A comparison of the Sensititre? MYCOTB panel and the agar proportion method for the susceptibility testing of Mycobacterium tuberculosis

The agar proportion method (APM) for determining Mycobacterium tuberculosis susceptibilities is a qualitative method that requires 21 days in order to produce the results. The Sensititre method allows for a quantitative assessment. Our objective was to compare the accuracy, time to results, and ease of use of the Sensititre method to the APM. 7H10 plates in the APM and 96-well microtiter dry MYCOTB panels containing 12 antibiotics at full dilution ranges in the Sensititre method were inoculated with M. tuberculosis and read for colony growth. Thirty-seven clinical isolates were tested using both methods and 26 challenge strains of blinded susceptibilities were tested using the Sensititre method only. The Sensititre method displayed 99.3% concordance with the APM. The APM provided reliable results on day 21, whereas the Sensititre method displayed consistent results by day 10. The Sensititre method provides a more rapid, quantitative, and efficient method of testing both first- and second-line drugs when compared to the gold standard. It will give clinicians a sense of the degree of susceptibility, thus, guiding the therapeutic decision-making process. Furthermore, the microwell plate format without the need for instrumentation will allow its use in resource-poor settings.     

Usefulness of Sofia Pneumococcal FIA® test in comparison with BinaxNOW® Pneumococcal test in urine samples for the diagnosis of pneumococcal pneumonia

The Sofia Pneumococcal FIA® test is a recently introduced immunofluorescent assay automatically read aimed to detect Streptococcus pneumoniae antigen in urine. The aim of this study was to evaluate the usefulness of SofiaFIA® urinary antigen test (UAT) in comparison with classical immunochromatographic BinaxNOW® test for the diagnosis of pneumococcal pneumonia (PP). Observational study was conducted in the Hospital Universitari Vall d’Hebron from December 2015 to August 2016. Consecutive adult patients diagnosed of pneumonia and admitted to the emergency department in whom UAT was requested were prospectively enrolled. Paired pneumococcal UAT was performed (BinaxNOW® and SofiaFIA®) in urine samples. To assess the performance of both tests, patients were categorized into proven PP (isolation of S. pneumoniae in sterile fluid) or probable PP (isolation of S. pneumoniae in respiratory secretion). Sensitivity, specificity, and concordance were calculated. A total of 219 patients with pneumonia were enrolled, of whom 14% had a proven or probable PP, 22% a non-pneumococcal etiology, and 64% an unidentified pathogen. Concordance between tests was good (κ?=?0.81). Sensitivity of SofiaFIA® and BinaxNOW® UAT was 78.6 and 50% for proven PP (p?=?0.124), and 74.2 and 58% for proven/probable PP (p?=?0.063). Specificity for both tests was 83.3 and 85.5% for proven and proven/probable PP. In patients without an identified pathogen, SofiaFIA® test was positive in 33 (23.6%) cases and BinaxNOW® in 25 (17.8%), so Sofia Pneumococcal FIA® detected 32.6% more cases than BinaxNOW® (p?=?0.001). Sofia Pneumococcal FIA® test showed an improved sensitivity over visual reading of BinaxNOW® test without a noticeable loss of specificity.     

Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – the EUCAST broth microdilution reference method for MIC determination

ScopeSeveral methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination for MTBC.MethodsThe EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation.RecommendationsThe EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 10⁵ CFU/mL suspension, obtained from a 10⁻² dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36° ± 1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 10³ CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03–0.12 for isoniazid, 0.12–0.5 for levofloxacin and 0.25–1 mg/L for amikacin.ConclusionsThe EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods will need to be calibrated against this reference method to be used with EUCAST breakpoints.     

