Effects of ultrasound on Drosophila—IV. Pulsed exposures of eggs

Drosophila larvae are damaged by exposures to low temporal average intensity pulsed ultrasound with peak intensities of 10–20 W/cm² (2 MHz). Eggs of the same organism are affected by exposures to 3 W/cm² c.w. ultrasound. This experiment shows that eggs become sensitive to high peak intensity (50–100 W/cm²) pulsed ultrasound only shortly before hatching. At this age the larvae have formed and have taken air into the respiratory system. This observation supports the postulate that the sites of action of the ultrasound are the small stabilized gas bodies within the organisms.     

Effects of short-duration exposures to 2MHz ultrasound on growth and mitotic index of Pisum sativum roots

Roots of Pisum sativum were exposed to 2 MHz ultrasound for durations from 0.006 to 60 sec. Growth was noticably reduced by exposure durations of 0.03 sec and longer (at a spatial peak intensity of 20 w/cm²) and mitotic index was noticably depressed by exposure durations of 0.06 sec and longer (at 30 W/cm²). Additional increments of ultrasound exposure were progressively less effective in further reducing growth and mitotic index. Thus, a major part of the damage from a one-minute ultrasound exposure appears to be produced within the first few seconds after the beam is turned on.     

The effects of continuous wave and pulsed ultrasound on rat thymocytes in vitro

Rat thymocytes in suspension were exposed to continuous wave and pulsed ultrasound at frequencies of 0.5 and 1 MHz. In qualitative confirmation of the observations of Chapman et al. (1979), cell lysis and cell survival were found to depend upon ultrasound intensity, frequency and time of exposure. Cell survival after 10 min, 2 W/cm², 0.5 MHz was approx. 40 percent of control. Pulsed exposures at 0.5 MHz with the same temporal average intensity but with a temporal peak intensity of 30 W/cm² showed no statistically significant decrease in survival in comparison with controls. The thymocyte observations provide no support for the belief that diagnostic ultrasound may cause biological effects in the fluids of the body.     

Absence of mutagenic effects of continuous and pulsed ultrasound in cultured (AL) human-hamster hybrid cells

Mutagenic effects of continuous and pulsed ultrasound were looked for using an in vitro assay system, the A_L hybrid, that is up to 100 times more sensitive for mutagens such as x-rays and neutrons than the assays used previously to evaluate ultrasound. Cells in suspension in rotated plastic test tubes were insonated with continuous wave ultrasound at I MHz, I_SPTP, = 0.62–40 W/cm² for 0–40 min. Cells attached in the central region of culture flasks received pulsed exposures at f_c = 2.5 MHz, PRF = 1 kHz, 2 and 8 cycles per pulse, with p_ = 1.2 MPa (I_SPTA = 31–180 mW/cm²) for 0–30 min. Although these exposures were cytotoxic (the plating efficiency was decreased to 65% by the highest doses), induction of mutation, if any occurred, was less than would be expected in this test system from 10–30 cGy of x-ray.     

Arthroscopic Ultrasound Technique for Simultaneous Quantitative Assessment of Articular Cartilage and Subchondral Bone: An In Vitro and In Vivo Feasibility Study

Traditional arthroscopic examination is subjective and poorly reproducible. Recently, we introduced an arthroscopic ultrasound method for quantitative diagnostics of cartilage lesions. Here we describe our investigation of the feasibility of ultrasound arthroscopy for simultaneous measurements of articular cartilage and subchondral bone. Human osteochondral samples (n = 13) were imaged using a clinical 9-MHz ultrasound system. Ultrasound reflection coefficients (R, IRC), the ultrasound roughness index (URI) and the apparent integrated backscattering coefficient (AIB) were determined for both tissues. Mechanical testing, histologic analyses and micro-scale computed tomography imaging were the reference methods. Ultrasound arthroscopies were conducted on two patients. The ultrasound reflection coefficient correlated with the Mankin score and Young's modulus of cartilage (|r| > 0.56, p < 0.05). Ultrasound parameters (R, IRC, AIB) for subchondral bone correlated with the bone surface/volume ratio (|r| > 0.70, p < 0.05) and trabecular thickness (|r| > 0.59, p < 0.05). Furthermore, R and subchondral bone mineral density were significantly correlated (|r| > 0.65, p < 0.05). Arthroscopic ultrasound examination provided diagnostically valuable information on cartilage and subchondral bone in vivo.     

