Discrepant Results Research Articles

Comparison of next generation technologies and bioinformatics pipelines for capsular typing of Streptococcus pneumoniae.

Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. In silico serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (n = 17; 14.3%), 14 (n = 10; 8.4%), and 15B/C (n = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.

The Hydrostatic Mass of A478: Discrepant Results from Chandra, NuSTAR, and XMM-Newton

Galaxy clusters are the most recently formed and most massive, gravitationally bound structures in the Universe. The number of galaxy clusters formed is highly dependent on cosmological parameters, such as the dark matter density, σ 8, and Ω m . The number density is a function of the cluster mass, which can be estimated from the density and temperature profiles of the intracluster medium under the assumption of hydrostatic equilibrium. The temperature of the plasma, hence its mass, is calculated from the X-ray spectra. However, effective-area calibration uncertainties in the soft band result in significantly different temperature measurements from various space-based X-ray telescopes. NuSTAR is potentially less susceptible to these issues than Chandra and XMM-Newton, having larger effective area, particularly at higher energies, enabling high-precision temperature measurements. In this work, we present analyses of Chandra, NuSTAR, and XMM-Newton data of A478 to investigate the nature of this calibration discrepancy. We find that NuSTAR temperatures are on average ∼11% lower than those of Chandra, and XMM-Newton temperatures are on average ∼5% lower than those of NuSTAR. This results in a NuSTAR mass at r 2500,Chandra of M ⊙, which is ∼10% lower than that of M 2500,Chandra and ∼4% higher than M 2500,XMM−Newton.

Validation of a real-time PCR panel for detection and quantification of nine pathogens commonly associated with canine infectious respiratory disease

Canine infectious respiratory disease (CIRD) is a complicated respiratory syndrome in dogs [1–3]. A panel PCR was developed [4] to detect nine pathogens commonly associated with CIRD: Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica; canine adenovirus type 2, canine herpesvirus 1, canine parainfluenza virus, canine distemper virus, canine influenza virus and canine respiratory coronavirus [5–16]. To evaluate diagnostic performance of the assay, 740 nasal swab and lung tissue samples were collected and tested with the new assay, and compared to an older version of the assay detecting the same pathogens except that it does not differentiate the two Mycoplasma species. Results indicated that the new assay had the same level of specificity, but with higher diagnostic sensitivity and had identified additional samples with potential co-infections. To confirm the new assay is detecting the correct pathogens, samples with discrepant results between the two assays were sequence-confirmed. Spiking a high concertation target to samples carrying lower concentrations of other targets was carried out and the results demonstrated that there was no apparent interference among targets in the same PCR reaction. Another spike-in experiment was used to determine detection sensitivity between nasal swab and lung tissue samples, and similar results were obtained.•A nine-pathogen CIRD PCR panel assay had identified 139 positives from 740 clinical samples with 60 co-infections;•High-concentration target does not have apparent effect on detecting low-concentration targets;•Detection sensitivity were similar between nasal swab and lung tissue samples.

Sleep in psoriasis: A meta-analysis

ObjectiveCurrently, there are discrepant results regarding the quantitative effect of psoriasis on sleep, which may, in part, be attributed to the use of non-standardised questionnaires. MethodsThe PubMed/Medline, Embase, and Cochrane databases were searched for cross-sectional, case-control or cohort studies that recruited patients with psoriasis and healthy controls and reported data regarding Pittsburgh Sleep Quality Index (PSQI) and the prevalence of sleep disturbance (SD) based on the PSQI, published from inception up to January 2023. Secondary outcomes included scores for the Insomnia Severity Index (ISI), Beck Depression Inventory (BDI), and Epworth Sleepiness Scale (ESS), and risk for restless legs syndrome (RLS). Meta-analyses using random-effects models were used for statistical analyses. ResultsFifteen studies including 1274 patients with psoriasis and 775 controls were analysed. A higher PSQI (weighted mean difference [WMD] = 3.397, P < 0.001, I2 = 84.2%) and a higher risk for SD (odds ratio [OR] = 6.640, P < 0.001, I2 = 67.5%) were observed in patients with psoriasis compared with controls. Subgroup analyses revealed a greater difference in PSQI score and/or risk for SD between patients with psoriasis and controls in subgroups of psoriatic arthritis, moderate-to-severe psoriasis, shorter psoriasis duration, and younger age. Moreover, patients with psoriasis exhibited higher ISI (WMD = 2.709, P < 0.001) and BDI scores (WMD = 4.565, P = 0.001), and risk for RLS (OR = 4.689, P = 0.01). However, there was no significant difference in ESS scores (WMD = -0.229, P = 0.77) compared with controls. ConclusionPsoriasis was associated with poor sleep quality and higher risk for SD, especially among patients with psoriatic arthritis, severe psoriasis, shorter duration of psoriasis, and younger age. Patients with psoriasis were also more likely to experience insomnia, RLS, and depression.

