Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens.

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics.

ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems.

After first detections of carbapenemase-producing Enterobacteriaceae (CPE) in animals, the European Union Reference Laboratory for Antimicrobial Resistance has provided a protocol for the isolation of carbapenemase-producing Escherichia (E.) coli from cecum content and meat. Up to now, only few isolates were recovered using this procedure. In our experience, the choice of the selective agar is important for the efficacy of the method. Currently, the use of the prevailing method fails to detect CPE that exhibit a low resistance against carbapenems. Thus, this study aims to evaluate the suitability of selective media with antibiotic supplements and commercial ChromID® CARBA agar for a reliable CPE detection. For comparative investigations, detection of freeze-dried carbapenemase-resistant bacteria was studied on different batches of the ChromID® CARBA agar as well as on MacConkey agar supplemented with 1 mg/L cefotaxime and 0.125 mg/L meropenem (McC+CTX+MEM). The suitability of the different media was assessed within a time of 25 weeks, starting at least six weeks before expiration of the media. Carbapenem-resistant isolates exhibiting a serine-based hydrolytic resistance mechanism (e.g., bla KPC genes) were consistently detected over 25 weeks on the different media. In contrast, carbapenemase producers with only slightly reduced susceptibility and exhibiting a zinc-catalyzed activity (e.g., bla VIM, bla NDM, and bla IMP) could only be cultivated on long-time expired ChromID® CARBA, but within the whole test period on McC+CTX+MEM. Thus, ChromID® CARBA agar appears to be not suitable for the detection of CPE with slightly increased minimum inhibitory concentrations (MIC) against carbapenems, which have been detected in German livestock and thus, are of main interest in the national monitoring programs. Our data are in concordance with the results of eleven state laboratories that had participated in this study with their ChromID® CARBA batches routinely used for the German CPE monitoring. Based on the determined CPE detection rate, we recommend the use of McC+CTX+MEM for monitoring purposes. This study indicates that the use of ChromID® CARBA agar might lead to an underestimation of the current CPE occurrence in food and livestock samples.

Evaluation of reticulocyte hemoglobin content (RETIC-HGB) for the diagnosis of iron-limited erythropoiesis in cats.

BACKGROUND: Decreased reticulocyte hemoglobin content (CHr) (Siemens ADVIA 2120) reflects iron-limited erythropoiesis (ILE). RETIC-HGB (IDEXX ProCyte Dx) is a novel marker of ILE for veterinary use. OBJECTIVES: We aimed to evaluate reference intervals (RIs) and the utility of RETIC-HGB and CHr in the diagnosis of feline ILE. MATERIALS AND METHODS: RIs were established in 59 healthy cats. Intra-assay coefficients of variation (CVs) and correlations between RETIC-HGB and CHr were assessed. Two hundred and seventy-five cats were classified as having ILE or not based on low plasma iron or low transferrin saturation along with anemia and/or altered RBC indices. CHr, RETIC-HGB, and serum amyloid A (SAA) were compared between the groups. The sensitivity and specificity of RETIC-HGB and CHr to diagnose ILE were analyzed to determine the RI lower limits. RESULTS: RIs for RETIC-HGB and CHr were 12.5-18.0 and 14.0-19.9 pg, respectively. The CV was 3% for both variables. RETIC-HGB and CHr were moderately correlated (rs = 0.59) with a bias of -1.2 picograms (pgs). Twenty of the 275 cats were classified as having ILE. Compared with non-ILE cats, ILE cats had significantly lower median RETIC-HGB (14.3 vs 15.2 pg, P = .0046) and mean CHr (14.7 vs 16.5 pg, P < .0001) values and significantly increased median SAA (44.6 vs 2.3 µg/dl, P < .0001) values. Using the lower RI limits resulted in a low sensitivity and relatively high specificity to diagnose ILE in cats. CONCLUSIONS: ILE was characterized by decreased CHr and RETIC-HGB; however, sensitivity was low. The moderate correlation between RETIC-HGB and CHr is likely due to species differences and different methodology.

Evaluation of reticulocyte hemoglobin content (RET-He) in the diagnosis of iron-deficient erythropoiesis in dogs.

BACKGROUND: Reticulocyte hemoglobin content provided by the Siemens ADVIA (CHr) is an established marker of iron deficiency. The IDEXX ProCyte Dx hematology analyzer now provides a similar variable, reticulocyte hemoglobin equivalent (RET-He). OBJECTIVES: The objective was to evaluate RET-He and its diagnostic utility in dogs, and to calculate a cutoff value for diagnosing iron-deficient erythropoiesis (IDE). Furthermore, the prevalence of RET-He values below this cutoff value was established. METHODS: One hundred and seventy-one CBCs of healthy dogs were used to establish a RI. Stability of RET-He was evaluated by repeated measurements over 48 hours (n = 10). The 25-run coefficient of variation (CV) was calculated, and correlation and bias between measurements of RET-He and CHr were assessed (n = 190). A cutoff value for diagnosing IDE was calculated. The utility of RET-He in the detection of IDE was evaluated in 123 dogs. The prevalence of low RET-He values was assessed retrospectively in a multicenter study (2012-2014) under participation of 7 veterinary clinics. RESULTS: Reticulocyte hemoglobin equivalent with an RI of 22.2 to 28.6 pg was statistically stable over 48 hours (P = .10). The CV was 1.8%. A fair correlation (ρ = 0.74) between RET-He and CHr with a small bias of -0.6 pg was found. The cutoff value for diagnosing IDE was 20.9 pg (sensitivity: 85%; specificity: 99%). The prevalence of RET-He values below 20.9 pg was 10.3% (1084/10,553 dogs). CONCLUSIONS: RET-He on the ProCyte Dx is a precise screening tool in dogs to detect iron-deficient erythropoiesis.

Canine reticulocyte hemoglobin content (RET-He) in different types of iron-deficient erythropoiesis.

BACKGROUND: Reticulocyte hemoglobin content (RET-He) is a diagnostic marker for iron deficiency (ID) in people and dogs. OBJECTIVES: The aim of our study was to evaluate the clinical utility of RET-He in the diagnosis of different causes of iron-deficient erythropoiesis (IDE). METHODS: Canine CBCs were separated into 2 groups according to RET-He values, < 20.9 pg or ≥ 20.9 pg. Erythrocyte and reticulocyte variables were compared between dogs with decreased and normal RET-He values. Additional data for a subgroup of dogs were collected, and dogs with low RET-He values were categorized as having ID, inflammatory disorders (INFL), portosystemic shunt (PSS), miscellaneous diseases (MISC), or combinations of these diseases (ID+INFL, ID+PSS). Hematologic variables were compared between dogs of the different disease groups. RESULTS: Overall, 10.3% (1084/10,553) of canine CBCs showed decreased RET-He values. Significant differences between dogs with decreased and normal RET-He values were found for all erythrocyte and reticulocyte variables. The majority (68.9%, 747/1084) of dogs with low RET-He values was anemic; 28.9% (216/747) of those anemic dogs had microcytosis and hypochromasia. In the subgroup of dogs, 8.9% (205/2306) had low RET-He values. According to their diagnosed diseases, anemic dogs (138/205) could be categorized as ID (17/138; 12.3%), INFL (16/138; 11.6%), PSS (30/138; 21.7%), ID+INFL (63/138; 45.7%), ID+PSS (8/138; 5.8%), and MISC (4/138; 2.9%). Distribution in nonanemic dogs (67/205) was similar, except for a lower number of dogs with PSS. CONCLUSIONS: Low RET-He values indicate IDE even in dogs with other CBC variables within the RIs.