Correlation of Trichosporon asahii Genotypes with Anatomical Sites and Antifungal Susceptibility Profiles: Data Analyses from 284 Isolates Collected in the Last 22 Years across 24 Medical Centers.

Trichosporon asahii is an opportunistic fungal pathogen that can cause severe infections with high mortality rates. Azole derivatives are the best-targeted therapy for T. asahii invasive infections, but azole-resistant isolates have been reported. To investigate peculiarities in the antifungal susceptibility profile (ASP) of T. asahii clinical isolates, we analyzed the genotype distribution, isolation sources, and ASP of 284 strains collected from 1997 to 2019 in different Brazilian medical centers. Species identification and genotype characterization were performed by analysis of the intergenic spacer (IGS1) region of the ribosomal DNA (rDNA). Antifungal susceptibility testing (AST) for amphotericin B and azoles was with the CLSI M27, 4th edition, microdilution broth method. Trends in the ASP of Brazilian T. asahii isolates were investigated using epidemiological cutoff values. Five different genotypes were found among the 284 isolates tested (G1, 76%; G3, 10%; G4, 3%; G5, 7%; and G7, 4%). The isolates were collected mainly from urine (55%) and blood/catheter tip samples (25%) where G1 was the most frequent genotype found (P < 0.05). The G7 isolates exhibited the highest MIC90 values for azoles compared to those for the other genotypes (P < 0.05). Genotype 7 isolates also contributed to the increasing rates of voriconazole non-wild-type isolates found in recent years (P = 0.02). No significant differences were found among the AST results generated by isolates cultured from different anatomical sites. Monitoring T. asahii genotype distributions and antifungal susceptibility profiles is warranted to prevent the spread of azole-resistant isolates.

Epidemiology, clinical aspects, outcomes and prognostic factors associated with Trichosporon fungaemia: results of an international multicentre study carried out at 23 medical centres.

BACKGROUND: Trichosporon fungaemia (TF) episodes have increased in recent years and mortality rates remain high despite the advances in the management of sepsis. New concepts about its clinical course, treatment and microbiology need to be investigated for the better management of this infection. OBJECTIVES: To describe the aetiology, natural history, clinical management and prognostic factors of TF. METHODS: TF episodes documented between 2005 and 2018 in 23 South American centres were retrospectively investigated by using a standard clinical form. Molecular identification, antifungal susceptibility testing and biofilm production were also performed. RESULTS: Eighty-eight TF episodes were studied. Patients had several underlying conditions, including haematological diseases (47.7%), post-operative status (34%), solid organ transplants (n = 7, 7.9%), among others. Seventy-three (82.9%) patients had a central venous catheter (CVC) at TF diagnosis. The 30 day mortality rate was 51.1%. Voriconazole-based therapy was given to 34 patients (38.6%), with a 30 day mortality rate of 38.2%. Multivariate predictors of 30 day mortality were age (OR 1.036), mechanical ventilation (OR 8.25) and persistent neutropenia (OR 9.299). CVC removal was associated with over 75% decreased risk of 30 day mortality (OR 0.241). Microbiological analyses revealed that 77.7% of the strains were identified as Trichosporon asahii, and voriconazole showed the strongest in vitro activity against Trichosporon spp. Most of the strains (63%) were considered medium or high biofilm producers. CONCLUSIONS: Older age, mechanical ventilation and persistent neutropenia were associated with poor prognosis. CVC may play a role in the pathogenicity of TF and its removal was associated with a better prognosis.

Rapid Detection of Carbapenemase Production Directly from Blood Culture by Colorimetric Methods: Evaluation in a Routine Microbiology Laboratory.