The Role of the Stimulus in Olfactory Plasticity.

Plasticity, the term we use to describe the ability of a nervous system to change with experience, is the evolutionary adaptation that freed animal behavior from the confines of genetic determinism. This capacity, which increases with brain complexity, is nowhere more evident than in vertebrates, especially mammals. Though the scientific study of brain plasticity dates back at least to the mid-19th century, the last several decades have seen unprecedented advances in the field afforded by new technologies. Olfaction is one system that has garnered particular attention in this realm because it is the only sensory modality with a lifelong supply of new neurons, from two niches no less! Here, we review some of the classical and contemporary literature dealing with the role of the stimulus or lack thereof in olfactory plasticity. We have restricted our comments to studies in mammals that have used dual tools of the field: stimulus deprivation and stimulus enrichment. The former manipulation has been implemented most frequently by unilateral naris occlusion and, thus, we have limited our comments to research using this technique. The work reviewed on deprivation provides substantial evidence of activity-dependent processes in both developing and adult mammals at multiple levels of the system from olfactory sensory neurons through to olfactory cortical areas. However, more recent evidence on the effects of deprivation also establishes several compensatory processes with mechanisms at every level of the system, whose function seems to be the restoration of information flow in the face of an impoverished signal. The results of sensory enrichment are more tentative, not least because of the actual manipulation: What odor or odors? At what concentrations? On what schedule? All of these have frequently not been sufficiently rationalized or characterized. Perhaps it is not surprising, then, that discrepant results are common in sensory enrichment studies. Despite this problem, evidence has accumulated that even passively encountered odors can "teach" olfactory cortical areas to better detect, discriminate, and more efficiently encode them for future encounters. We discuss these and other less-established roles for the stimulus in olfactory plasticity, culminating in our recommended "aspirations" for the field going forward.

An audit of elevated serum testosterone in females with discrepant results

An audit of elevated serum testosterone in females with discrepant results

Spurious elevation of serum alpha-fetoprotein level due to assay interference-challenges in managing discrepant results

Spurious elevation of serum alpha-fetoprotein level due to assay interference-challenges in managing discrepant results

Assessment of retinal and choroidal vessel density and nerve fibre layer thickness changes after orbitotomy in patients with severe non-active thyroid orbitopathy: a prospective study.

To evaluate the optical coherence tomography angiogram changes in non-active severe thyroid-related ophthalmopathy patients after cosmetic bone decompression. Eighteen patients (25 eyes) with severe not active not compressive (NANC) TED who were candidates for decompression surgery for cosmetic reasons were included in this study, and a 3 × 3mm macular scan was used to measure vessel density and RNFL thickness. Whole macular vessel density in its superficial, deep and choriocapillaris layers was evaluated. The following data were extracted for each of layers: superior and inferior hemispheres, fovea, parafoveal vessel density, its superior and inferior hemispheres, and temporal, superior, nasal and inferior quadrant. The mean RPC increased postoperatively, which was statistically significant in small vessels of peripapillary area (p-value = 0.045). The mean RNFL thickness decreased after surgery and it was statistically significant in the peripapillary (p-value = 0.032) and Inferior-Hemifield area (p-value = 0.036). The choriocapillaris changes were significant in Superior-Hemifield (p-value = 0.031) and Fovea (p-value = 0.03). Thyroid-associated orbitopathy patients have a tendency to decrease vascular density and correlated with disease activity more than stage of orbitopathy. There was not a strong and even discrepant result in linkage of RNFL thickness and other optic nerve function tests and TED patient status and it is needed to do studies with more epidemiologic power and same methodology of study to be more comparable.