A controlled comparison of the BacT/ALERT® 3D and VIRTUO™ microbial detection systems

The performance of the next-generation BacT/ALERT® VIRTUO? Microbial Detection System (VIRTUO?, bioMérieux Inc., Hazelwood, MO) was compared to the BacT/ALERT® 3D Microbial Detection System (3D, bioMérieux Inc., Durham, NC) using BacT/ALERT® FA Plus (FA Plus), BacT/ALERT® PF Plus (PF Plus), BacT/ALERT® FN Plus (FN Plus), BacT/ALERT® Standard Aerobic (SA), and BacT/ALERT® Standard Anaerobic (SN) blood culture bottles (bioMérieux Inc., Durham, NC). A seeded limit of detection (LoD) study was performed for each bottle type in both systems. The LoD studies demonstrated that both systems were capable of detecting organisms at nearly identical levels [<10 colony-forming units (CFU) per bottle], with no significant difference. Following LoD determination, a seeded study was performed to compare the time to detection (TTD) between the systems using a panel of clinically relevant microorganisms inoculated at or near the LoD with 0, 4, or 10 mL of healthy human blood. VIRTUO? exhibited a faster TTD by an average of 3.5 h, as well as demonstrated a significantly improved detection rate of 99.9% compared to 98.8% with 3D (p-value <0.05).     

Assessment of the Phoenix™ automated system and EUCAST breakpoints for antimicrobial susceptibility testing against isolates expressing clinically relevant resistance mechanisms

EUCAST breakpoint criteria are being adopted by automatic antimicrobial susceptibility testing systems. The accuracy of the Phoenix Automated System in combination with 2012 EUCAST breakpoints against recent clinical isolates was evaluated. A total of 697 isolates (349 Enterobacteriaceae, 113 Pseudomonas spp., 25 Acinetobacter baumannii, 11 Stenotrophomonas maltophilia, 95 Staphylococcus aureus, 6 coagulase negative staphylococci, 77 enterococci and 21 Streptococcus pneumoniae) with defined resistance phenotypes and well-characterized resistance mechanisms recovered in Spain (n = 343) and Italy (n = 354) were tested. Comparator antimicrobial susceptibility testing data were obtained following CLSI guidelines. Experimental agreement (EA), defined as MIC agreement ±1 log₂ dilution, category agreement (CA) and relative discrepancies (minor (mD), major (MD) and very major discrepancies (VMD)) were determined. The overall EA and CA for all organism-antimicrobial agent combinations (n = 6.294) were 97.3% and 95.2%, respectively. mD, MD and VMD were 4.7%, 1.3% and 2.7%, all of them in agreement with the ISO (ISO20776–2:2007) acceptance criteria for assessment of susceptibility testing devices. VMD were mainly observed in amoxicillin-clavulanate and cefuroxime in Enterobacteriaceae and gentamicin in Pseudomonas aeruginosa, whereas MD were mainly observed in amoxicillin-clavulante in Enterobacteriaceae. mD were mainly observed in Enterobacteriaceae but distributed in different antimicrobials. For S. aureus and enterococci relative discrepancies were low. The Phoenix system showed accuracy assessment in accordance with the ISO standards when using EUCAST breakpoints. Inclusion of EUCAST criteria in automatic antimicrobial susceptibility testing systems will facilitate the implementation of EUCAST breakpoints in clinical microbiology laboratories.     

The use of E-test for the drug susceptibility testing of Mycobacterium tuberculosis – A solution or an illusion?

