Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis.

Among emerging non-albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, and UPC2 Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.

Multidrug Transporters and Alterations in Sterol Biosynthesis Contribute to Azole Antifungal Resistance in Candida parapsilosis.

While much is known concerning azole resistance in Candida albicans, considerably less is understood about Candida parapsilosis, an emerging species of Candida with clinical relevance. We conducted a comprehensive analysis of azole resistance in a collection of resistant C. parapsilosis clinical isolates in order to determine which genes might play a role in this process within this species. We examined the relative expression of the putative drug transporter genes CDR1 and MDR1 and that of ERG11. In isolates overexpressing these genes, we sequenced the genes encoding their presumed transcriptional regulators, TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3 and ERG11 in these isolates to find mutations that might contribute to this phenotype in this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and Mdr1 contribute directly to azole resistance and suggest that their overexpression is due to activating mutations in the genes encoding their transcriptional regulators. We also observed that the Y132F substitution in ERG11 is the only substitution occurring exclusively among azole-resistant isolates, and we correlated this with specific changes in sterol biosynthesis. Finally, sterol analysis of these isolates suggests that other changes in sterol biosynthesis may contribute to azole resistance in C. parapsilosis.

Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus.

PURPOSE: Systemic lupus erythematous (SLE) is a systemic autoimmune inflammatory disease with both genetic and epigenetic etiologies. Evidence suggests that deregulation of specific genes through epigenetic mechanisms may be a contributing factor to SLE pathology. There is increasing evidence that DNA methyltransferase activity may be involved. This study demonstrated modulation in expression of DNA methyltransferases (DNMTs) according to ethnicity in patients diagnosed with SLE. Furthermore, differential expression in one of the DNMTs was found in a subset of lupus patients on dehydroepiandrosterone (DHEA) therapy. METHODS: Real-time PCR analyses of DNMT1, DNMT3A and DNMT3B in peripheral blood mononuclear cells from a cohort of African American and European American lupus and non-lupus women were conducted. Also, global DNA methylation was assessed using the MethylFlash(TM) methylated quantification colorimetric assay. RESULTS: Significant increase in DNMT3A (p < 0.001) was shown in lupus patients when compared to age-matched healthy controls. This increase was associated with a higher SLEDI index. More striking was that expression levels for African American (AA) women were higher than European American women in the lupus populations. A subset of AA women on DHEA therapy showed a significant decrease (p < 0.05) in DNMT3A expression in comparison to lupus patients not on the therapy. DHEA is an androgenic steroid found in low levels in the serum of lupus patients. Supplementation of this hormone has been shown to be beneficial to some lupus patients. DHEA was not shown to effect DNMT1 or DNMT3B expression. Increased expression was also noted in DNMT3B (p < 0.05) in lupus patients compared to age-matched healthy controls. However, no significant difference was noted in DNMT1 (p = 0.2148) expression between lupus patients and healthy controls. Although increases were detected in de novo methyltransferases, a global decrease (p < 0.001) in 5-methycytosine was observed in lupus patients when compared to age-matched healthy controls. CONCLUSION: These findings suggest that epigenetic changes may play a critical role in the manifestations of the disease observed among ethnic groups, particularly African American women who often have a higher incidence of lupus. DHEA therapy effects on DNMT3A expression in AA women warrant further investigation in a larger population.

Beta-lactam target attainment and associated outcomes in patients with bloodstream infections.