Comprehensive assessment of mismatch repair and microsatellite instability status in molecular classification of endometrial carcinoma

Objective: To explore the concordance and causes of different mismatch repair (MMR) and microsatellite instability (MSI) detection results in endometrial carcinoma (EC) molecular typing. Methods: A total of 214 EC patients diagnosed from January 2021 to April 2023 were selected at the Department of Pathology, Peking University Third Hospital. The immunohistochemistry (IHC) results of MMR protein were reviewed. Tumor specific somatic mutations, MMR germline mutations, microsatellite scores and tumor mutation burden (TMB) were detected by next-generation sequencing (NGS) with multi-gene panel. Methylation-specific PCR was used to detect the methylation status of MLH1 gene promoter in cases with deficient MLH1 protein expression. In cases with discrepant results between MMR-IHC and MSI-NGS, the MSI status was detected again by PCR (MSI-PCR), and the molecular typing was determined by combining the results of TMB and MLH1 gene promoter methylation. Results: (1) In this study, there were 22 cases of POLE gene mutation subtype, 55 cases of mismatch repair deficient (MMR-d) subtype, 29 cases of p53 abnormal subtype, and 108 cases of no specific molecular profile (NSMP). The median age at diagnosis of MMR-d subtype (54 years old) and the proportion of aggressive histological types (40.0%, 22/55) were higher than those of NSMP subtype [50 years old and 12.0% (13/108) respectively; all P<0.05]. (2) Among 214 patients, MMR-IHC test showed that 153 patients were mismatch repair proficient (MMR-p), 49 patients were MMR-d, and 12 patients were difficult to evaluate directly. MSI-NGS showed that 164 patients were microsatellite stable (MSS; equal to MMR-p), 48 patients were high microsatellite instability (MSI-H; equal to MMR-d), and 2 patients had no MSI-NGS results because the effective sequencing depth did not meet the quality control. The overall concordance between MMR-IHC and MSI-NGS was 94.3% (200/212). All the 12 discrepant cases were MMR-d or subclonal loss of MMR protein by IHC, but MSS by NGS. Among them, 10 cases were loss or subclonal loss of MLH1 and (or) PMS2 protein. Three discrepant cases were classified as POLE gene mutation subtype. In the remaining 9 cases, 5 cases and 3 cases were confirmed as MSI-H and low microsatellite instability (MSI-L) respectively by MSI-PCR, 6 cases were detected as MLH1 gene promoter methylation and 7 cases demonstrated high TMB (>10 mutations/Mb). These 9 cases were classified as MMR-d EC. (3) Lynch syndrome was diagnosed in 27.3% (15/55) of all 55 MMR-d EC cases, and the TMB of EC with MSH2 and (or) MSH6 protein loss or associated with Lynch syndrome [(71.0±26.2) and (71.5±20.1) mutations/Mb respectively] were significantly higher than those of EC with MLH1 and (or) PMS2 loss or sporadic MMR-d EC [(38.2±19.1) and (41.9±24.3) mutations/Mb respectively, all P<0.01]. The top 10 most frequently mutated genes in MMR-d EC were PTEN (85.5%, 47/55), ARID1A (80.0%, 44/55), PIK3CA (69.1%, 38/55), KMT2B (60.0%, 33/55), CTCF (45.5%, 25/55), RNF43 (40.0%, 22/55), KRAS (36.4%, 20/55), CREBBP (34.5%, 19/55), LRP1B (32.7%, 18/55) and BRCA2 (32.7%, 18/55). Concurrent PTEN, ARID1A and PIK3CA gene mutations were found in 50.9% (28/55) of MMR-d EC patients. Conclusions: The concordance of MMR-IHC and MSI-NGS in EC is relatively high.The discordance in a few MMR-d EC are mostly found in cases with MLH1 and (or) PMS2 protein loss or MMR protein subclonal staining caused by MLH1 gene promoter hypermethylation. In order to provide accurate molecular typing for EC patients, MLH1 gene methylation, MSI-PCR, MMR gene germline mutation and TMB should be combined to comprehensively evaluate MMR and MSI status.

