Performance evaluation of machine-assisted interpretation of Gram stains from positive blood cultures.

Manual microscopy of Gram stains from positive blood cultures (PBCs) is crucial for diagnosing bloodstream infections but remains labor intensive, time consuming, and subjective. This study aimed to evaluate a scan and analysis system that combines fully automated digital microscopy with deep convolutional neural networks (CNNs) to assist the interpretation of Gram stains from PBCs for routine laboratory use. The CNN was trained to classify images of Gram stains based on staining and morphology into seven different classes: background/false-positive, Gram-positive cocci in clusters (GPCCL), Gram-positive cocci in pairs (GPCP), Gram-positive cocci in chains (GPCC), rod-shaped bacilli (RSB), yeasts, and polymicrobial specimens. A total of 1,555 Gram-stained slides of PBCs were scanned, pre-classified, and reviewed by medical professionals. The results of assisted Gram stain interpretation were compared to those of manual microscopy and cultural species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparison of assisted Gram stain interpretation and manual microscopy yielded positive/negative percent agreement values of 95.8%/98.0% (GPCCL), 87.6%/99.3% (GPCP/GPCC), 97.4%/97.8% (RSB), 83.3%/99.3% (yeasts), and 87.0%/98.5% (negative/false positive). The assisted Gram stain interpretation, when compared to MALDI-TOF MS species identification, also yielded similar results. During the analytical performance study, assisted interpretation showed excellent reproducibility and repeatability. Any microorganism in PBCs should be detectable at the determined limit of detection of 105 CFU/mL. Although the CNN-based interpretation of Gram stains from PBCs is not yet ready for clinical implementation, it has potential for future integration and advancement.

Applicability and performance of EUCAST's rapid antimicrobial susceptibility testing (RAST) on primarily sterile body fluids in blood culture bottles in laboratory routine with total lab automation.

Optimisation of microbiological diagnostics in primarily sterile body fluids is required. Our objective was to apply EUCAST's RAST on primarily sterile body fluids in blood culture bottles with total lab automation (TLA) and to compare results to our reference method Vitek2 in order to report susceptibility results earlier. Positive blood culture bottles (BACTEC™ Aerobic/Anaerobic/PEDS) inoculated with primarily sterile body fluids were semi-automatically subcultured onto Columbia 5% SB agar, chocolate agar, MacConkey agar, Schaedler/KV agar and Mueller-Hinton agar. On latter, cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin were added. After 6 h, subcultures and RAST were imaged and MALDI-TOF MS was performed. Zone sizes were digitally measured and interpreted following RAST breakpoints for blood cultures. MIC values were determined using Vitek2 panels. During a 1-year period, 197 Staphylococcus aureus, 91 Enterococcus spp., 38 Escherichia coli, 11 Klebsiella pneumoniae and 8 Pseudomonas aeruginosa were found. Categorical agreement between RAST and MIC was 96.5%. Comparison showed no very major errors, 2/7 (28.6%) and 1/7 (14.3%) of major errors for P. aeruginosa and meropenem and ciprofloxacin, 1/9 (11.1%) for K. pneumoniae and ciprofloxacin, 4/69 (7.0%) and 3/43 (5.8%) for Enterococcus spp. and vancomycin and ampicillin, respectively. Minor errors for P. aeruginosa and meropenem (1/8; 12.8%) and for E. coli and ciprofloxacin (2/29; 6.5%) were found. 30/550 RAST measurements were within area of technical uncertainty. RAST is applicable and performs well for primarily sterile body fluids in blood culture bottles, partially better than blood-based RAST. Official EUCAST evaluation is needed.

Evaluation of EUCAST rapid antimicrobial susceptibility testing (RAST) for positive blood cultures in clinical practice using a total lab automation.