The aim of this study was to evaluate the two rapid colorimetric methods (CNPt-Direct and Blue-Carba) for the detection of carbapenemase production directly from blood culture in a routine microbiology laboratory. The methods were initially evaluated on spiked blood cultures with 61 carbapenemase-positive isolates. Afterwards, they were used in blood cultures (314 samples were evaluated) obtained from patients in a routine microbiology laboratory during a period of 6 months. The colorimetric methods were compared to the conventional culture of blood. The results of the spiked blood cultures indicated that both colorimetric methods presented positive results for the vast majority (95%) of the isolates harboring KPC, NDM, and IMP genes. However, the assay failed to detect many GES- and OXA-48-like-positive isolates (65% positive results). In the second part of the study, a total of 314 blood cultures from patients were evaluated, and 33 yielded Enterobacteriaceae isolates resistant to meropenem (30 isolates were positive for carbapenemases according to PCR). The colorimetric tests correctly detected 24 out of the 30 carbapenemase-positive isolates directly from the blood vial (80% positive results). Overall positive percent agreement and negative percent agreement were 80% and 100%, respectively. The colorimetric assays are simple and cost-effective methods that can be implemented in a routine microbiology laboratory, diminishing the time necessary to detect carbapenemase-producing isolates from 24 to 48 h to 3 to 5 h. Moreover, according to our results, the positive colorimetric test results do not need to be confirmed and can be immediately provided to the attending physician.

Relationship between intracranial pressure and antifungal agents levels in the CSF of patients with cryptococcal meningitis.

The purpose of this study was to evaluate the influence of intracranial hypertension in the cerebrospinal fluid (CSF) levels of amphotericin B and fluconazole levels of patients with cryptococcal meningitis. CSF samples and intracranial pressure were obtained by means of routine punctures performed at days 1, 7, and 14 of therapy, respectively. Amphotericin B and fluconazole CSF levels were measured by HPLC method as previously described. The minimum inhibitory concentration for amphotericin B, fluconazole, 5΄flucytosine, and voriconazole of each Cryptococcus isolate was performed according to CLSI. The predominant Cryptococcus species found was C. neoformans, and the major underlying condition was AIDS. Only one CSF sample had a detectable level for amphotericin B during the 14 days of therapy. Fluconazole CSF levels progressively increased from day 1 to day 14 of therapy for most cases. Fluconazole levels in the CSF were above the minimum inhibitory concentrations (MICs) for Cryptococcus during the initial 14 days of antifungal therapy. Variations of intracranial pressure did not affect amphotericin B and fluconazole levels in the CSF. The generalized estimating correlation (GEE) and Spearman correlation test (SCT) showed no significant correlation between the amphotericin B or fluconazole concentrations in the CSF and intracranial pressure (P = .953 and P = .093, respectively for GEE test and P = .477 and P = .847, respectively, for SCT). Combination therapy of amphotericin B with fluconazole was effective in 60% of the patients considering CSF cultures were negative in 9 of 15 patients after 14 days of therapy. Further studies are necessary to evaluate the role of intracranial hypertension on the therapeutic efficacy of different antifungal agents in patients with cryptococcal meningitis.

Triazole Resistance Is Still Not Emerging in Aspergillus fumigatus Isolates Causing Invasive Aspergillosis in Brazilian Patients.

Aspergillus fumigatus azole resistance has emerged as a global health problem. We evaluated the in vitro antifungal susceptibility of 221 clinical A. fumigatus isolates according to CLSI guidelines. Sixty-one isolates exhibiting MICs at the epidemiological cutoff value (ECV) for itraconazole or above the ECV for any triazole were checked for CYP51A mutations. No mutations were documented, even for the isolates (1.8%) with high voriconazole MICs, indicating that triazoles may be used safely to treat aspergillosis in Brazil.

Rapid identification,fluconazole and micafungin susceptibility testing of Candida species from blood culture by a short incubation method.

This study aimed to develop and validate a rapid method for identification by MALDI-TOF system and determination of the susceptibility to Fluconazole and Micafungin by broth microdilution among Candidaspecies causing bloodstream infections. Subcultures from blood culture bottles were incubated for 5 hours (+/- 1h) and used to perform the tests, so that the turnaround time of rapid identification and susceptibility profile was about 5 and 24 hours, respectively. The rapid identification showed agreement of 92.05 %. Regarding the rapid broth microdilution for Fluconazole and Micafungin, the agreement was 97.06 % (p<0.001) and 100 % (p<0.001), and the Kappa coefficient was 0.91 (p<0.001) and 1.0 (p<0.001), respectively. To conclude, both rapid methods showed to be reproducible, inexpensive, easy to perform and time-saving. Thus, these methodologies could be useful to guide and adjust empirical antifungal therapy.

