Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy.

Background: Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Methods: Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. Results: We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. Conclusions: We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining.

Multiple stains have been historically utilized in electron microscopy to provide proper contrast and superior image quality enabling the discovery of ultrastructures. However, the use of these stains in microbiological viability assessment has been limited. Phosphotungstic acid (PTA) staining is a common negative stain used in scanning electron microscopy (SEM). Here, we investigate the feasibility of a new SEM-PTA assay, aiming to determine both viable and dead microbes. The optimal sample preparation was established by staining bacteria with different PTA concentrations and incubation times. Once the assay conditions were set, we applied the protocol to various samples, evaluating bacterial viability under different conditions, and comparing SEM-PTA results to culture. The five minutes 10% PTA staining exhibited a strong distinction between viable micro-organisms perceived as hypo-dense, and dead micro-organisms displaying intense internal staining which was confirmed by high Tungsten (W) peak on the EDX spectra. SEM-PTA viability count after freezing, freeze-drying, or oxygen exposure, were concordant with culture. To our knowledge, this study is the first contribution towards PTA staining of live and dead bacteria. The SEM-PTA strategy demonstrated the feasibility of a rapid, cost-effective and efficient viability assay, presenting an open-view of the sample, and providing a potentially valuable tool for applications in microbiome investigations and antimicrobial susceptibility testing.

Observational study of factors associated with morbidity and mortality from COVID-19 in Lebanon, 2020-2021.

BACKGROUND: The COVID-19 pandemic claimed millions of lives worldwide without clear signs of abating despite several mitigation efforts and vaccination campaigns. There have been tremendous interests in understanding the etiology of the disease particularly in what makes it severe and fatal in certain patients. Studies have shown that COVID-19 patients with kidney injury on admission were more likely to develop severe disease, and acute kidney disease was associated with high mortality in COVID-19 hospitalized patients. METHODS: This study investigated 819 COVID-19 patients admitted between January 2020-April 2021 to the COVID-19 ward at a tertiary care center in Lebanon and evaluated their vital signs and biomarkers while probing for two main outcomes: intubation and fatality. Logistic and Cox regressions were performed to investigate the association between clinical and metabolic variables and disease outcomes, mainly intubation and mortality. Times were defined in terms of admission and discharge/fatality for COVID-19, with no other exclusions. RESULTS: Regression analysis revealed that the following are independent risk factors for both intubation and fatality respectively: diabetes (p = 0.021 and p = 0.04), being overweight (p = 0.021 and p = 0.072), chronic kidney disease (p = 0.045 and p = 0.001), and gender (p = 0.016 and p = 0.114). Further, shortness of breath (p<0.001), age (p<0.001) and being overweight (p = 0.014) associated with intubation, while fatality with shortness of breath (p<0.001) in our group of patients. Elevated level of serum creatinine was the highest factor associated with fatality (p = 0.002), while both white blood count (p<0.001) and serum glutamic-oxaloacetic transaminase levels (p<0.001) emerged as independent risk factors for intubation. CONCLUSIONS: Collectively our data show that high creatinine levels were significantly associated with fatality in our COVID-19 study patients, underscoring the importance of kidney function as a main modulator of SARS-CoV-2 morbidity and favor a careful and proactive management of patients with elevated creatinine levels on admission.

A challenging modified measles outbreak in vaccinated healthcare providers.

BACKGROUND: Modified measles is rarely reported and thought to be an attenuated, less transmissible form of measles. The occupational safety and management of previously immunized healthcare providers (HCP) facing the global reemergence of measles is controversial and unclear.Aim: We report a measles outbreak with an unusual presentation among our vaccinated HCP at Saint George Hospital University Medical Center (SGHUMC) in Lebanon that occurred during a nationwide measles epidemic. METHODS: We recorded cases at SGHUMC, a 333-bed tertiary-care center, from April 2018 to June 2018. We established a measles clinic for investigating all febrile patients. HCP exposure was linked to influx of index cases through our Emergency Department. Modified measles was defined as any variation in the classic presentation with a pinpoint/vesicular rash, documented exposure and evidence of prior immunity. We performed serology testing to diagnose and/or document immunity and implemented outbreak controls measures including PPE, airborne isolation, and mass notification. FINDINGS: We diagnosed 8 inpatients with classic measles, and 9 affected HCP. We diagnosed 8 HCP with modified measles. One previously immunized HCP developed classic measles despite being immunized and having a positive IgG titer. Our contact tracing revealed a total of 96 exposed HCP with 27 HCP showing non-specific signs of viral illness. We required all the 9 affected HCP to undergo home isolation. CONCLUSION: We believe it is a top priority to achieve adequate measles immunity, especially among HCP that are at the frontline of healthcare systems. This necessitates revisiting vaccination schedules and achieving seroprotective titers to reclaim proper herd immunity.

