The use of Kudoh method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia.

BACKGROUND: Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly. OBJECTIVE: To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method's performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates. METHOD: 75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30th December 2017 and 25th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2. RESULT: Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar's test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages. CONCLUSION: The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment.

Validation of rapid detection methods for Salmonella enterica in green chile.

The objective of this study is to validate the US Food and Drug Administration (FDA) rea-time polymerase chain reaction (qPCR) assay, the Neogen Amplified Nucleic Single Temperature Reaction (ANSR) assay, and the Vitek ImmunoDiagnostic Assay System (VIDAS) SLM procedure against the FDA cultural procedure for Salmonella detection in green chile pepper. Green chile was artificially contaminated with Salmonella according to the FDA guidelines (FDA. Guidelines for the Validation of Microbiological Methods for the FDA Foods Program, 3rd Edition. 2019. www.fda.gov/media/83812/download?attachment (17 March 2024, date last accessed)) at a fractional recovery level (where 50%-25% tests positive and at a level +1 log greater for each organism tested). Enriched samples were tested directly by the ANSR Salmonella test and by qPCR, and were subcultured into Rappaport-Vassiliadis and tetrathionate brilliant green broth for cultural detection and qPCR. For the VIDAS-SLM assay, the selective enrichments were further cultured in M broth before testing. Presumptive salmonellae were confirmed with biochemical tests, serology, and qPCR. All three rapid assays were compared favorably with the FDA-BAM (Bacteriological Analytical Manual) method. No significant differences at P < .05 were found between the procedures using McNemar's χ2 test. The three procedures were found to be rapid and reliable alternatives to cultural detection of Salmonella enterica in green chile.

Comparison of three rapid diagnostic tests for bloodstream infections using Benefit-risk Evaluation Framework (BED-FRAME).

Rapid diagnostic tests (RDTs) for bloodstream infections have the potential to reduce time to appropriate antimicrobial therapy and improve patient outcomes. Previously, an in-house, lipid-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) method, Fast Lipid Analysis Technique (FLAT MS), has shown promise as a rapid pathogen identification method. In this study, FLAT MS for direct from blood culture identification was evaluated and compared to FDA-cleared identification methods using the Benefit-risk Evaluation Framework (BED-FRAME) analysis. FLAT MS was evaluated and compared to Bruker Sepsityper and bioMérieux BioFire FilmArray BCID2 using results from a previous study. For this study, 301 positive blood cultures were collected from the University of Maryland Medical Center. The RDTs were compared by their sensitivities, time-to-results, hands-on time, and BED-FRAME analysis. The overall sensitivity of all platforms compared to culture results from monomicrobial-positive blood cultures was 88.3%. However, the three RDTs differed in their accuracy for identifying Gram-positive bacteria, Gram-negative bacteria, and yeast. Time-to-results for FLAT MS, Sepsityper, and BioFire BCID2 were all approximately one hour. Hands-on times for FLAT MS, Sepsityper, and BioFire BCID2 were 10 (±1.3), 40 (±2.8), and 5 (±0.25) minutes, respectively. BED-FRAME demonstrated that each RDT had utility at different pathogen prevalence and relative importance. BED-FRAME is a useful tool that can used to determine which RDT is best for a healthcare center.

Use of a chromogenic medium with and without selective enrichment to screen for carbapenemase-producing Enterobacterales (CPE) from canine and feline fecal specimens during an outbreak of NDM-5-producing Escherichia coli.

Carbapenemase-producing Enterobacterales (CPE) are one of the most urgent threats to human healthcare globally. Descriptions of CPE outbreaks in veterinary hospitals suggest the need for screening strategies for CPE from companion animals. Our aim was to optimize a chromogenic agar method with and without selective enrichment to isolate CPE from companion animal feces in an ongoing outbreak of New Delhi metallo-ß-lactamse-5 Escherichia coli. A limit of detection (LOD) assay for spiked canine and feline feces was performed for both methods using a carbapenamase-producing E. coli (24213-18); the LOD (1.5 × 103 cfu/g of feces) was equivalent to that reported for human fecal specimens. We screened 1,247 companion animal fecal specimens for carriage of CPE by 1) direct plating to chromogenic agar and 2) plating to chromogenic agar following selective enrichment. Twenty-one specimens were positive for CPE by both direct culture and enrichment culture. No specimens were positive with selective enrichment and negative by direct culture. A selective enrichment step did not result in any increased recovery of CPE from companion animals, which suggests that enrichment broth may not be necessary for outbreak surveillance testing. It is important to continue to validate methods for the detection of CPE in companion animals as outbreaks become more common in veterinary facilities.