Comparison of MALDI-TOF MS and VITEK 2 system for laboratory diagnosis of Granulicatella and Abiotrophia species causing invasive infections

Granulicatella and Abiotrophia spp. were known as nutritionally variant streptococci (NVS). Such strains have caused major diagnostic difficulties due to fastidious culturing and unspecific colony morphology. The present study is aimed at comparing the performance of laboratory available diagnostic methods for NVS isolates and determining the antimicrobial susceptibility of these isolates. Fourteen clinical invasive isolates, consisting of 10 Granulicatella adiacens, 1 Granulicatella elegans, and 3 Abiotrophia defectiva were in parallel analyzed by 2 matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, i.e., Bruker MS and Vitek MS, as well as Vitek 2 for the species determination. 16S rRNA gene sequencing was applied as a reference method. The Vitek MS gave correct identification for all 14 isolates. The Bruker MS could correctly identify 8/10 G. adiacens, 0/1 G. elegans, and 3/3 A. defectiva isolates at the first analysis occasion, and all 14 isolates became identifiable after repeated tests. The Vitek 2 system could identify 6/10 G. adiacens, 1/1 G. elegans, and 2/3 A. defectiva isolates at the species level. Antimicrobial susceptibilities of 11 antibiotics were determined by Etest. Resistance against ciprofloxacin, ceftriaxone, rifampicin, and tetracycline were observed in 4, 10, 4, and 1 isolates, respectively. In conclusion, MALDI-TOF MS is a useful tool for the rapid diagnosis of NVS. Phenotypic testing by Vitek 2 is only partially effective for the accurate identification of such strains. The emergence of resistant NVS isolates indicates the necessity of monitoring antimicrobial susceptibilities of such uncommon pathogens.     

Comparison of VITEK2, MALDI-TOF MS,and 16S rDNA sequencing for identification of Myroides odoratus and Myroides odoratimimus

The genus Myroides comprises the 2 medically relevant species Myroides odoratus and Myroides odoratimimus that are rare opportunistic pathogens and cause infections in immunocompromised patients. A fast identification of Myroides is of importance because these bacterial strains show multiple resistance against antibiotics and therefore limit treatment options. They are associated, for instance, with urinary tract infections, sepsis, meningitis, pneumonia, and infectious cellulitis. Since more and more Myroides spp. are being described, additional potentially pathogenic bacteria may be identified in the future demanding the need for fast and reliable identification methods at species level. However, to date, only molecular approaches meet these demands. In this study, we, therefore, attempt to define an appropriate method other than DNA fingerprinting that will permit a comparable efficacy and, possibly, a more economical strain identification. For this purpose, we compared 2 widely used automated diagnostic systems (VITEK 2 [bioMérieux, Nürtingen, Germany] and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) [Bruker Daltonics, Bremen, Germany]) and correlated the results to 16S rDNA sequencing data. In total, we analyzed 22 strains collected in the course of routine diagnostics. In this study, we demonstrate that VITEK 2 reliably identifies the genus Myroides but cannot differentiate between M. odoratimimus and M. odoratus. In contrast to this, both MALDI-TOF MS and 16S rDNA sequencing efficiently distinguish between the 2 species.     

