Determination of clozapine and norclozapine in dried plasma spot and dried blood spot by liquid chromatography-tandem mass spectrometry.

The use of alternative blood sampling strategies in clozapine (CLZ) therapeutic drug monitoring (TDM) aims to facilitate collection and improve drug therapy and adherence. This study aimed to develop and validate two methods for the determination CLZ and norclozapine (NOR) in dried blood spots (DBS) and dried plasma spots (DPS) by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The analytes were extracted from one 10 microliter volumetric DBS disc punch and from one 6 mm DPS disc punch with methyl tert-butyl ether: methanol (1:9, v/v) and injected into the HPLC-MS/MS with Atmospheric pressure chemical ionization (APCI) source. Separation was performed in a phenyl column, with mobile phase ammonium formate 1 mM pH 4.0 with methanol in gradient mode. The method was linear from 50 to 1500 ng/ml (r > 0.99), with accuracy between 98% and 105% in DBS and 91-101% in DPS, and intra- and inter-assay CV% from 5.23% to 9.35% in DBS and 2.22-11.36% in DPS for both analytes. The matrix effect was compensated by the internal standard, between - 5.1-6.89% in DBS and - 2.45-5.74% in DPS. The average extraction efficiency was 63-67% for CLZ and 58-69% for NOR with no significant impact of hematocrit (HCT). The analytes were stable in the dried matrices stored up to 42 °C for 26 days. The method was applied in the evaluation of clozapine therapy in 13 schizophrenic patients with mean serum levels of 401 ng/ml (43-914 ng/ml). Only 38% were within the therapeutic range, 46% below and 23% above. CLZ and NOR concentrations in dried samples were highly correlated to serum levels, with greater accuracy for DPS compared to DBS (97 versus 89%, and 99 versus 131%, for CLZ and NOR, respectively). Our data support the use of DBS and DPS as alternative sampling strategies in CLZ therapeutic drug monitoring, with satisfactory performance and logistics advantages.

A four-year follow-up survey of antimicrobial resistance among Acinetobacter baumannii complex from inpatients in Southern Brazil.

OBJECTIVES: To determine the trends of antimicrobial resistance for Acinetobacter baumannii complex isolates recovered from inpatients over a 4-year follow-up survey. METHODS: A total of 659 A baumannii complex isolates were recovered from hospitalized patients in Porto Alegre and its metropolitan area, Southern Brazil, from 2017 to 2020. Susceptibility profile was determined for ampicillin/sulbactam, amikacin, gentamicin, imipenem, meropenem, minocycline, polymyxin B and tigecycline. RESULTS: Overall, PMB was the most active agent against the set of A baumannii isolates over the period. Although stable, a high resistance rate was observed. CONCLUSIONS: Our results shown the presence of an extensively-drug resistant A baumannii complex isolates over the past four years. Polymyxin B has been the only antimicrobial agent that remain with a good in vitro activity. Strict surveillance and infection control measures are mandatory.

Impact of the blue-carba rapid test for carbapenemase detection on turnaround time for an early therapy against Pseudomonas aeruginosa.

BACKGROUND: To determine the turnaround time from a blue-carba result until a final microbiological report (bacterial identification plus antimicrobial susceptibility profile) and to infer the impact of an early therapeutic intervention based on the blue-carba results. METHODS: Pseudomonas aeruginosa isolates were recovered from hospitalized patients from Porto Alegre, Brazil, and tested by blue-carba test. Time required for a blue-carba result, right after the sample processing, was compared with those required to get final report (specie identification and antimicrobial susceptibility profile) Isolates blue-carba positive were tested by phenotypically and genotypically for Klebsiella pneumoniae carbapenemase and metallo-ß-lactamase genes. RESULTS: A total of 199 isolates were analyzed and 23 (11.6%) were blue-carba positive and harboring the blaSPM-1-like gene. Fifty-two (26.1%) isolates were blue-carba negative but resistant to meropenem and/or imipenem. Polymyxin B and ceftolozane/tazobactam (this latter except for SPM-1 producers) were 100% active for all P. aeruginosa isolates, a blue-carba test allow an earlier intervention or adequacy of therapy. CONCLUSIONS: Early adequacy can be promoted by blue-carba test for 11.6% of SPM-1-producing P. aeruginosa isolates, polymyxin B could be prior associated and ceftolozane/tazobactam withdrawn from therapy. For the remaining isolates, empirical therapy involving ceftolozane/tazobactam can be maintained with greater likelihood of adequacy. An active communication between laboratory and clinical services is necessary to better explore these earlier blue-carba results, significantly reducing the time for a first intervention.

Evaluation of a polymyxin drop test for polymyxin resistance detection among non-fermentative gram-negative rods and enterobacterales resistant to carbapenems.

To assess the performance of the drop test for polymyxin B resistance detection among Enterobacterales and non-fermentative gram-negative rods resistant to carbapenems. Seven hundred and fifteen carbapenem-resistant isolates were tested: 628 Enterobacterales species and 87 non-fermentative gram-negative rods. For the polymyxin drop test, concentrations range from 0.25 to 8.0 µg/mL. Broth microdilution, as gold standard, was performed using in-house-prepared panels and interpreted according to the CLSI guidelines. Results were interpreted in terms of categorical agreements and discrepancies. Accuracy for a drop of polymyxin B at 2.0, 4.0 and 8.0 was calculated as better cutoff for resistance determination. No very major error was observed among all isolates, and 95.5% of agreement was observed among Enterobacterales, particularly for Klebsiella pneumoniae. A higher accuracy (95.1%) was obtained when a single drop of polymyxin B at 4.0 µg/mL was applied. Polymyxin drop test presented >95% of categorical agreement, without very major errors, for KPC-producing K. pneumoniae isolates. An accuracy of 95.1% was obtained with a single drop at 4.0 µg/mL polymyxin B. Polymyxin B drop is an easy and feasible test and may allow a reduction on the turnaround time for polymyxin resistance detection and impacting on early implementation of accurate therapeutic interventions.