Evaluation of PhenoMATRIX and PhenoMATRIX PLUS for the screening of MRSA from nasal and inguinal/perineal swabs using chromogenic media.

The objective of this study was to assess the clinical performances of PhenoMATRIX and PhenoMATRIX PLUS for the screening of methicillin-resistant Staphylococcus aureus (MRSA) from nasal and inguinal/perineal ESwabs using chromogenic media. The automated performances were compared to the manual reading. Additionally, we evaluated PhenoMATRIX PLUS for the automatic release of the negative results to the Laboratory Information System (LIS) and the automatic discharge of the negative plates from the incubators. A total of 6,771 non-duplicate specimens were used by PhenoMATRIX as a machine learning model. The validation of these settings was performed on 17,223 non-duplicate specimens. The MRSA positivity rate was 5% (866/17,223). Validated settings were then used by PhenoMATRIX PLUS on another 1,409 non-duplicate specimens. The sensitivities of PhenoMATRIX and PhenoMATRIX PLUS were 99.8% [95% confidence interval (CI), 99.2%-99.9%] and 100% (95% CI, 92.1%-100%), respectively. The specificities of PhenoMATRIX and PhenoMATRIX PLUS were 99.1% (95% CI, 99.0%-99.2%) and 95.2% (95% CI, 93.8%-96.1%), respectively. All the 1,297 MRSA-negative specimens analyzed by PhenoMATRIX PLUS were automatically released and sent to the LIS immediately after availability of the culture image on the WASPLab (100% accuracy). All negative media plates were automatically discarded. PhenoMATRIX PLUS decreases the time spent by technologists on negative plates and ensures optimal usage of the incubators' capacity.

Sensitive detection of viable Cronobacter sakazakii by bioluminescent reporter phage emitting stable signals with truncated holin.

As outbreaks of foodborne illness caused by the opportunistic pathogen Cronobacter sakazakii (Cs) continue to occur, particularly in infants consuming powdered infant formula (PIF), the need for sensitive, rapid, and easy-to-use detection of Cs from food and food processing environments is increasing. Here, we developed bioluminescent reporter bacteriophages for viable Cs-specific, substrate-free, rapid detection by introducing luciferase and its corresponding substrate-providing enzyme complex into the virulent phage ΦC01. Although the reporter phage ΦC01_lux, constructed by replacing non-essential genes for phage infectivity with a luxCDABE reporter operon, produced bioluminescence upon Cs infection, the emitted signal was quickly decayed due to the superior bacteriolytic activity of ΦC01. By truncating the membrane pore-forming protein holin and thus limiting its function, the bacterial lysis was delayed and the resultant engineered reporter phage ΦC01_lux_Δhol could produce a more stable and reliable bioluminescent signal. Accordingly, ΦC01_lux_Δhol was able to detect at least an average of 2 CFU/ml of Cs artificially contaminated PIF and Sunsik and food contact surface models within a total of 7 h of assays, including 5 h of pre-enrichment for Cs amplification. The sensitive, easy-to-use, and specific detection of live Cs with the developed reporter phage could be applied as a novel complementary tool for monitoring Cs in food and food-related environments for food safety and public health.

[Evaluation of two phenotypic techniques and a commercial multiplex molecular panel for the detection of different carbapenemases]. / Evaluación de dos técnicas fenotípicas y un panel molecular multiplex comercial para la detección de diferentes carbapenemasas.

OBJECTIVE: The prevalence of carbapenemase-producing Enterobacterales has increased in recent years and is considered an important public health problem. METHODS: A total of 106 clinical samples were analyzed by different carbapenemase detection techniques: inhibition discs (ID), immunochromatographic test (ICT) and a genotypic method, comparing them with a multiplex RT-PCR as a reference method. RESULTS: Overall, all 3 techniques exceeded 90% sensitivity, although with differences in the performance of some of them by carbapenemase type. DI had low specificity (62%) for OXA-48, while with TIC the sensitivity for NDM-type metallo-beta-lactamase (93%) was slightly lower than for OXA-48 (95%). The best results were obtained with the genotypic technique (100% overall performance). CONCLUSIONS: Despite the lower sensitivity of TICs (especially in NDM carbapenemases) compared to molecular techniques, with the modification of the protocol we managed to increase this sensitivity and, together with the lower price, simplicity and speed, it makes this technique a good screening option.

