0.062-2 mg/l minocycline, 3.37, 1-32 mg/l erythromycin, and 0.112, 0.031-1 mg/l rokitamycin. Since rokitamycin appears to be the more bactericidal from the four antibiotics tested, clinical studies in sexually transmitted diseases are indicated.     

Demonstration of an inoculum effect on the estimation of the minimal inhibitory concentration of LY 146032

This work has been performed to study the inoculum effect on the in vitro activity of LY 146032, a new lipopeptide antibiotic. A statistical analysis (X2 and t test) of the data concerning the MIC of different bacterial groups has been carried out to appreciate the phenomenon. The activity of vancomycin has been compared. A major inoculum effect has been detected for LY 146032. It must be taken into account to evaluate the activity of this new antibiotic.     

Cobblestone area measuring (CAM) assay: a new way of assessing the potential of human haemopoietic stem cells

A new in vitro way of scoring the potential (proliferative- differentiative) of human haemopoietic stem cells (HHSC) is presented. We have applied it in the investigation of HHSC behaviour in normal bone marrow specimens under culture conditions. This system describes a modification of some existing culture assays for primitive HHSC function, and proliferation of the cobblestone area forming cell (CAFC) was determined by counting the cobblestone areas (CA) produced every week. The main steps of the assay are: (a) culture of MNC fraction on pre-established fibroblastic confluent layers from the M2. 10B4 mouse cell line; (b) weekly counting of CA over a period of 5 weeks; (c) weekly in situ observation of CA morphology; (d) enumeration of secondary progenitors per CA, in the 5th week. The CAM index, expressing the value of the CA kinetic line slope and the number of secondary progenitors per CA were evaluated as factors indicating HHSC proliferative and differentiative potential, respectively. This culture system has the potential to serve as an in vitro assay of human stem cell transplantation. It could be applied to evaluating the potential of HHSC contained in haemopoietic collections of diverse origin, only in combination with measurements of the starting number of CAFC.This revised version was published online in April 2005 with corrections to the authors email address.     

Paracetamol analysis: evaluation of a new kit for enzymatic assay

Paracetamol in serum was assayed by a new enzymatic method, and the results compared with a high performance liquid chromatographic method. Results by the two procedures agreed well (t = 0.05). The correlation coefficient was 0.999, and the slope and intercept by Deming analysis were 0.975 and 0.003 mmol/l respectively. The enzymatic method represents a simple, accurate, precise and not too costly method with several advantages over many currently used techniques. It is eminently suitable for the smaller laboratory, and for use out of normal hours.     

Cobblestone area measuring (CAM) assay: a new way of assessing the potential of human haemopoietic stem cells.

A new in vitro way of scoring the potential (proliferative- differentiative) of human haemopoietic stem cells (HHSC) is presented. We have applied it in the investigation of HHSC behaviour in normal bone marrow specimens under culture conditions. This system describes a modification of some existing culture assays for primitive HHSC function, and proliferation of the cobblestone area forming cell (CAFC) was determined by counting the cobblestone areas (CA) produced every week. The main steps of the assay are: (a) culture of MNC fraction on pre-established fibroblastic confluent layers from the M2.10B4 mouse cell line; (b) weekly counting of CA over a period of 5 weeks; (c) weekly in situ observation of CA morphology; (d) enumeration of secondary progenitors per CA, in the 5th week. The CAM index, expressing the value of the CA kinetic line slope and the number of secondary progenitors per CA were evaluated as factors indicating HHSC proliferative and differentiative potential, respectively. This culture system has the potential to serve as an in vitro assay of human stem cell transplantation. It could be applied to evaluating the potential of HHSC contained in haemopoietic collections of diverse origin, only in combination with measurements of the starting number of CAFC.     