Our objective was to evaluate EUCAST's 'rapid antimicrobial susceptibility testing' (RAST) directly from positive blood culture that delivers antimicrobial results within 6 h for Staphylococcus aureus, Enterococcus spp., Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, using total lab automation. Zone diameters from RAST were compared with MIC results. Furthermore, its influence on time to report was investigated. RAST was performed to all positive aerobic and anaerobic blood culture bottles by subculturing them, i.e. onto Mueller-Hinton agar and adding six antibiotic discs covering Gram-negative and Gram-positive therapy (cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin). RAST was automatically imaged after 6 h. Zone sizes were measured using a TLA software tool and interpreted according to EUCAST clinical breakpoints. Bacteria were identified using MALDI-TOF MS and MIC results were determined using Vitek2 panels. Categorial agreement between agar diffusion and MIC results was investigated. Additionally, time to RAST and time to Vitek were compared for 100 isolates (20 per species). Between November 2018 and April 2019, 3313 positive mono-bacterial blood culture bottles were collected of which 894 bottles with RAST-validated species were investigated. Among these bottles, 2029 individual antibiotic measurements were compared with MIC results from Vitek2 and 14 very major, 28 major and 12 minor errors were found. A median reduction of 17:30 h in time to report was observed. Introduction of RAST with automatic TLA imaging function could reduce time to report by 17:30 h. Excellent accordance between zone diameter and MIC results, particularly for cefoxitin, vancomycin and meropenem, was observed, but drawbacks due to ATU were seen.

Reincubation of culture-negative urines for an additional 20 hours does not identify additional UTI cases.

Introduction. The question of whether a single day of incubation is sufficient for urine cultures has been a matter of debate.Aim. The aim of this study was to investigate the potential benefit of prolonged incubation for initially culture-negative urines.Methodology. Eight hundred and twelve urine specimens with no growth after incubation for 20 h were incubated for an additional 20 h to detect slower growing uropathogenic organisms.Results. This study included a considerable number of urine cultures from immunocompromised and/or kidney-transplanted patients. For 99.9 % of the specimens, there was no difference in the interpretation of results.Conclusion. Twenty hours of incubation did not have any negative effect on the detection of uropathogens.

Analysis of Streptococcus pneumoniae using Fourier-transformed infrared spectroscopy allows prediction of capsular serotype.

Determination of the capsule type of clinical isolates of Streptococcus pneumoniae is a prerequisite for epidemiological studies and further vaccine development. The Quellung reaction for serotyping is expensive and mostly done in reference centres. We wanted to evaluate whether Fourier-transformed infrared (FT-IR) spectroscopy is suitable for capsular type analysis and prediction of pneumococcal serotypes. We used the IR-Biotyper™ (Bruker) to create a database containing the spectra of 120 strains from invasive disease. The strains covered the 24 vaccine serotypes contained in the 13-valent conjugate vaccine (PCV13) and the 23-valent polysaccharide vaccine (PSV23). Hierarchical clustering analysis was performed. Finally, two different classification sets were created (PCV13 and PSV23). They were used to predict the serotype of 168 different challenge strains (invasive and non-invasive disease) covering 48 different serotypes (vaccine and non-vaccine types). FT-IR spectra from pneumococci (1300-800 cm-1) clustered along their serotype as determined by the Quellung reaction (120 strains, 24 different serotypes). Strains with unknown serotype fell within the cluster of the correct serotype, as long as the latter was represented in the database (168 strains, 48 different serotypes). Concordance between the Quellung reaction and FT-IR spectroscopy was excellent (kappa ≥ 0.75). FT-IR spectroscopy is a fast and cost-effective method to predict the capsular serotype of pneumococci.

In vitro activity of newer antimicrobials against penicillin non-susceptible strains of Streptococcus pneumoniae.