Assessment of the impact of centralized bioMérieux BACT/ALERT® VIRTUO® blood culture system (VIRTUO) implementation on outcomes in patients with gram-negative bacteremia.

BACKGROUND: We evaluated pre- and postimplementation of Virtuo on outcome in patients with gram-negative bacteremia using a quasiexperimental time-in-motion design. METHODS: Becton Dickinson BACTEC™ 9000 series (Bactec) (2018) and Virtuo system (2020) were utilized in a decentralized and centralized process, respectively. Data collected in August-December in 2018 and 2020 were analyzed with SPSS (ver 28). RESULTS: For 185 patients in each time period, patient age, gender, length of hospitalization were not different. However, blood culture (BC) volume was significantly lower in 2020 (7.1 ± 2.6 mL) compared to 2018 (8.9 ± 1.9 mL). Time from BC draw and time from pathogen identification (ID) to treatment change were both significantly faster in 2020 (52.9 ± 38.3 hours; 15.1 ± 27.4 hours), compared to 2018 (65.0 ± 46.3 hours; 23.8 ± 33.8), respectively. CONCLUSIONS: Replacement of decentralized Bactec with centralized Virtuo, resulted in significant improvement in management of patients gram-negative bacteremia.

A real-time analysis of protein transport via the twin arginine translocation pathway in response to different components of the protonmotive force.

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

Resist Acineto rapid immunological test for the detection of acquired carbapenemase producers among Acinetobacter spp.

The Resist Acineto from Coris Bioconcept is a novel immunochromatographic test for detection of the major acquired carbapenemases (OXA-23, OXA-40, OXA-58, and NDM) identified in Acinetobacter spp. This rapid and easy-to-perform test showed an excellent specificity and sensitivity, with positive and negatives predictive values of 100% in both cases.

SERS-based rapid susceptibility testing of commonly administered antibiotics on clinically important bacteria species directly from blood culture of bacteremia patients.

Bloodstream infections are a growing public health concern due to emerging pathogens and increasing antimicrobial resistance. Rapid antibiotic susceptibility testing (AST) is urgently needed for timely and optimized choice of antibiotics, but current methods require days to obtain results. Here, we present a general AST protocol based on surface-enhanced Raman scattering (SERS-AST) for bacteremia caused by eight clinically relevant Gram-positive and Gram-negative pathogens treated with seven commonly administered antibiotics. Our results show that the SERS-AST protocol achieves a high level of agreement (96% for Gram-positive and 97% for Gram-negative bacteria) with the widely deployed VITEK 2 diagnostic system. The protocol requires only five hours to complete per blood-culture sample, making it a rapid and effective alternative to conventional methods. Our findings provide a solid foundation for the SERS-AST protocol as a promising approach to optimize the choice of antibiotics for specific bacteremia patients. This novel protocol has the potential to improve patient outcomes and reduce the spread of antibiotic resistance.

Evaluation of a novel Clostridioides difficile-selective growth broth under aerobic conditions.

A newly developed Clostridioides difficile-selective growth broth, which can be cultured under aerobic conditions, was found to have a sensitivity/specificity (98%/89%) comparable to conventional anaerobic culture methods. This might be a powerful tool for diagnosing Clostridioides difficile infection in resource-limited regions and health care settings in the future.

Impact of randomized blinded rechecking program on the performance of the AFB Microscopy Laboratory Network in Uganda: a decadal retrospective study.