Análisis visual y semicuantitativo del 6-[18F]FDOPA PET/TC en pacientes con tumores cerebrales ante la sospecha de recurrencia tumoral versus radionecrosis

IntroductionAmino acid PET is a tool recommended by the main neuroimaging societies in the differential diagnosis between radionecrosis (RNC) and tumour recurrence (TR) in brain tumours, but its use in our country is still limited. The aim of this work is to present our experience with 6-[18F]FDOPA PET/CT (FDOPA) in brain tumours (primary and M1), comparing these results with other published results. Material and methodsRetrospective study of 62 patients with suspected tumour recurrence (TR): 42 brain metastases (M1) and 20 primary, who underwent FDOPA. Images were analysed visually and semi-quantitatively, obtaining SUVmax and SUVmaxlesion/SUVmaxstriatum (L/S) and SUVmaxlesion/SUVmaxcortex (L/C) ratios. The diagnostic validity of PET was analysed and the best performing cut-off points were calculated. PET results were compared with clinical-radiological follow-up and/or histopathology. ResultsTR was identified in 49% of M1 and 76% of brain primaries. The best performing FDOPA interpretation was visual and semi-quantitative, with a sensitivity and specificity in primaries of 94% and 80% and in M1s of 96% and 72% respectively. The cut-off points with the best diagnostic performance were L/C1.44 in M1 and L/C1.55 in primaries. There are discrepant results with other published results. ConclusionFDOPA PET/CT is a useful tool in the differential diagnosis between recurrence and RNC in brain tumours. It is needed a standardization to contribute to homogenise FDOPA results a inter-centre level.

Visual and semi-quantitative analysis of 6-[18F]FDOPA PET/CT in patients with brain tumors and suspected tumor recurrence versus radionecrosis

IntroductionAmino acid PET is a tool recommended by the main neuroimaging societies in the differential diagnosis between radionecrosis (RNC) and umour recurrence (TR) in brain tumours, but its use in our country is still limited. The aim of this work is to present our experience with 6-[18F]FDOPA PET/CT (FDOPA) in brain tumours (primary and M1), comparing these results with other published results. Material and methodsRetrospective study of 62 patients with suspected tumour recurrence (TR): 42 brain metastases (M1) and 20 primary, who underwent FDOPA. Images were analysed visually and semi-quantitatively, obtaining SUVmax and SUVmaxlesion/SUVmaxstriatum (L/S) and SUVmaxlesion/SUVmaxcortex (L/C) ratios. The diagnostic validity of PET was analysed and the best performing cut-off points were calculated. PET results were compared with clinical-radiological follow-up and/or histopathology. ResultsTR was identified in 49% of M1 and 76% of brain primaries. The best performing FDOPA interpretation was visual and semi-quantitative, with a sensitivity and specificity in primaries of 94% and 80% and in M1s of 96% and 72% respectively. The cut-off points with the best diagnostic performance were L/C1.44 in M1 and L/C1.55 in primaries. There are discrepant results with other published results. ConclusionFDOPA PET/CT is a useful tool in the differential diagnosis between recurrence and RNC in brain tumours. It is needed a standardization to contribute to homogenise FDOPA results a inter-centre level.