Trends of Multidrug-Resistant Pathogens, Difficult to Treat Bloodstream Infections, and Antimicrobial Consumption at a Tertiary Care Center in Lebanon from 2015-2020: COVID-19 Aftermath.

INTRODUCTION: We studied the trend of antimicrobial resistance and consumption at Saint George Hospital University Medical Center (SGHUMC), a tertiary care center in Beirut, Lebanon, with a focus on the SARS-CoV-2 pandemic. MATERIALS AND METHODS: We calculated the isolation density/1000 patient-days (PD) of the most isolated organisms from 1 January 2015-31 December 2020 that included: E. coli (Eco), K. pneumoniae (Kp), P. aeruginosa (Pae), A. baumannii (Ab), S. aureus (Sau), and E. faecium (Efm). We considered March-December 2020 a surrogate of COVID-19. We considered one culture/patient for each antimicrobial susceptibility and excluded Staphylococcus epidermidis, Staphylococcus coagulase-negative, and Corynebacterium species. We analyzed the trends of the overall isolates, the antimicrobial susceptibilities of blood isolates (BSI), difficult-to-treat (DTR) BSI, carbapenem-resistant Enterobacteriaceae (CRE) BSI, and restricted antimicrobial consumption as daily-defined-dose/1000 PD. DTR implies resistance to carbapenems, beta-lactams, fluoroquinolones, and additional antimicrobials where applicable. RESULTS AND DISCUSSION: After applying exclusion criteria, we analyzed 1614 blood cultures out of 8314 cultures. We isolated 85 species, most commonly Eco, at 52%. The isolation density of total BSI in 2020 decreased by 16%: 82 patients were spared from bacteremia, with 13 being DTR. The isolation density of CRE BSI/1000 PD decreased by 64% from 2019 to 2020, while VREfm BSI decreased by 34%. There was a significant decrease of 80% in Ab isolates (p-value < 0.0001). During COVID-19, restricted antimicrobial consumption decreased to 175 DDD/1000 PD (p-value < 0.0001). Total carbapenem consumption persistently decreased by 71.2% from 108DDD/1000 PD in 2015-2019 to 31 DDD/1000 PD in 2020. At SGHUMC, existing epidemics were not worsened by the pandemic. We attribute this to our unique and dynamic collaboration of antimicrobial stewardship, infection prevention and control, and infectious disease consultation.

Carbapenem resistant organisms: A 9-year surveillance and trends at Saint George University Medical Center.

INTRODUCTION: Carbapenem resistant organisms (CRO) constitute a large group of bacteria with different mechanisms of resistance and recently increasing global incidence. This rise has ambiguous dynamics and essential local epidemiologic data is lacking. MATERIALS AND METHODS: In this retrospective study at the 400-bed Saint George Hospital (SGH) in Beirut, Lebanon, we retrieved electronic laboratory records of all intrinsic and acquired CRO isolates from January 1, 2010 until June 30, 2018. Isolation density was calculated as: number of isolates/1000PD. Analysis carried out using WHOnet with a trend time series analysis. RESULTS: During the study period, a total of 2150 non-duplicate CRO were isolated. While Acinetobacter baumanii (AB), Pseudomonas aeruginosa (CRPa), and Stenotrophomonas maltophilia (Sm) constituted 85% of total CRO in the study period, the carbapenem resistant enterobacteriaceae (CRE) rose from few sporadic cases before 2016 to a solid 32% of total CRO in 2018. Our most concrete trends were as follows. The rate of AB bacteremia was at an average of 0.114/1000 PD from 2011 to 2014. In 2015, a sudden doubling of AB bacteremia to 0.23/1000 PD. In 2017, there was a significant decrease to 0.113/1,000PD (p < 0.0001) to reach 0.097/1000PD in 2018 with a continuously declining trend. The peak of Sm bacteremia was in 2016 at 0.121/1000PD after which it significantly decreased by 21% in 2017 to disappear in 2018 (p < 0.0001). There were no significant trends observed in the isolation density of the CRPa group from 2010 until June 2018. Klebsiella pneumonia (CRKp) bacteremia was isolated first in 2013, then in 2016 and continued to rise (p = 0.028). In 2017, carbapenem resistant KP bacteremia rate doubled to 0.05/1,000PD from 0.024/1000 (p = 0.0139). CONCLUSION: In conclusion, this 9-year study at SGH depicts the major trends and dynamics of local CRO isolation, mainly A. baumanii, P. aeruginosa and CRKp. Further efforts are warranted both locally and internationally for a richer understanding of this trend. Bearing in mind that understanding antimicrobial resistance is a complex, multifaceted process that is only feasible when all its aspects are combined: molecular, phenotypic and clinical.