Background: Since the first isolation of Streptococcus pneumoniae with low penicillin susceptibility in the 1960s, resistant strains have spread over the globe, causing substantial problems in the treatment of pneumococcal infections. However, in Germany, rates of non-susceptibility are still below 5%. Methods: Since 2009 clinical pneumococcal strains have been collected at the Center for Infectious Diseases, Heidelberg University Hospital, Germany. In this study, 56 of these strains were chosen due to their decreased penicillin susceptibility (minimal inhibitory concentration (MIC)≥0.12 µg/mL). Sixteen of these strains even showed an MIC of ≥2 µg/mL. We examined the in vitro activity of newer antimicrobials known to be active against Gram-positive bacteria. For this purpose MICs of ceftaroline, ceftobiprole, dalbavancin, delafloxacin, eravacycline, tedizolid, and telavancin were determined and evaluated. Results: All of the 7 antimicrobial agents inhibited pneumococcal growth at concentrations of 0.5 µg/mL or lower. Currently, clinical breakpoints are only available for two substances, ceftaroline and ceftobiprole. According to these breakpoints, all MICs were below the susceptibility breakpoint; however, there was a correlation between high penicillin MICs (≥2 µg/mL) and MICs near the ceftaroline and ceftobiprole susceptibility breakpoint. The other agents showed very promising effects against all tested strains with the lowest MIC90 of 0.002 µg/mL for telavancin. Conclusion: Consequently, this study demonstrates the promising in vitro activity of newer antimicrobials against penicillin non-susceptible strains of S. pneumoniae.

Shorter Incubation Times for Detecting Multi-drug Resistant Bacteria in Patient Samples: Defining Early Imaging Time Points Using Growth Kinetics and Total Laboratory Automation.

BACKGROUND: The transition from manual processing of patient samples to automated workflows in medical microbiology is challenging. Although automation enables microbiologists to evaluate all samples following the same incubation period, the essential incubation times have yet to be determined. We defined essential incubation times for detecting methicillin-resistant Staphylococcus aureus (MRSA), multi-drug resistant gram-negative bacteria (MDRGN), and vancomycin-resistant enterococci (VRE). METHODS: We monitored the growth kinetics of MRSA, MDRGN, and VRE between two and 48 hours on chromogenic media to establish the time points of first growth, single colony appearance, and typical morphology for 10², 104, 106, and 108 colony forming units/mL. Subsequently, we imaged plates inoculated with 778 patient samples after 20, 24, and 36 hours. RESULTS: The first growth, single colony appearance, and typical morphology time points were inoculum-dependent. First growth appeared after 6-18 hours, 4-18 hours, and 8-48 hours for MRSA, MDRGN, and VRE, respectively, and single colonies appeared at 12-18 hours, 6-20 hours, and 12-48 hours, respectively. Typical morphology was visible at 14-22 hours and 12-48 hours for MRSA and VRE, but was not determined for MDRGN. By examining patient samples, ≥98% of MRSA and MDRGN were visible 20 hours after the start of incubation. Following 24 hours of incubation, only 79.5% of VRE were clearly visible on the respective plates. CONCLUSIONS: An incubation time of 20 hours is sufficient for detecting MRSA and MDRGN. VRE growth is much slower and requires additional imaging after 36 hours.

Laboratory Automation in Clinical Microbiology.

Laboratory automation is currently the main organizational challenge for microbiologists. Automating classic workflows is a strenuous process for the laboratory personnel and a huge and long-lasting financial investment. The investments are rewarded through increases in quality and shortened time to report. However, the benefits for an individual laboratory can only be estimated after the implementation and depending on the classic workflows currently performed. The two main components of automation are hardware and workflow. This review focusses on the workflow aspects of automation and describes some of the main developments during recent years. Additionally, it tries to define some terms which are related to automation and specifies some developments which would further improve automated systems.

Susceptibility Testing of Bacteria Using Maldi-Tof Mass Spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was introduced into the microbiological routine more than 10 years ago. Since then it has almost replaced biochemical identification. It is unrivaled in terms of accuracy and cost. From a laboratory's perspective it would be an ideal method to replace classic susceptibility testing, that is Kirby-Baur agardiffusion or determination of minimal inhibitory concentrations (MICs). First reports on possible assays for susceptibility testing are more than 10 years old. However, the developments during the last 5 years were substantial. This review focuses with some exceptions on the progress, which was achieved during the last decade.

