Retrospective validation of MetaSystems' deep-learning-based digital microscopy platform with assistance compared to manual fluorescence microscopy for detection of mycobacteria.

This study aimed to validate Metasystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module (Neon Metafer) with assistance on respiratory and pleural samples, compared to conventional manual fluorescence microscopy (MM). Analytical parameters were assessed first, followed by a retrospective validation study. In all, 320 archived auramine-O-stained slides selected non-consecutively [85 originally reported as AFB-smear-positive, 235 AFB-smear-negative slides; with an overall mycobacterial culture positivity rate of 24.1% (77/320)] underwent whole-slide imaging and were analyzed by the Metafer Neon AFB Module (version 4.3.130) using a predetermined probability threshold (PT) for AFB detection of 96%. Digital slides were then examined by a trained reviewer blinded to previous AFB smear and culture results, for the final interpretation of assisted digital microscopy (a-DM). Paired results from both microscopic methods were compared to mycobacterial culture. A scanning failure rate of 10.6% (34/320) was observed, leaving 286 slides for analysis. After discrepant analysis, concordance, positive and negative agreements were 95.5% (95%CI, 92.4%-97.6%), 96.2% (95%CI, 89.2%-99.2%), and 95.2% (95%CI, 91.3%-97.7%), respectively. Using mycobacterial culture as reference standard, a-DM and MM had comparable sensitivities: 90.7% (95%CI, 81.7%-96.2%) versus 92.0% (95%CI, 83.4%-97.0%) (P-value = 1.00); while their specificities differed 91.9% (95%CI, 87.4%-95.2%) versus 95.7% (95%CI, 92.1%-98.0%), respectively (P-value = 0.03). Using a PT of 96%, MetaSystems' platform shows acceptable performance. With a national laboratory staff shortage and a local low mycobacterial infection rate, this instrument when combined with culture, can reliably triage-negative AFB-smear respiratory slides and identify positive slides requiring manual confirmation and semi-quantification. IMPORTANCE: This manuscript presents a full validation of MetaSystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module using a probability threshold of 96% including accuracy, precision studies, and evaluation of limit of AFB detection on respiratory samples when the technology is used with assistance. This study is complementary to the conversation started by Tomasello et al. on the use of image analysis artificial intelligence software in routine mycobacterial diagnostic activities within the context of high-throughput laboratories with low incidence of tuberculosis.

Approach to Assessment of New Swabs and Viral Transport Media for SARS-CoV-2 Testing.

In light of the present pandemic of novel coronavirus disease 2019 (COVID-19) and the unprecedented high demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing worldwide, there are shortages of established specimen collection devices for respiratory viral testing for diagnostic microbiology laboratories. This creates the need to validate unverified collection devices from manufacturers that may not be a registered supplier for medical devices. As clinical laboratories do not routinely perform quality control of established collection devices, there is a need to have a systematic, robust approach to the assessment of substitute unregistered collection swabs and viral transport media (VTM). A discussion of the aspects requiring consideration when determining the suitability and implementation of new collection devices is presented. These specific assessment criteria include an inspection of device integrity, determination of swab and VTM sterility and in vitro performance, VTM stability, and examination of the clinical performance of the device. This method was used in a front-line medical microbiology laboratory on swabs and VTM from an unregistered manufacturer, with suboptimal results that precluded implementation. As the pandemic continues, it will be important for diagnostic laboratories to adopt a flexible and streamlined approach to maintaining adequate supply chains for testing reagents and materials.

Potential influence of rapid diagnostics on timeliness of management decisions for patients with positive blood cultures.

Background: The Accelerate Pheno system (AXDX) provides rapid identification (ID; 90 minutes) and antimicrobial susceptibility testing (AST; approximately 7 hours) from positive blood culture (BC) bottles. We assessed the potential of AXDX results to influence more timely antibiotic interventions with a convenience sample of 158 positive BCs. Methods: BCs with a mono-microbial Gram stain likely to be on the AXDX panel were run in parallel with the standard of care (SOC). Using results from the SOC, the medical microbiologist on call (MMOC) noted interventions made at the time of BC Gram stain and when ID and AST results were available. The timing of MMOC intervention was noted and compared with fastest potential SOC time and AXDX time. Results: Of 158 specimens selected for analysis, 144 were evaluable. ID was available 11.9 hours and AST 27.7 hours faster than SOC. Correct ID was provided for 85.2% of specimens and AST for 59.0% of specimens, with 97.5% essential agreement compared with the SOC. One hundred and thirteen clinical interventions were made on 100 specimens: 54.9% were narrowing; 33.6%, escalation; 6.2%, consultation with ID; and 3.5%, further investigation. If AXDX data had been used immediately once available, interventions would have been possible 24 hours earlier for ID interventions and 39 hours earlier for AST results. Conclusions: Results from rapid diagnostic panels such as AXDX have the potential to support timely antimicrobial de-escalation and other decisions to benefit patients, especially if paired with stewardship interventions.