OBJECTIVES: To evaluate the association between early and cumulative beta-lactam pharmacokinetic/pharmacodynamic (PK/PD) parameters and therapy outcomes in bloodstream infection (BSI). METHODS: Adult patients who received cefepime, meropenem, or piperacillin/tazobactam for BSI and had concentrations measured were included. Beta-lactam exposure was generated and the time that free concentration remained above the minimum inhibitory concentration (fT>MIC) and four multiples of MIC (fT>4 × MIC) were calculated for times 0-24 h and 0-7 days of therapy. Multiple regression analysis was performed to evaluate the impact of PK/PD on microbiological and clinical outcomes. RESULTS: A total of 204 patients and 213 BSI episodes were included. The mean age was 58 years and weight 83 kg. Age, Sequential Organ Failure Assessment (SOFA) score, haemodialysis, Pitt bacteraemia score, and hours of empiric antibiotic therapy were significantly associated with certain outcomes and retained in the final model. In multiple regression analysis, fT>4 × MIC at 0-24 h and 0-7 days was a significant predictor of negative blood culture on day 7 (P=0.0161 and 0.0068, respectively). In the time-to-event analysis, patients who achieved 100% fT>4 × MIC at 0-24 h and 0-7 days had a shorter time to negative blood culture compared with those who did not (log-rank P=0.0004 and 0.0014, respectively). No significant associations were identified between PK/PD parameters and other outcomes, including improvement in symptoms at day 7 and 30-day mortality. CONCLUSION: Early and cumulative achievement of fT>4 × MIC was a significant predictor of microbiological outcome in patients with BSI.

Cefepime Daily Exposure and the Associated Impact on the Change in Sequential Organ Failure Assessment Scores and Vasopressors Requirement in Critically Ill Patients Using Repeated-Measures Mixed-Effect Modeling.

IMPORTANCE: Sepsis and septic shock are major healthcare problems that need early and appropriate management. OBJECTIVES: To evaluate the association of daily cefepime pharmacokinetic/pharmacodynamic (PK/PD) parameters with change in Sequential Organ Failure Assessment (SOFA) score and vasopressors requirement. DESIGN SETTING AND PARTICIPANTS: This is a retrospective study. Adult ICU patients who received cefepime for Gram-negative pneumonia or bloodstream infection (BSI) and had cefepime concentrations measured were included. Daily cefepime exposure was generated and PK/PD parameters calculated for patients. Repeated-measures mixed-effect modeling was used to evaluate the impact of PK/PD on the outcomes. MAIN OUTCOMES AND MEASURES: Change in daily SOFA score and vasopressors requirement. RESULTS: A total of 394 and 207 patients were included in the SOFA and vasopressors analyses, respectively. The mean (±sd) age was 55 years (19) and weight 81 kg (29). For the change in SOFA score, daily SOFA score, mechanical ventilation, renal replacement therapy, and number of vasopressors were included. In the vasopressors analysis, daily SOFA score, day of therapy, and hydrocortisone dose were significant covariates in the final model. Achieving cefepime concentrations above the minimum inhibitory concentration (MIC) (T>MIC) for 100% of the dosing interval was associated with 0.006 µg/kg/min decrease in norepinephrine-equivalent dose. Cefepime PK/PD did not have an impact on the daily change in SOFA score. CONCLUSIONS AND RELEVANCE: Achieving 100% T>MIC was associated with negligible decrease in vasopressors requirement in ICU patients with Gram-negative pneumonia and BSI. There was no impact on the change in SOFA score.

A waterfall of susceptibility results: impact of microbiology testing cascade for MDROs.

Cascade reporting is an antimicrobial stewardship strategy which selectively reports broad-spectrum antibiotic susceptibility when narrower-spectrum agents are resistant. Three regional laboratories, which provide microbiology services for HCA Healthcare hospitals in Florida, implemented a standardized antibiotic testing cascade for multi-drug resistant organisms. This study evaluated the impact of the testing cascade on time to susceptibility results for carbapenem-resistant Gram-negative organisms. Appropriateness of manual susceptibility test selection was also assessed. Compliance with the testing cascade was 56%. Appropriateness of manual tests in the pre- and post-intervention groups was 87% (257/297) and 96% (310/323), respectively (P < 0.0001). Median time to first manual test result was 23.1 hours (IQR: 19.8-25.2 hours) in the pre-intervention group and 23.9 hours (IQR: 20.2-27.0 hours) in the post-intervention group (P = 0.11). Implementation of a testing cascade did not result in faster manual susceptibility results, however there was a significant increase in testing appropriateness.

COVID-19: The Culprit, the People and Lessons Learned.