Detection of MRSA in nasal swabs-marked reduction of time to report for negative reports by substituting classical manual workflow with total lab automation.

In 2016, the workflow for MRSA detection in nasal swabs was changed from a classic-manual workflow to an automated workflow using total lab automation (TLA; BD Kiestra). This change entailed a reduction of the incubation time from 2 days to 20 h and reading of plates on weekdays and weekends instead of weekdays only. The workflow alteration did not include the introduction of 24/7. We wanted to follow up on the consequences for the times to report (TTR). We compared the TTR of all nasal swabs, which were sent for MRSA detection from June until August in 2015 (workflow-classic-manual) and in 2016 (workflow-automated). We calculated median TTR and interquartile ranges for the three possible reporting outcomes (negative, MRSA-known, MRSA-new) per day and workflow. A multivariable linear regression modeled the exposure variables workflow, day, and reporting outcome on TTR including interaction variables. The quantity and reasons for a TTR longer than 3 days were analyzed. During both 3-month periods, a total of 16,111 reports were issued (2015:7620; 2016:8491). The median TTR for negative reports was 48:28 (hh:mm) in 2015 and 23:58 in 2016. In the linear regression, all exposure variables had a strong and highly significant (p < 0.001) influence on the TTR. The number of reports with a TTR longer than 3 days shrank from 2418 (2015) to 60 (2016). The workflow alteration halved the median TTR for negative reports and the number of reports with a TTR longer than 3 days was reduced by 97.5%.

Significant increase in cultivation of Gardnerella vaginalis, Alloscardovia omnicolens, Actinotignum schaalii, and Actinomyces spp. in urine samples with total laboratory automation.

While total laboratory automation (TLA) is well established in laboratory medicine, only a few microbiological laboratories are using TLA systems. Especially in terms of speed and accuracy, working with TLA is expected to be superior to conventional microbiology. We compared in total 35,564 microbiological urine cultures with and without incubation and processing with BD Kiestra TLA for a 6-month period each retrospectively. Sixteen thousand three hundred thirty-eight urine samples were analyzed in the pre-TLA period and 19,226 with TLA. Sixty-two percent (n = 10,101/16338) of the cultures processed without TLA and 68% (n = 13,102/19226) of the cultures processed with TLA showed growth. There were significantly more samples with two or more species per sample and with low numbers of colony forming units (CFU) after incubation with TLA. Regarding the type of bacteria, there were comparable amounts of Enterobacteriaceae in the samples, slightly less non-fermenting Gram-negative bacteria, but significantly more Gram-positive cocci, and Gram-positive rods. Especially Alloscardivia omnicolens, Gardnerella vaginalis, Actinomyces spp., and Actinotignum schaalii were significantly more abundant in the samples incubated and processed with TLA. The time to report was significantly lower in the TLA processed samples by 1.5 h. We provide the first report in Europe of a large number of urine samples processed with TLA. TLA showed enhanced growth of non-classical and rarely cultured bacteria from urine samples. Our findings suggest that previously underestimated bacteria may be relevant pathogens for urinary tract infections. Further studies are needed to confirm our findings.

Identification of Streptococcus pneumoniae: Development of a Standardized Protocol for Optochin Susceptibility Testing Using Total Lab Automation.

Purpose. Optochin susceptibility is one parameter used in the laboratory to identify Streptococcus pneumoniae. However, a single standardized procedure does not exist. Optochin is included neither in the current EUCAST breakpoint tables nor in the CLSI performance standards for antimicrobial susceptibility testing. We wanted to establish an evidence-based protocol for optochin testing for our Total Lab Automation. Methods. We tested seven different agars and four different reading time points (7 h, 12 h, 18 h, and 24 h). To accommodate for serotype diversity, all tests were done with 99 different strains covering 34 different serotypes of S. pneumoniae. We calculated a multivariable linear regression using data from 5544 inhibition zones. Results. Reading was possible for all strains at 12 h. Agar type and manufacturer influenced the size of the inhibition zones by up to 2 mm and they varied considerably depending on serotype (up to 3 mm for serotype 3). Depending on agar and reading time point, up to 38% of inhibition zones were smaller than the cut-off of 14 mm; that is, the result of the test was false-negative. Conclusions. Shortening incubation time from 24 h to 12 h for optochin susceptibility testing is feasible. Agar and incubation time have to be chosen carefully to avoid false-negative results.