Aim: To evaluate E-test as a tool for rapid determination of drug susceptibility against the conventional LJ method focusing on reliability, expense, ease of standardization and performance of the technique in low resource settings. Materials and Methods: A total of 74 clinical isolates (2004-2005) of Mycobacterium tuberculosis were tested using E-test for susceptibility to streptomycin (STM), isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) by E-strip and LJ (LJPM) proportion methods. Results: The LJPM method, the gold standard, detected resistance against STM in 16.2%, INH in 40.5%, RIF in 18.9% and EMB in 27% cases. In comparison, the resistance values showed by E-test was 66.67% for STM, 57.14% for INH 71.43% for RIF and 80% for EMB. The susceptible correlation was 90.32% for STM, 73.91% for INH, 93.33% for RIF and 59.26% for EMB. E-test correctly identified only eight of the 12 (66.6%) MDR isolates and wrongly identified four isolates which were not MDR. The overall agreement between the two methods was only 48.6%. Resistant isolates showed false positive resistance observed while using E-strip towards all the drugs. Conclusion: E-strips are not quite feasible as a replacement for LJ-proportion method on a large scale due to high risk of cross contamination, laboratory infection, expense associated with it and high false positive resistance observed to all first line drugs. However, the good correlation observed for RIF between the two methods indicates that E-test could contribute to the role in rapid screening of MDR TB isolates as rifampicin mutations are invariably observed in MDR TB isolates.     

Standardization of a bottle assay—an indigenous method for laboratory and field monitoring of insecticide resistance and comparison with WHO adult susceptibility test

The WHO adult susceptibility test is in use for insecticide resistance monitoring. Presently, materials are being imported from the Universiti Sains Malaysia, Malaysia and sometimes it is cost prohibitive. As an alternative, we present here a method of bottle bioassay using indigenous material. Different aspects related to the assay were studied and validated in the field. Bottle assay was standardized in the laboratory by using locally sourced material and laboratory-maintained insecticide-susceptible Anopheles stephensi and Aedes aegypti strains against technical grade deltamethrin and cyfluthrin insecticides dissolved in ethanol in a range of different concentrations. The frequency of use of the deltamethrin-coated bottles and shelf-life were determined. Discriminating dose for deltamethrin and cyfluthrin was 10 μg against An. stephensi and 2 μg against Ae. aegypti females. Insecticide-coated bottles stored at 25 to 35 °C can be used for three exposures within 7 days of coating. The study carried out in the laboratory was validated on wild caught An. culicifacies in the states of Odisha and Chhattisgarh against deltamethrin-coated bottles in comparison to WHO adult susceptibility test. Results of the study indicated that deltamethrin-coated bottles were effective up to three exposures within 7 days of coating for field population and 100 % mortality was recorded within 35 min as observed in laboratory studies for field collected susceptible population. Also in the WHO adult susceptibility test, 100 % knock-down within 35 min and 100 % mortality after 24 h holding period were observed in susceptible population, while in it was 50 % knock-down in 1 h and 64 % mortality after 24 h holding period for resistant population (50 % mortality in bottle assay in 60 min). The bottle assay can be used as an alternative to the WHO adult susceptibility test both in the laboratory and field for monitoring insecticide resistance in mosquito vectors using locally sourced material.     

Head-to-head comparison of the diagnostic accuracies of BD Veritor™ System RSV and Quidel® Sofia® RSV FIA systems for respiratory syncytial virus (RSV) diagnosis

BackgroundRespiratory syncytial virus (RSV) is one of the most common causes of severe lower respiratory tract disease among infants and young children. BD Veritor™ System RSV (BD) and Quidel^® Sofia^® RSV FIA (QD) are the new generation lateral flow digital immunoassay (DIA) tests with an instrumented read for the qualitative detection of RSV viral antigens.ObjectiveTo compare the diagnostic accuracies of BD and QD for RSV detection using fresh nasopharyngeal aspirates and nasopharyngeal swab specimens collected in universal transport media during 2013–2014 respiratory season.Study designThe two DIA tests were performed simultaneously on randomly selected specimens on a weekly basis during the RSV season until 200 fresh remnant specimens were enrolled. Real-time RT-PCR assay results were used to compare and evaluate the performance of both RSV DIA assays.ResultsAmong 200 specimens tested, RSV real-time RT-PCR assay detected RSV in 104 samples, while QD detected 84 samples and BD detected 74 samples as positive. The overall sensitivity for detection of RSV in comparison to PCR was 71.15% (61.3–79.4) for BD and 80.77% (71.6–87.6) for QD system (P = 0.36). The specificity was 100% (95.2–100) for both systems. The work flow analysis revealed that the overall specimen processing time was significantly lower for BD as compared with the QD assay.ConclusionsIn comparison with the real-time PCR, the QD system showed a higher sensitivity than that of the BD system, but the difference did not reach statistical significance (P = 0.36). Both BD and QD systems were found comparable in terms of specificity.     