Description COVID-19 has had a palpable impact on everyone from losing jobs to losing loved ones. It has altered our social dynamics and disturbed the world economy. We should all learn something from this challenging time. This article elaborates on three lessons learned by two brothers who grew up in Rwanda right after the 1994 genocide against the Tutsi, where more than one million people died in 100 days. One, Dr. Kayihura Manigaba, is currently responding to the COVID-19 pandemic as a clinical pharmacy manager and as an infectious diseases pharmacy specialist at a hospital in Florida, U.S, and the other, Mukundwa Gael, is finishing his pharmacy doctorate training in Tennessee, U.S. Every year, on average, three new human pathogen species are discovered in different geographical locations, and the majority of them are viruses. This implies that we will continue to face crisis secondary to a new microbe, and we have no idea where it will begin and whether the community of origin will be capable of containing it from reaching the rest of the world. In this paper, the authors emphasize the importance of collaboration in identifying, preventing, and treating health threats. In addition to sharing lessons such as the impact of fear on crisis response, and how to rise to the occasion during a crisis, the authors intend to motivate readers to believe in their unique skills that are needed to advance our society as we define our new world during and post COVID-19 pandemic.

Remdesivir-Warfarin Interaction: A Case Report.

Description A greater than 65-year-old Caucasian woman receiving long-term anticoagulation with warfarin for atrial fibrillation experienced a sudden rise in an international normalized ratio (INR) after she was started on remdesivir for management of 2019 Novel Coronavirus (COVID-19). Patient INR was maintained within the target therapeutic range of 2-3 with a warfarin dose of 11 mg/week before starting remdesivir. After 2 days of remdesivir therapy, the patient's INR increased significantly and remained elevated during the 5 day course of remdesivir therapy. Patient required an interruption of her warfarin therapy for 7 days, and her INR did not return to the targeted therapeutic INR range of 2-3 until day 5 from the last dose of remdesivir, despite no warfarin administration. A comprehensive PubMed/MEDLINE search did not find published literature documenting interaction between warfarin and remdesivir. We describe the first case report, to our knowledge, documenting a potential drug interaction between warfarin and remdesivir. The authors found that there is a probable interaction between warfarin and remdesivir when applying the Adverse Drug Reaction Probability Scale, Naranjo Scale. To reduce the risk of bleeding associated with excessive anticoagulation, clinicians should closely monitor INR, and adjust the warfarin dose accordingly when patients are receiving remdesivir and warfarin concomitantly.

Training a Drug to Do New Tricks: Insights on Stability of Meropenem Administered as a Continuous Infusion.

BACKGROUND: The antibiotic armamentarium used to combat multi-drug resistant organisms (MDROs) include carbapenems. Continuous infusion (CI) dosing is frequently employed to maximize beta-lactam efficacy; however, use of meropenem CI has been limited due to concerns with product instability. OBJECTIVE: The primary objective of this study was to quantify meropenem serum concentrations to reflect drug stability when administered as CI over 8- or 12-h exchanges. In addition, a stability experiment was performed to further establish meropenem integrity over 12 h. The secondary objectives were to assess the ability of meropenem to achieve target pharmacokinetic/pharmacodynamic (PK/PD) exposures relative to the minimum inhibitory concentration (MIC) of the pathogen, and to determine clinical cure. METHODS: This was a retrospective, observational study on use of CI meropenem (infused either over 8- or 12- h) at a 1% concentration. The stability experiment was conducted on 1% meropenem at room temperature. RESULTS: In 22 patients, a median meropenem daily dose of 6 g/day (range 2-6 g/day) resulted in a median serum concentration of 17.8 mg/L (interquartile range, 9.3-27.8 mg/L). In 95% of cases, meropenem delivered as CI resulted in free drug concentrations at or above the MIC of the pathogen for the entire dosing interval. Clinical cure was achieved in 80% of patients included in this review. The stability experiment revealed negligible drug degradation at the end of the 12-h dosing interval. CONCLUSIONS: The data from this study provides compelling evidence for the use of meropenem as CI utilizing either a 12- or 8-h exchange process.