Streptococcus pneumoniae as an agent of urinary tract infections - a laboratory experience from 2010 to 2014 and further characterization of strains.

Streptococcus pneumoniae is a rare cause of urinary tract infection. Between January 2010 and December 2014, 26 urine samples from 18 different patients contained S. pneumoniae at the Department for Infectious Diseases, University Hospital of Heidelberg. Patient age varied between three and 72 years. 13 patients were male and five were female. Past medical histories of 16 patients were available. Eight patients had a past medical history of renal transplant and four patients had other renal dysfunctions. Further analyses of the isolates revealed that the aspect of colonies is more resembling S. mitis than invasive isolates of S. pneumoniae. Optochin disk diameters tend to be 14 mm or smaller. Identification using MALDI-TOF or VITEK2 identification cards was accurate. Only 2 isolates showed a decreased susceptibility towards penicillin (MIC = 0.5 mg/L). Eight different serotypes were identified using a PCR approach as well Neufeld-Quellungs reaction.

Multicentre investigation of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in German hospitals.

Aim of this study was to determine the incidence and molecular epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Germany. E. coli and K. pneumoniae isolates from clinical samples which were non-susceptible to carbapenems were collected in laboratories serving 20 hospitals throughout Germany from November 2013 to April 2014. The isolates were tested for the presence of carbapenemases by PCR and phenotypic methods and typed by multilocus sequence typing. Risk factors including a previous hospitalization abroad were analysed. Carbapenemases were detected in 24 isolates from 22 patients out of 464,514 admissions. Carbapenemases included OXA-48 (n=14), KPC-2 (n=8) and NDM-1 (n=2). Except for two K. pneumoniae isolates with ST101, all OXA-48 producing strains belonged to different clones. In contrast, half of KPC-2 producing K. pneumoniae were of ST258 and both NDM-1 producing strains were of ST11. Compared to carbapenem-susceptible controls, patients with carbapenemase-producing strains differed by a significantly higher proportion of males, a higher proportion of isolates from wound samples and a more frequent previous stay abroad in univariate analysis. This multicentre study demonstrated an incidence of carbapenemase-producing E. coli and K. pneumoniae from clinical samples in Germany of 0.047 cases per 1000 admissions. OXA-48 was more frequent than KPC-2 and NDM-1 and showed a multiclonal background.

Are atrophic long-bone nonunions associated with low-grade infections?

Impaired fracture healing, especially when associated with bacterial infection, is a severe complication following long-bone fractures and requires special treatment. Because standard diagnostic techniques might provide falsely negative results, we evaluated the sonication method for detection of bacteria on implants of patients with fracture nonunions. A total of 49 patients with a nonunion (group NU) and, for comparison, 45 patients who had undergone routine removal of osteosynthetic material (group OM), were included in the study. Five different diagnostic methods (culture of tissue samples, culture of intraoperative swabs, histopathology of tissue samples, culture of sonication fluid, and 16S ribosomal DNA polymerase chain reaction of sonication fluid) were compared and related to clinical data. Among the diagnostic tests, culture of sonication fluid demonstrated by far the highest detection rate of bacteria (57%) in group NU, and rather unexpectedly 40% in group OM. Culture of sonication samples also revealed a broad spectrum of bacteria, in particular Propionibacterium spp. In conclusion, our results indicate that more bacteria can be detected on implants of patients with atrophic nonunions of long-bone fractures by means of the sonication procedure, which provides a valuable additional diagnostic tool to decide on a surgical procedure (eg, two-step procedure) and to further specify antimicrobial therapy.