Comparison of the TruView infant EVO2 PCD™ and C-MAC video laryngoscopes with direct Macintosh laryngoscopy for routine tracheal intubation in infants with normal airways

OBJECTIVE:

Videolaryngoscopy has mainly been developed to facilitate difficult airway intubation. However, there is a lack of studies demonstrating this method''s efficacy in pediatric patients. The aim of the present study was to compare the TruView infant EVO2 and the C-MAC videolaryngoscope with conventional direct Macintosh laryngoscopy in children with a bodyweight ≤10 kg in terms of intubation conditions and the time to intubation.

METHODS:

In total, 65 children with a bodyweight ≤10 kg (0-22 months) who had undergone elective surgery requiring endotracheal intubation were retrospectively analyzed. Our database was screened for intubations with the TruView infant EVO2, the C-MAC videolaryngoscope, and conventional direct Macintosh laryngoscopy. The intubation conditions, the time to intubation, and the oxygen saturation before and after intubation were monitored, and demographic data were recorded. Only children with a bodyweight ≤10 kg were included in the analysis.

RESULTS:

A total of 23 children were intubated using the C-MAC videolaryngoscope, and 22 children were intubated using the TruView EVO2. Additionally, 20 children were intubated using a standard Macintosh blade. The time required for tracheal intubation was significantly longer using the TruView EVO2 (52 sec vs. 28 sec for C-MAC vs. 26 sec for direct LG). However, no significant difference in oxygen saturation was found after intubation.

CONCLUSION:

All devices allowed excellent visualization of the vocal cords, but the time to intubation was prolonged when the TruView EVO2 was used. The absence of a decline in oxygen saturation may be due to apneic oxygenation via the TruView scope and may provide a margin of safety. In sum, the use of the TruView by a well-trained anesthetist may be an alternative for difficult airway management in pediatric patients.     

Is there a role for fatty acid synthase in the diagnosis of prostatic adenocarcinoma?: A comparison with AMACR

Our aim was to compare the usefulness of fatty acid synthase (FASn) with that of α-methylacyl coenzyme-A racemase (AMACR) in the diagnosis of prostatic adenocarcinoma. The expression of these 2 markers was compared in a tissue microarray containing 62 foci of benign glands and 36 foci of prostatic adenocarcinoma. Similar to AMACR, there was significantly higher FASn expression in adenocarcinoma compared with that in benign glands. The optimal accuracy rate and area under curve (AUC) by receiver operating characteristic analysis for FASn were not significantly different from those for AMACR (accuracy, 80% vs 87%; AUC, 0.942 vs 0.956; P for both, > .05). Moreover, in cases with coexistent malignant and benign glands on the same core, FASn could selectively distinguish a proportion of cases (17/21 [81%]) similar to using AMACR. We conclude that FASn may aid in the diagnosis of prostatic adenocarcinoma, at least to supplement AMACR as another positive marker of carcinoma and potentially increase diagnostic accuracy.     

Cytome micronucleus assays with a metabolically competent human derived liver cell line (Huh6): A promising approach for routine testing of chemicals?