Historique: Le système Accelerate Pheno (AXDX) permet de procéder à une identification rapide (ID; 90 minutes) et à des tests de susceptibilité antimicrobienne (AST; environ sept heures) à partir de bouteilles d'hémoculture (BH) positives. À l'aide d'un échantillon de commodité de 158 BH positives, les auteurs ont évalué le potentiel de résultats du système AXDX pour favoriser des interventions antibiotiques plus opportunes. Méthodologie: Les auteurs ont comparé les BH présentant une coloration de Gram monomicrobienne susceptible de se trouver sur le panel AXDX avec la norme de soins (NdS). À l'aide des résultats de la NdS, le microbiologiste médical sur appel (MMSA) a consigné les interventions effectuées au moment de la coloration de Gram de la BH et lorsque les résultats de l'ID et de l'AST étaient disponibles. Le moment de l'intervention du MMSA était consigné et comparé avec la durée de la NdS au potentiel le plus rapide et la durée de l'AXDX. Résultats: Des 158 échantillons sélectionnés en vue d'être analysés, 144 étaient évaluables. L'ID était disponible 11,9 heures et l'AST, 27,7 heures plus rapidement que la NdS. L'ID exacte était fournie pour 85,2 % des échantillons et l'AST exacte, pour 59,0 % des échantillons, selon une entente essentielle de 97,5 % par rapport à la NdS. Cent treize interventions ont été effectuées sur 100 échantillons : 54,9 % visaient à réduire le spectre, 33,6 %, à accroître la médication, 6,2 %, à demander une consultation avec l'ID et 3,5 %, à obtenir des explorations plus approfondies. Si les données de l'AXDX avaient été utilisées dès l'obtention des résultats, il aurait été possible d'agir 24 heures plus rapidement pour les interventions d'ID et 39 heures plus rapidement pour les résultats de l'AST. Conclusions: Les résultats des panels diagnostiques rapides comme l'AXDX ont le potentiel de favoriser une désescalade antimicrobienne et d'autres décisions au profit des patients, surtout s'ils s'associent à des interventions de gestion.

Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport.

We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions.

Performance assessment of urine flow cytometry (UFC) to screen urines to reflex to culture in immunocompetent and immunosuppressed hosts.

PURPOSE: Urine flow cytometry (UFC) is an automated method to quantify bacterial and white blood cell (WBC) counts. We aimed to determine whether a threshold for these parameters can be set to use UFC as a sensitive screen to predict which urine samples will subsequently grow in culture. METHODOLOGY: Urines submitted to our microbiology laboratory at a tertiary care centre from 22 July 2015-17 February 2016 underwent UFC (Sysmex UF-1000i) analysis, regular urinalysis and urine culture. Positive urine cultures were defined as growth ≥104 c.f.u. ml-1 of organisms associated with urinary tract infections. The correlation of UFC bacterial and WBC counts with urine culture was assessed using receiver operating characteristics curves. The sensitivity (SN), specificity (SP), negative predictive values (NPVs), positive predictive values (PPVs) and false negative rate (FNR) were calculated at various thresholds in immunocompetent and immunosuppressed patients. RESULTS: A total of 15 046 urine specimens were submitted, of which 14 908 were analysable in the study. The average time to UFC result from receipt in the laboratory was 0.76 h (+/-1.04). The test performance at a set threshold of UFC bacteria ≥20 or WBC >5 was: SN=96.0 %, SP=39.2 %, PPV=47.0 %, NPV=94.5 % and FNR=4.0 %. This threshold eliminates 26 % of urine cultures. Immunosuppressed hosts had a lower sensitivity of 90.6 % and a higher FNR of 9.4 %. CONCLUSIONS: UFC is a rapid and sensitive method to screen out urine samples that will subsequently be negative and to reflex urines to culture that will subsequently grow. UFC results are available within 1 h from receipt and enable the elimination of culture when the set threshold is not met.

Certolizumab pegol does not bind the neonatal Fc receptor (FcRn): Consequences for FcRn-mediated in vitro transcytosis and ex vivo human placental transfer.

Antibodies to tumor necrosis factor (anti-TNF) are used to treat inflammatory diseases, which often affect women of childbearing age. The active transfer of these antibodies across the placenta by binding of the Fc-region to the neonatal Fc receptor (FcRn) may result in adverse fetal or neonatal effects. In contrast to other anti-TNFs, certolizumab pegol lacks an Fc-region. The objective of this study was to determine whether the structure of certolizumab pegol limits active placental transfer. Binding affinities of certolizumab pegol, infliximab, adalimumab and etanercept to human FcRn and FcRn-mediated transcytosis were determined using in vitro assays. Human placentas were perfused ex vivo to measure transfer of certolizumab pegol and positive control anti-D IgG from the maternal to fetal circulation. FcRn binding affinity (KD) was 132nM, 225nM and 1500nM for infliximab, adalimumab and etanercept, respectively. There was no measurable certolizumab pegol binding affinity, similar to that of the negative control. FcRn-mediated transcytosis across a cell layer (mean±SD; n=3) was 249.6±25.0 (infliximab), 159.0±20.2 (adalimumab) and 81.3±13.1ng/mL (etanercept). Certolizumab pegol transcytosis (3.2±3.4ng/mL) was less than the negative control antibody (5.9±4.6ng/mL). No measurable transfer of certolizumab pegol from the maternal to the fetal circulation was observed in 5 out of 6 placentas that demonstrated positive-control IgG transport in the ex vivo perfusion model. Together these results support the hypothesis that the unique structure of certolizumab pegol limits its transfer through the placenta to the fetus and may be responsible for previously reported differences in transfer of other anti-TNFs from mother to fetus.