BACKGROUND: Smear microscopy has remained the initial diagnostic test for presumptive tuberculosis (TB) patients in health facilities without the World Health Organization (WHO) recommended rapid diagnostic tools. In the Uganda TB laboratory network, the technique remains the only tool to monitor response to treatment among drug susceptible TB patients, with the country currently having over 1,600 microscopy TB testing units. It has been evidenced that acid-fast bacilli (AFB) microscopy's yield highly depends on the staining technique and reading ability of the laboratory personnel. For the quality of TB testing in the country, the TB control program set up a Randomized Blinded Rechecking (RBRC) program in 2008 to monitor the testing performance of laboratories to continuously improve the reliability and efficiency of results. This is the first study to determine the effectiveness and impact of the RBRC program on the performance of the participating laboratories in Uganda. METHODS: This was a retrospective cross-sectional study based on a record review of the RBRC's annual results compilations between January 2008 and December 2017. RESULTS: Between January 2008 and December 2017, a total of 265,523 smears were re-checked during the RBRC program. The number of enrolled laboratories in the RBRC program rose from 660 to 2008 to 1,406 in 2017. The RBRC program resulted in a statistically significant reduction in microscopy errors, with false positives decreasing from 12.8% to 2008 to 7.6% in 2017, false positive errors decreasing from 10 to 6.3%, false negative errors decreasing from 2.9 to 0.7%, quantification errors decreasing from 6.0 to 1.8%, and the overall sensitivity of smear microscopy compared to the controllers increased with statistical significance from 93 to 97%. CONCLUSION: The study reveals an overall significant error reduction and an improved sensitivity of smear microscopy upon continuous implementation of the RBRC program in an AFB microscopy TB laboratory network. Implementation of a RBRC program is crucial and essential to maintaining a reliable TB laboratory service that can facilitate accurate diagnosis and offset the disadvantages of using smear microscopy.

Evaluation of a Novel Benchtop Tool for Acceleration of Sample Preparation for MALDI-TOF Mass Spectrometry.

During the past decade, MALDI-TOF mass spectrometry (MS) has become a standard method for identification of bacteria and yeasts. Nonetheless, further optimization of the identification process is important to streamline workflows and save resources. This study evaluated the application of a multipurpose benchtop tool, MBT FAST Shuttle IVD, for accelerated drying of liquid assay components (matrix, formic acid, and/or sample) on a MALDI target. A total of 50 bacterial and fungal isolates were subjected to three different sample preparation procedures prior to the identification by MALDI-TOF MS: direct transfer (DT), extended direct transfer (eDT), and protein extraction (PE). Compared to conventional drying at room temperature, the preparation was performed with standardized heating of the MALDI target on the MBT FAST Shuttle. During DT, eDT, and PE, 56.7% (P < 0.001), 56.8% (P < 0.001), and 52.8% (P < 0.001) of time for matrix drying were saved by using the MBT FAST Shuttle, respectively. Applying the MBT FAST Shuttle, 57.5% (P < 0.001) of time for drying of formic acid were saved for eDT and 57.5% (P < 0.001) of time for sample drying were saved for PE. A significant improvement of the identification rates and scores was observed with MBT FAST Shuttle for eDT (P = 0.001) and PE (P = 0.008) methods, while the effect on identification quality for DT was not statistically significant (P = 0.16). In conclusion, the use of the MBT FAST Shuttle shortened the drying time of assay components by about a half for all preparation methods. Moreover, positive effect on identification success was observed.

Comparison of 3 diagnostic platforms for identification of bacteria and yeast from positive blood culture bottles.

Managing bloodstream infections requires fast and accurate diagnostics. Culture-based diagnostic methods for identification from positive blood culture require 24-hour subculture, potentially delaying time to appropriate therapy. Positive blood cultures were collected (n = 301) from September 2021 to August 2022 at the University of Maryland Medical Center. Platforms compared were BioFire® BCID2, Sepsityper®, and short-term culture. For monomicrobial cultures, FilmArray® BCID2 identified 88.3% (241/273) of pathogens. Rapid Sepsityper® identified 76.9% (210/273) of pathogens. Sepsityper® extraction identified 82.4% (225/273) of pathogens. Short-term culture identified 83.5% (228/273) of pathogens. For polymicrobial cultures, Sepsityper®, short-term culture, and BioFire® BCID2 had complete identifications at 10.7% (3/28), 0%, and 92.9% (26/28), respectively. Time-to-results for Rapid Sepsityper®, Sepsityper® extraction, BioFire® BCID2, and Short-term culture were 35, 52, 65, and 306 minutes, respectively. Performance of these platforms can reduce time-to-results and may help effectively treat bloodstream infections faster. Accuracy, time-to-result, and hands-on time are important factors when evaluation diagnostic platforms.

Shiga toxin-producing Escherichia coli (STEC) stool multiplex PCR can replace culture for clinical diagnosis and follow-up.