One of the main problems of in vitro genotoxicity tests is the inadequate representation of drug metabolizing enzymes in most indicator cell lines which are currently used. We identified recently a human derived liver cell line (Huh6) which detected induction of DNA damage by representatives of different groups of promutagens without enzyme mix and showed that these cells are more suitable in terms of reproducibility and sensitivity as other currently used liver derived lines. We developed a protocol for micronucleus (MN) cytome assays with these cells and validated the procedure in experiments with representatives of different groups of directly and indirectly acting genotoxic carcinogens (MMS, cisplatin, PhIP, IQ, NDMA, B(a)P, AFB1, etoposide, and H₂O₂). The optimal cytochalasin B concentration in combination with 48 hr treatment was found to be 1.5 μg/mL and leads to a cytokinesis block proliferation index in the range between 1.7 and 2.0. The morphological characteristics of different nuclear anomalies which reflect DNA damage (MN, nuclear bridges, and buds) and their baseline frequencies in untreated cells were characterized, and the rates which are required to cause significant effects were calculated. All compounds caused dose dependent induction of MN when the cells were treated for 24 hr, longer and shorter exposure times were less effective. Experiments with different serum levels (fetal bovine serum [FBS]) showed that 10% FBS in the medium (instead of 4%) causes a substantial increase of the sensitivity of the cells. Our results indicate that the new protocol is a promising approach for routine testing of chemicals. Environ. Mol. Mutagen. 60: 134–144, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.     

Components of family history associated with women's disease perceptions for cancer: A report from the Family Healthware™ Impact Trial

PurposeTo determine the specific components of family history and personal characteristics related to disease perceptions about breast, colon, and ovarian cancers.MethodsBaseline, cross-sectional data on 2,505 healthy women aged 35–65 years enrolled from 41 primary care practices in the cluster-randomized Family Healthware™ Impact Trial, assessed for detailed family history and perceived risk, perceived severity, worry, and perceived control over getting six common diseases including breast, colon, and ovarian cancers.ResultsParticipants provided family history information on 41,841 total relatives. We found evidence of underreporting of paternal family history and lower perceived breast cancer risk with cancer in the paternal versus maternal lineage. We observed cancer-specific perceived risks and worry for individual family history elements and also found novel “spillover” effects where a family history of one cancer was associated with altered disease perceptions of another. Having a mother with early-onset breast or ovarian cancer was strongly associated with perceived risk of breast cancer. Age, parenthood, and affected lineage were associated with disease perceptions and ran counter to empiric risks.ConclusionsUnderstanding patients' formulation of risk for multiple diseases is important for public health initiatives that seek to inform risk appraisal, influence disease perceptions, or match preventive interventions to existing risk perceptions.     

Performance comparison of a flow cytometry immunoassay for intracellular cytokine staining and the QuantiFERON® SARS-CoV-2 test for detection and quantification of SARS-CoV-2-Spike-reactive-IFN-γ-producing T cells after COVID-19 vaccination

European Journal of Clinical Microbiology & Infectious Diseases - We compared the performance of an in-house-developed flow cytometry assay for intracellular cytokine staining (FC-ICS) and a...     

A simple test for the detection of KPC and metallo-β-lactamase carbapenemase-producing Pseudomonas aeruginosa isolates with the use of meropenem disks supplemented with aminophenylboronic acid,dipicolinic acid and cloxacillin

We evaluated the ability of the combination disk test (CDT) and the Modified Hodge Test (MHT) to discriminate between various carbapenemase-producing Pseudomonas aeruginosa isolates (KPC, n = 36; metallo-β-lactamase (MBL), n = 38) and carbapenemase non-producers (n = 75). For the CDT, the optimal inhibitor concentrations and cut-off values were: 600 µg of 3-aminophenylboronic acid (APB) per disk (an increment of ≥4 mm), 1000 µg of dipicolinic acid (DPA) per disk (an increment of ≥5 mm) and 3000 µg of cloxacillin per disk (an increment of ≥3 mm). APB had excellent sensitivity (97%) and specificity (97%) for the detection of KPC enzymes. DPA detected MBL enzymes with a sensitivity and specificity of 97% and 81%, respectively. The MHT resulted in a low sensitivity (78%) and specificity (57%). The CDT could be very useful in daily practice to provide fast and reliable detection of KPC and MBL carbapenemases among P. aeruginosa isolates.