Comparison of the Verigene Clostridium difficile, Simplexa C. difficile Universal Direct,BD MAX Cdiff,and Xpert C. difficile assays for the detection of toxigenic C. difficile

We compared the Verigene Clostridium difficile test (Nanosphere, Northbrook, IL, USA), the Simplexa C. difficile Universal Direct (Focus Diagnostics, Cypress, CA, USA), the BD MAX Cdiff (Becton Dickinson, Franklin Lakes, NJ, USA), and the Xpert C. difficile (Cepheid, Sunnyvale, CA, USA) assays for the detection of toxigenic C. difficile. One hundred and ninety deidentified, remnant diarrheal specimens were included in this study. After resolution of discordant results by toxigenic culture, the Xpert C. difficile assay displayed the highest sensitivity (100%), with a specificity of 98.8%. The sensitivity and specificity were 95.2% and 99.4% and 87% and 100% for the Verigene CDF and Simplexa Universal Direct assays, respectively. Finally, the BD MAX assay showed a sensitivity of 87% and a specificity of 98.8%. Despite differences in the overall performance of these assays, these results support the routine use of these platforms for the detection of toxigenic C. difficile in the clinical laboratory.     

Comparison of a conventional and nested PCR for diagnostic confirmation and genotyping of Orientia tsutsugamushi

A nested polymerase chain reaction (PCR) targeting the 56-kDa antigen gene is currently the most commonly used molecular technique for confirmation of scrub typhus and genotyping of Orientia tsutsugamushi. In this study, we have compared the commonly used nested PCR (N-PCR) with a single-step conventional PCR (C-PCR) for amplification and genotyping. Eschar samples collected from 24 patients with scrub typhus confirmed by IgM enzyme-linked immunosorbent assay were used for DNA extraction following which amplifications were carried out using nested and C-PCR methods. The amplicons were sequenced and compared to other sequences in the database using BLAST. Conventional PCR showed a high positivity rate of 95.8% compared to the 75% observed using N-PCR. On sequence analysis, the N-PCR amplified region showed more variation among strains than the C-PCR amplified region. The C-PCR, which is more economical, provided faster and better results compared to N-PCR.     

Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR,and the VITEK2 system for identification of Acinetobacter clinical isolates

Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and bla_OXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter.     

Comparison of polycarbonate and cellulose acetate membrane filters for isolation of Campylobacter concisus from stool samples

One thousand seven hundred ninety-one diarrheic stool samples were cultivated for Campylobacter spp. We found a high prevalence of Campylobacter concisus with use of a polycarbonate filter (n = 114) compared to a cellulose acetate filter (n = 79) (P < .0001). The polycarbonate filter is superior to the commonly used cellulose acetate filter for detection of C. concisus.     

Comparison of 3 Etest methods and time-kill assay for determination of antimicrobial synergy against carbapenemase-producing Klebsiella species

Increasing global antibiotic resistance has resulted in more use of antibiotic combinations. There is a lack of a gold standard for in vitro testing of these combinations for synergy or antagonism. Time-kill assay (TKA) may be used but is labor intensive and not practical for clinical use. Etest® synergy methods are more rapid and easier to perform, but there is no agreement regarding which method is best. We tested 31 clinical genetically unique Klebsiella pneumoniae carbapenemase-producing Klebsiella isolates with the combination of meropenem and polymyxin B by TKA and 3 Etest methods, each in triplicate: Method 1, MIC:MIC; Method 2, direct overlay; and Method 3, cross. Overall, testing with Etest synergy methods showed the following agreement with TKA: Method 1: 25/31 (80.6%), Method 2: 7/31 (22.6%), and Method 3: 8/31 (25.8%). The MIC:MIC method had the highest agreement (80.6%, κ = 0.59, P < 0.001) and should be evaluated more extensively.     

Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile

This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting.     

Comparison of EUCAST and CLSI broth microdilution methods for the susceptibility testing of 10 Systemically active antifungal agents when tested against Candida spp.