FRI148 Adrenal Vein Sampling: Intra-protocol Hormonal Variability

Abstract Disclosure: C. Lee: None. Z. Salman: None. J. Shields: None. A.F. Turcu: None. Background: Primary aldosteronism (PA) might arise from one or both adrenal glands. Treatment of PA relies on determining the relative contribution of aldosterone from the two adrenal glands. Adrenal vein sampling (AVS) is essential to identifying patients with lateralized PA (LPA), who benefit from surgery. AVS procedure and interpretation protocols differ between centers. Variability in AVS lateralization has been primarily attributed to cosyntropin use and lateralization index (LI) thresholds. However, data on intra-protocol variability are lacking. Methods: We retrospectively reviewed the clinical records of patients with PA who underwent AVS at University of Michigan between January 2015 and December 2022. In our center, AVS is performed both before and after cosyntropin stimulation, and 6 sets of paired adrenal vein and peripheral serum are obtained: 3 at baseline, and 3 between 5-30 min after cosyntropin stimulation. We included patients who had successful adrenal vein catheterization and valid data from 6 time points. A LI≥4 was considered indicative of LPA. We assessed the rates of inconsistent AVS results within baseline and cosyntropin stimulated samples as proportion of sets in which 1/3 LIs provided different subtyping results than the other 2/3. Results: Of 361 patients included, 107 (29.6%) patients had at least one inconsistent LI. Of these, 74 (20%) patients had an inconsistent LI within the basal sets, 45 (12%) within the cosyntropin stimulated sets, and 12 (3.3%) patients had a discrepant result in both baseline and cosyntropin stimulated sets. The highest outlier rates were observed in the first basal sample (30.3%), followed by the third basal sample (21.9%), the first post-cosyntropin sample (16.0%), and the last post-cosyntropin sample (13.5%). Patients with one intra-protocol outlier were younger (mean age 52.2 ± 11.5 vs. 56.2 ± 10.8 years), but there were no sex, race distribution, or BMI differences between the two groups. Conclusions: The intra-procedural AVS data variability is almost twice as high in basal vs. post-cosyntropin stimulation sampling, particularly in the initial set, likely mediated by acute ACTH fluctuations during the procedure. Our results emphasize the importance of obtaining multiple AVS data sets, in order to best inform the decision for or against surgery. Presentation: Friday, June 16, 2023

Discrepant results of the total pacing prematurity in orthodromic reciprocating tachycardia with right bundle branch block.

The total pacing prematurity (TPP) is useful for distinguishing orthodromic reciprocating tachycardia (ORT) from atrioventricular nodal re-entrant tachycardia, but it may not be effective in patients with right bundle branch block (RBBB). We faced this challenge in an elderly woman, as RBBB and a prolonged transseptal conduction made it difficult to diagnose the tachycardia using the TPP. It is important to consider the presence or absence of RBBB when evaluating the results of the TPP.

Map-based cosmology inference with weak lensing – information content and its dependence on the parameter space

ABSTRACT Field-level inference is emerging as a promising technique for optimally extracting information from cosmological data sets. Previous analyses have shown field-based inference produces tighter parameter constraints than power spectrum analyses. However, estimates of the detailed quantitative gain in constraining power differ. Here, we demonstrate the gain in constraining power depends on the parameter space being constrained. As a specific example, we find that lognormal field-based analysis of an LSST Y1-like mock data set only marginally improves constraints relative to a 2-point function analysis in Lambda cold dark matter (ΛCDM), yet it more than doubles the constraining power of the data in the context of wCDM models. This effect reconciles some, but not all, of the discrepant results found in the literature. Our results suggest the importance of using a full systematics model when quantifying the information gain for realistic field-level analyses of future data sets.

Longitudinal study of patients with discrepant results in CLIFT and a solid-phase dsDNA antibody assay: does a gold standard dsDNA assay exist?

ObjectiveAntidouble-stranded DNA (dsDNA) antibodies are essential for diagnosis and follow-up of systemic lupus erythematous (SLE). To ensure the best diagnostic approach, most healthcare laboratories opt for a combination of highly...

A multicenter evaluation of Copan's Colibrí™, an automated instrument for MALDI TOF MS target application for bacterial identification

The Colibrí™ is a new instrument that automates picking and placement of colonies on target plates for MALDI identification. This study compared the performance of the Colibrí™ to standard manual spotting using the VITEK® MS for bacterial identification. Colonies were selected from cultures of urine, wound, respiratory, and positive blood cultures. The Colibrí™ sampled the colonies, transferred them to a MALDI target slide, and overlayed each spot with matrix. Manual spotting was then performed using the same or similar colonies. A total of 444 bacteria were compared. Identification was achieved with both methods for 432 organisms with only 2 discrepant results, overall agreement of 99.54%. Twelve organisms (2.70%) gave no identification using Colibrí™. The Colibrí™ provides automation to a manual process with a high accuracy. Use of the Colibrí™ instrumentation provides an opportunity to reallocate technologist time to more complicated tasks and provides complete traceability from plating to organism identification.