Shiga toxin (stx)-producing Escherichia coli (STEC) causes potentially severe gastrointestinal infections. Due to its public health importance, control measures are required, and carriers may need to refrain from work or daycare when the risk of spread to vulnerable people is high. We evaluated the use of direct stool multiplex PCR compared to culture for primary STEC diagnostics and for follow-up in order to update the national guidelines for STEC monitoring. We analyzed primary and follow-up samples of 236 STEC PCR-positive cases at HUSLAB, Helsinki, Finland in 2016-2017, altogether 858 samples. All STEC PCR-positive samples were inoculated on non-selective chromogenic agar plates. Culture positivity was confirmed from culture sweeps by PCR. 211 (89%) of the cases were culture positive in their primary sample. Of all primary and follow-up samples, 499 were PCR positive and of these 450 (90%) were culture positive. PCR-negative follow-up samples were available from 125 cases. Of these, 88 cases were followed for at least three consecutive PCR-negative samples. Two cases (2%) had culture-positive sample(s) after two consecutive PCR-negative samples. The median time for STEC clearance was 22-23 days. The laboratory-developed multiplex PCR test used in this study is a reliable method for STEC diagnostics and follow-up in a clinical laboratory. When non-selective methodology is used, the majority of PCR-positive samples (90%) are also culture positive. Furthermore, only two cases (2%) in our material had two consecutive PCR-negative samples followed by positive samples. Consequently, to demonstrate the clearance from STEC infection, we consider two PCR-negative follow-up samples sufficient. The Finnish national guidelines for STEC monitoring have been updated accordingly.

Recovery rates of mycobacterium from suspected extra-pulmonary tuberculosis patients using liquid culture at a tertiary referral centre of India.

Tuberculosis still remains a serious public health problem in developing countries. Rapid isolation of mycobacteria is critical for accurate diagnosis and management of tuberculosis. In the present study BACTEC MGIT 960 system was evaluated against Lowenstein Jensen (LJ) medium for isolation of mycobacteria from different extra-pulmonary specimens (N = 371). The samples were processed using NaOH-NALC method and inoculated in BACTEC MGIT and on LJ medium. The BACTEC MGIT 960 system detected 93 (25.06%) samples positive for acid fast bacilli and by LJ only 38 samples (10.24%) was positive. Furthermore, total 99 (26.68%) samples were detected positive by both the culture methods. The mean turnaround time to detection of mycobacteria by MGIT 960 were significantly less (12.4 days) as compared with LJ (22.76 days). In conclusion, BACTEC MGIT 960 system is more sensitive and rapid culture system for isolation of mycobacteria. However LJ culture method also suggested to further increase the detection rate of EPTB cases.

Proof of concept for multiplex detection of antibodies against Chlamydia species in chicken serum using a bead-based suspension array with peptides as antigens.

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

Evaluation of a new rapid immunochromatographic assay for the detection of GES-producing Gram-negative bacteria.

BACKGROUND: As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. OBJECTIVES: Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type ß-lactamases. METHODS: The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various ß-lactamases grown on Mueller-Hinton (MH) agar, chromogenic, and chromogenic/selective media. RESULTS: The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type ß-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. CONCLUSIONS: The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type ß-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters.

DropCarba - An automated magnetic digital microfluidic platform for rapid phenotypic testing of carbapenemase-producing Gram-negative bacilli.

Carbapenemase-producing Gram-negative bacilli (CPGNB) is a type of antibiotic-resistant pathogens that often lead to severe clinical consequences. Phenotypic tests, such as Carba NP and blue Carba, are able to detect the resistant mechanism and provide rapid detection of carbapenemase producers to potentially guide personalized therapy. However, these tests require relatively tedious hands-on fluidic operations, and the assay format is ill-suited for automation and parallelization for improved throughput. In this study, we report an automated magnetic digital microfluidics-based platform, known as DropCarba, for parallel CPGNB detection in droplets. It automates the entire carbapenemase testing process and eliminates the need for almost all hands-on fluidic operations, which ensures high consistency and minimizes human errors with a simple "press-and-go" operation. DropCarba was validated with a large number of bacterial isolates of various Enterobacterales species (200 strains in total with 100 CPGNB and 100 non-resistant strains) in a blinded manner, and the results agree well with the benchmark Carba NP. DropCarba, with its full automation, simple operation, reduced reagent consumption, parallelization processing, and scalable manufacturing, will greatly improve CPGNB screening and make a valuable contribution to our fight against antibiotic resistance.