The antifungal broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was compared with Clinical and Laboratory Standards Institute (CLSI) BMD method M27-A3 for amphotericin B, flucytosine, anidulafungin, caspofungin, micafungin, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole susceptibility testing of 357 isolates of Candida. The isolates were selected from global surveillance collections to represent both wild-type (WT) and non-WT MIC results for the azoles (12% of fluconazole and voriconazole results were non-WT) and the echinocandins (6% of anidulafungin and micafungin results were non-WT). The study collection included 114 isolates of Candida albicans, 73 of C. glabrata, 76 of C. parapsilosis, 60 of C. tropicalis, and 34 of C. krusei. The overall essential agreement (EA) between EUCAST and CLSI results ranged from 78.9% (posaconazole) to 99.6% (flucytosine). The categorical agreement (CA) between methods and species of Candida was assessed using previously determined CLSI epidemiological cutoff values. The overall CA between methods was 95.0% with 2.5% very major (VM) and major (M) discrepancies. The CA was >93% for all antifungal agents with the exception of caspofungin (84.6%), where 10% of the results were categorized as non-WT by the EUCAST method and WT by the CLSI method. Problem areas with low EA or CA include testing of amphotericin B, anidulafungin, and isavuconazole against C. glabrata, itraconazole, and posaconazole against most species, and caspofungin against C. parapsilosis, C. tropicalis, and C. krusei. We confirm high level EA and CA (>90%) between the 2 methods for testing fluconazole, voriconazole, and micafungin against all 5 species. The results indicate that the EUCAST and CLSI methods produce comparable results for testing the systemically active antifungal agents against the 5 most common species of Candida; however, there are several areas where additional steps toward harmonization are warranted.     

Comparison of the analytic sensitivities of a recombinant immunoblot assay and the radioimmune precipitation assay for the detection of antibodies to Trypanosoma cruzi in patients with Chagas disease

The diagnosis of chronic Chagas disease usually is made by detecting antibodies to Trypanosoma cruzi, the protozoan parasite that causes this illness. A highly sensitive and specific immunoblot assay developed by us showed a higher analytic sensitivity than the radioimmune precipitation assay, which is used widely as a confirmatory test.     

Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia

Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM−, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia.     

Comparison of the Vitek 2 yeast susceptibility system with CLSI microdilution for antifungal susceptibility testing of fluconazole and voriconazole against Candida spp., using new clinical breakpoints and epidemiological cutoff values

A commercially available, fully automated yeast susceptibility test system (Vitek 2; bioMérieux, Marcy d’Etoile, France) was compared in 3 different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference microdilution (BMD) method by testing 2 quality control strains, 10 reproducibility strains, and 425 isolates of Candida spp. against fluconazole and voriconazole. Reference CLSI BMD MIC endpoints and Vitek 2 MIC endpoints were read after 24 hours and 9.1–27.1 hours incubation, respectively. Excellent essential agreement (within 2 dilutions) between the reference and Vitek 2 MICs was observed for fluconazole (97.9%) and voriconazole (96.7%). Categorical agreement (CA) between the 2 methods was assessed using the new species-specific clinical breakpoints (CBPs): susceptible (S) ≤2 μg/mL, susceptible dose-dependent (SDD) 4 μg/mL, and resistant (R) ≥8 μg/mL for fluconazole and Candida albicans, Candida tropicalis, and Candida parapsilosis and ≤32 μg/mL (SDD), ≥64 μg/mL (R) for Candida glabrata; S ≤0.12 μg/mL, SDD 0.25–0.5 μg/mL, R ≥1 μg/mL for voriconazole and C. albicans, C. tropicalis, and C. parapsilosis, and ≤0.5 μg/mL (S), 1 μg/mL (SDD), ≥2 μg/mL (R) for Candida krusei. The epidemiological cutoff value (ECV) of 0.5 μg/mL for voriconazole and C. glabrata was used to differentiate wild-type (WT; MIC ≤ ECV) from non-WT (MIC > ECV) strains of this species. Due to the lack of CBPs for the less common species, the ECVs for fluconazole and voriconazole, respectively, were used for Candida lusitaniae (2 μg/mL and 0.03 μg/mL), Candida dubliniensis (0.5 μg/mL and 0.03 μg/mL), Candida guilliermondii (8 μg/mL and 0.25 μg/mL), and Candida pelliculosa (4 μg/mL and 0.25 μg/mL) to categorize isolates of these species as WT and non-WT. CA between the 2 methods was 96.8% for fluconazole and 96.5% for voriconazole with less than 1% very major errors and 1.3–3.0% major errors. The Vitek 2 yeast susceptibility system remains comparable to the CLSI BMD reference method for testing the susceptibility of Candida spp. when using the new (lower) CBPs and ECVs.