Reeling in the Whirlpool galaxy: Distance to M 51 clarified through Cepheids and the type IIP supernova 2005cs

Context. The distance to the Whirlpool galaxy, M 51, is still debated, even though the galaxy has been studied in great detail. Current estimates range from 6.02 to 9.09 Mpc, and different methods yield discrepant results. No Cepheid distance has been published for M 51 to date. Aims. We aim to estimate a more reliable distance to M 51 through two independent methods: Cepheid variables and their period-luminosity relation, and an augmented version of the expanding photosphere method (EPM) on the type IIP supernova SN 2005cs, which exploded in this galaxy. Methods. For the Cepheid variables, we analysed a recently published Hubble Space Telescope catalogue of stars in M 51. By applying filtering based on the light curve and colour-magnitude diagram, we selected a high-quality sample of M 51 Cepheids to estimate the distance through the period-luminosity relation. For SN 2005cs, an emulator-based spectral fitting technique was applied, which allows for the fast and reliable estimation of the physical parameters of the supernova atmosphere. We augmented the established framework of EPM with these spectral models to obtain a precise distance to M 51. Results. The two resulting distance estimates are DCep = 7.59 ± 0.30 Mpc and D2005cs = 7.34 ± 0.39 Mpc using the Cepheid period-luminosity relation and the spectral modelling of SN 2005cs, respectively. This is the first published Cepheid distance for this galaxy. The obtained values are precise to 4–5% and are fully consistent within 1σ uncertainties. Because these two estimates are completely independent, they can be combined for an even more precise estimate, which yields DM 51 = 7.50 ± 0.24 Mpc (3.2% uncertainty). Conclusions. Our distance estimates agree with most of the results obtained previously for M 51, but they are more precise than the earlier counterparts. However, they are significantly lower than the TRGB estimates, which are often adopted for the distance to this galaxy. The results highlight the importance of direct cross-checks between independent distance estimates so that systematic uncertainties can be quantified. Because of the large discrepancy, this finding can also affect distance-sensitive studies and their discussion for objects within M 51, as well as the estimation of the Hubble constant through the type IIP standardizable candle method, for which SN 2005cs is a calibrator object.

А.ҚҰНАНБАЙҰЛЫ ЖӘНЕ М.КӨПЕЙҰЛЫНЫҢ КӨЗҚАРАСТАРЫНДАҒЫ ДІНИ ИДЕЯЛАР

Мақалада қазақ мәдениетінің өкілдері А.Құнанбайұлы, Ж.Көпейұлының шығармаларындағы негізге алынды. Еңбектеріндегі ортақ мәселелер қарастырылады. Қазақ даласындағы діни ағартушылық арнап, оның ішінде А.Құнанбайұлы, Ж.Көпейұлының көзқарастарындағы діни идеяларды салыстырмалы талдау жасалды. Ойшылдардың негізгі көтерген мәселелері ағартушылық, діндегі қате түсініктерге, адамзатқа ортақ гуманизм секілді мәселелерді көтерді. Қазіргі қоғамда Жүсіп Көпейұлының шығармаларына қате түсінік беріліп отырғанын көріп отырмыз. Бұл ғылыми ізденісте сондай олқылықтардың орнын толтырып, осы тақырыпқа аясында өз тұжырымдарымызды беріп отырмыз

Genotypic and phenotypic comparison of drug resistance profiles of clinical multidrug-resistant Mycobacterium tuberculosis isolates using whole genome sequencing in Latvia

BackgroundMultidrug-resistant tuberculosis (MDR–TB) remains a major public health problem in many high tuberculosis (TB) burden countries. Phenotypic drug susceptibility testing (DST) take several weeks or months to result, but line probe assays and Xpert/Rif Ultra assay detect a limited number of resistance conferring gene mutations. Whole genome sequencing (WGS) is an advanced molecular testing method which theoretically can predict the resistance of M. tuberculosis (Mtb) isolates to all anti-TB agents through a single analysis.MethodsHere, we aimed to identify the level of concordance between the phenotypic and WGS-based genotypic drug susceptibility (DS) patterns of MDR–TB isolates. Overall, data for 12 anti-TB medications were analyzed.ResultsIn total, 63 MDR–TB Mtb isolates were included in the analysis, representing 27.4% of the total number of MDR–TB cases in Latvia in 2012–2014. Among them, five different sublineages were detected, and 2.2.1 (Beijing group) and 4.3.3 (Latin American-Mediterranean group) were the most abundant. There were 100% agreement between phenotypic and genotypic DS pattern for isoniazid, rifampicin, and linezolid. High concordance rate (> 90%) between phenotypic and genotypic DST results was detected for ofloxacin (93.7%), pyrazinamide (93.7%) and streptomycin (95.4%). Phenotypic and genotypic DS patterns were poorly correlated for ethionamide (agreement 56.4%), ethambutol (85.7%), amikacin (82.5%), capreomycin (81.0%), kanamycin (85.4%), and moxifloxacin (77.8%). For capreomycin, resistance conferring mutations were not identified in several phenotypically resistant isolates, and, in contrary, for ethionamide, ethambutol, amikacin, kanamycin, and moxifloxacin the resistance-related mutations were identified in several phenotypically sensitive isolates.ConclusionsWGS is a valuable tool for rapid genotypic DST for all anti-TB agents. For isoniazid and rifampicin phenotypic DST potentially can be replaced by genotypic DST based on 100% agreement between the tests. However, discrepant results for other anti-TB agents limit their prescription based solely on WGS data. For clinical decision, at the current level of knowledge, there is a need for combination of genotypic DST with modern, validated phenotypic DST methodologies for those medications which did not showed 100% agreement between the methods.