Serotyping of pneumococci: evaluation of the genetic approach and performance with clinical samples.

Determination of pneumococcal serotypes depends on a successful culture and the Quellung's reaction. However, in 2006, the capsular sequences of 90 different pneumococcal capsular loci were published, thus making "genetic" serotyping via PCR possible. We wanted to determine the reliability of the published primers for the 13 serotypes included in pneumococcal conjugated vaccine 13 (PCV13) with pneumococcal isolates from Germany. We used a multiplex PCR approach and agarose gel detection of amplicons. Three hundred ninety well-characterized strains of Streptococcus pneumoniae and 46 clinical samples were used in the study. A 100% concordance was achieved between PCR and Quellung's reaction. In 7 clinical samples with a PCR positive for S. pneumoniae, we could determine a serotype included in PCV13.

Evaluation of implant sonication as a diagnostic tool in implant-associated infections.

Infections of implants pose a severe problem in the field of orthopedic surgery, because they can cause bone degradation with subsequent loosening of the implant. The discrimination between septic implant loosening and aseptic loosening can be a challenge, and hence novel diagnostic methods have been introduced to improve the detection of bacteria. Because a major problem is their firm adherence to implants due to biofilm formation, sonication has been introduced, followed by identification of bacteria by culture or genetic methods. In this study, we compared the results obtained after sonication pretreatment with those of microbiological testing of tissue samples and histopathological evaluation of the same tissue. Furthermore, we related the results obtained following sonication to the clinical diagnosis of septic or aseptic implant loosening, respectively. Sonication of explanted devices also enhances the likelihood of detecting bacterial growth in patients who were considered "aseptic" based on the clinical evaluation.

Prosthetic infection: improvement of diagnostic procedures using 16S ribosomal deoxyribonucleic acid polymerase chain reaction.

PURPOSE: Prosthetic infection is the worst complication in joint arthroplasty. The diagnostic procedure is time consuming and in many cases unrewarding. The aim of this investigation was to raise the sensitivity of the diagnostic procedure. METHODS: Altogether, 229 implants were removed from 229 patients. Complete data from 157 patients could be analysed. On explantation of the respective arthroplasty, tissue was removed, puncture fluid aspirated and biofilm scratched from the implant surface with a surgical knife. Specimens were investigated with conventional culture methods and with 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) and sequencing. RESULTS: In 123 cases, no pathogen could be identified by routine culture methods. In three of these culture-negative cases, bacteria could be identified with 16S rDNA sequencing of the removed biofilm. In 34 cases, bacteria could be identified with culture methods. In two of these cases, sequencing detected additional pathogens. CONCLUSIONS: The process of 16S ribosomal deoxyribonucleic acid polymerase chain reaction (rDNA PCR) and sequencing of biofilm removed from the explanted prosthesis is an important addition to conventional culture methods in prosthetic joint infection. Polymerase chain reaction detects additional pathogens and improves diagnostic sensitivity. The examination of tissue, puncture fluid and biofilm should be performed in cases of prosthesis loosening and explantation.

Using matrix-assisted laser desorption ionization-time of flight mass spectrometry to detect carbapenem resistance within 1 to 2.5 hours.

In recent years, the percentage of carbapenem-resistant bacteria has increased at an alarming pace and become a major threat for patient survival. Carbapenemase-induced carbapenem resistance can be confirmed through the detection of carbapenem degradation using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This method works for strains carrying NDM-1, VIM-1, VIM-2, KPC-2, and different IMP enzymes.

Streptococcus pneumoniae in urinary tracts of children with chronic kidney disease.

Streptococcus pneumoniae is not commonly considered an agent of urinary tract infections. We report 3 children with urinary tract abnormalities who had high numbers of S. pneumoniae in their urine (≥104 CFU/mL) and varying clinical symptoms.