Endometrioid adenocarcinoma of the uterus with a minimal deviation invasive pattern

AIMS: Minimal deviation adenocarcinoma of endometrioid type is a rare pathological entity. We describe a variant of typical endometrioid adenocarcinoma associated with minimal deviation adenocarcinoma of endometrioid type. METHODS AND RESULTS: One 'pilot' case of minimal deviation adenocarcinoma of endometrioid type associated with typical endometrioid adenocarcinoma was encountered at our institution in 2001. A second case of same type was received in consultation. We reviewed 168 consecutive hysterectomy specimens diagnosed with 'endometrioid adenocarcinoma' specifically to identify areas of minimal deviation adenocarcinoma of endometrioid type. Immunohistochemistry was done with the following antibodies: MIB1, p53, oestrogen receptor (ER), progesterone receptor (PR), cytokeratin 7 (CK7), cytokeratin 20 (CK20), carcinoembryonic antigen (CEA), and vimentin (VIM). Four additional cases of minimal deviation adenocarcinoma of endometrioid type were identified. All six cases of minimal deviation adenocarcinoma of endometrioid type were associated with superficial endometrioid adenocarcinoma. In two cases with a large amount of minimal deviation adenocarcinoma of endometrioid type, the cervix was involved. The immunoprofile of two representative cases was ER+, PR+, CK7+, CK20-, CEA-, VIM+. MIB1 immunostaining of four cases revealed little proliferative activity of the minimal deviation adenocarcinoma of endometrioid type glandular cells (0-1%) compared with the associated 'typical' endometrioid adenocarcinoma (20-30%). The same four cases showed no p53 immunostaining in minimal deviation adenocarcinoma of endometrioid type compared with a range of positive staining in the associated endometrioid adenocarcinoma. CONCLUSIONS: Minimal deviation adenocarcinoma of endometrioid type more often develops as a result of differentiation from typical endometrioid adenocarcinoma than de novo. Due to its deceptively benign microscopic appearance, minimal deviation adenocarcinoma of endometrioid type may be overlooked and may lead to incorrect assessment of tumour depth and pathological stage. There was a tendency for tumour with a large amount of minimal deviation adenocarcinoma of endometrioid type to invade the cervix.     

Nutritional requirements of shigellae for growth in a minimal medium.

Most (about 81%) of the clinical isolates of shigellae that were tested failed to grow in a minimal medium. Of the auxotrophic isolates belonging to the four Shigella species, 98% grew in a minimal medium containing methionine, nicotinic acid, and tryptophan. The combination of methionine and tryptophan appears to be an obligatory requirement for Shigella dysenteriae serotype 1 strains, while the combination of nicotinic acid and tryptophan appears to be obligatory for serotype 2. Requirements which varied in other isolates were, however, genetically stable, indicating that the auxotypes may be useful as epidemiological markers. Cultures of shigellae in liquid minimal medium containing the above three supplements showed rapid growth and gave reasonably high cell yields.     

A new radiological method for assessing long bone growth in the small laboratory animal

The growth of the immature rat tibia has been measured following five different growth stimulating procedures. These comprised proximal periosteal release, proximal periosteal release with insertion of dissimilar metal wire, repeated proximal periosteal release, distal periosteal release and full periosteal stripping of the diaphysis. The new radiographic method of measurement employed, utilizes a photographic technique. A reproducible measurement of the rate of long bone growth in the small experimental animal is possible.     

Development of a serum-based Taqman real-time PCR assay for diagnosis of invasive aspergillosis

We compared an Aspergillus fumigatus-specific Taqman real-time PCR assay for the diagnosis of invasive aspergillosis (IA) with the enzyme-linked immunosorbent assay Platelia Aspergillus method. Patients with proven or probable IA had positive results with at least one of the two methods. The PCR method seems to be more specific, and a combination of the two should improve the diagnosis of IA.     

Covert laparoscopic cholecystectomy:a new minimally invasive technique

To further improve our developed transumbilical endoscopic surgery (TUES), we developed a completely covert laparoscopic cholecystectomy (LC). Twelve cases of LC were recruited for this new approach. First, a 10-mm trocar was placed above the umbilicus for inserting the laparoscope. Two 5-mm trocars were then placed near the right and left ends of the superior margin of the suprapubic hair. After the 5-mm 30° laparoscope was shifted to the left suprapubic trocar, the harmonic scalper, electric hook, and grasper were inserted either through the 10-mm umbilical trocar or through the right suprapubic trocar. All gallbladders were successfully removed without intraoperative complications. The mean operating time was 28.5 ± 5.7 min (range 20-45 min). All patients felt well after surgery and did not need postoperative analgesia. They resumed free oral intake 6h after the procedure. All patients were satisfied with the appearance of the incisions, which were completely hidden in the umbilicus and suprapubic hair. The approach we developed has overcome both external instrument interference around the umbilicus and the loss of triangulation in the operative field. It is relatively simpler than a typical TUES and offers better cosmetic results.