B-302 Increased False-Positives vs Increased Interference Flags: Using the AU 5800 to Compare Two Ethyl Glucuronide Assays

Abstract Background Ethyl glucuronide (EtG) is a metabolite of ethanol that can be detected in urine for up to three to five days following consumption of alcohol. Urine specimens are routinely screened for EtG to assess ethanol exposure for compliance and/or abuse with presumptive positive samples being confirmed using quantitative Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). LC-MS/MS is costly and labor intensive. Therefore, providing a sensitive and specific screening option for EtG in urine is beneficial, with it being more cost-effective, efficient, and widely available. Evaluation of the performance characteristics of different EtG assays can aid in identifying an assay that most closely agrees with LC-MS/MS. Methods To evaluate agreement, 100 residual urine specimens (approximately 50 positive/50 negatives identified using ARK Ethyl Glucuronide Assay) were thawed, mixed, and analyzed. Specimen were analyzed using ARK and Immunalysis Ethyl Glucuronide Urine HEIA on the Beckman Coulter AU 5800. Discrepant results were investigated using LC-MS/MS. Additionally, 15 false-positive samples (identified positive by ARK but confirmed negative by LC-MS/MS) were evaluated on both ARK and Immunalysis assays. Lastly, 7 samples were analyzed on ARK and Immunalysis because of the inability to quantitate by LC-MS/MS due to interfering substance. Results were evaluated using EP Evaluator and Microsoft Excel. Results Of the 100 samples analyzed, 8 samples were identified as discrepant between the two immunoassays. These 8 discrepant samples tested positive on ARK and negative on Immunalysis, resulting in an 83.3% positive agreement and a 100.0% negative agreement between ARK and Immunalysis. For these 8 discrepant samples, LC-MS/MS confirmed 7 samples negative and 1 sample positive suggesting that using ARK would result in 14.6% of presumptive positives confirming negative. Of the initial 100 samples, 7 samples resulted with interference flags by Immunalysis, indicating the presence of an unknown interfering substance within the sample. Of these samples with interference flags, 3 were among the 8 discrepant samples. Evaluation of identified false-positive samples (n = 15) by ARK resulted in 90.9% negative agreement for Immunalysis compared to 0.0% negative agreement by ARK. Of these 15 false-positive samples tested, 3 had interference flags identified using Immunalysis. Between agreement samples (n = 100) and false-positive samples (n = 15), a total of 10 interference flags were identified by Immunalysis, 6 of which were observed to have discrepant results between ARK and Immunalysis. ARK identified no interference flags. Interference was confirmed for 7 samples using LC-MS/MS, with Immunalysis detecting interference in 57% of those samples, and ARK reported no interference flags. In total, LC-MS/MS results were obtained for 37 samples, of which, only 2 false-positive and 1 false-negative samples were observed using Immunalysis. Conclusion While the assays offered by ARK and Immunalysis are similar in that they screen for EtG, we observed the accuracy between the two assays to be quite different, when compared to LC-MS/MS. Using ARK reagents would result in greater number of presumptive positives. Of note, the instrument flags accompanying Immunalysis results appear to be indicative of an interfering substance that ARK is not detecting and then subsequently reports as (falsely) positive.