The indoor air as a potential determinant of the frequency of invasive aspergillosis in the intensive care.

Invasive aspergillosis (IA) seems to be an emerging condition in intensive care units (ICUs). However, little attention has been given to the role of environmental factors that could increase the risk for IA in the ICU. The objective of this study was to determine the concentration of airborne fungi in three Brazilian ICUs, in an attempt to correlate fungal burden with the frequency of Aspergillus spp isolation from clinical samples of patients hospitalised in these units. During a 1-year period we quantitatively evaluated the presence of fungi in the air of three ICUs in Porto Alegre, Brazil. The quantity of fungi was correlated with environmental factors. Only one of the ICUs studied showed equal concentrations of Aspergillus conidia in the indoor air, in comparison with the outdoor environment. All cases of Aspergillus colonisation and IA cases observed during the study occurred in that particular ICU. Environmental factors have a direct influence on fungal spore concentration in the air in ICUs, as well as air filtration systems in air conditioners. Fungal contamination of the indoor air may influence the frequency of AI in ICU patients.

Sporotrichosis in renal transplant patients.

The current report describes two renal transplant recipients who presented with sporotrichosis. In addition, the authors review the general aspects of sporotrichosis in renal transplant recipients reported in the literature. Sporotrichosis is a rare fungal infection in transplant patients and has been reported primarily in renal transplant recipients not treated with antifungal prophylaxis. Extracutaneous forms of sporotrichosis without skin manifestations and no previous history of traumatic injuries have been described in such patients and are difficult to diagnose. Renal transplant recipients with sporotrichosis described in the present report were successfully treated with antifungal therapy including amphotericin B deoxycholate, lipid amphotericin B formulations, fluconazole and itraconazole.

Le présent rapport décrit deux greffés du rein qui ont présenté une sporotrichose. De plus, les auteurs ont examiné les aspects généraux de la sporotrichose chez des greffés du rein signalés dans les publications. La sporotrichose, une infection fongique rare chez les greffés, s'observe surtout chez des greffés du rein qui ne reçoivent pas de prophylaxie antifongique. Des formes extracutanées de sporotrichose, sans manifestations cutanées et sans antécédents de lésions traumatiques, ont été décrites chez ces patients, mais elles sont difficiles à diagnostiquer. Les greffés du rein atteints de sporotrichose décrits dans le présent rapport ont été traités avec succès à l'aide d'un antifongique, y compris le désoxycholated d'amphotéricine B, les formulations lipidiques d'amphotéricine B, le fluconazole et l'itraconazole.

The impact of serum vancomycin levels and minimum inhibitory concentrations of methicillin-resistant Staphylococcus aureus on mortality in patients with nosocomial pneumonia.

BACKGROUND: Vancomycin is the treatment of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections; however, treatment failure is not uncommon, even when the minimum inhibitory concentration (MIC) of the MRSA strain is within the susceptible range for vancomycin. OBJECTIVE: To describe the relationship between molecular markers such as the mecA and agrII genes, serum vancomycin levels and vancomycin MICs, and the 30-day mortality rate of patients with nosocomial MRSA pneumonia in an intensive care unit (ICU). METHODS: The present study was a prospective cohort study including all patients with MRSA hospital-acquired pneumonia or ventilator-associated pneumonia who were admitted to the ICU of a tertiary care hospital between June 2009 and December 2011. The MIC for vancomycin was determined using the E-test and broth microdilution methods. Variables analyzed included age, sex, comorbid conditions, serum vancomycin trough concentration, the Acute Physiology and Chronic Health Evaluation II (APACHE) score and the presence of the agrII gene. The primary outcome was mortality at 30 days. RESULTS: Thirty-six (42.4%) patients died within 30 days of the index MRSA culture. A multiple regression analysis that included the variables of MIC (determined using the E-test or broth microdilution methods), APACHE II score, serum vancomycin level and the presence of agrII revealed that only the APACHE II score was related to the 30-day mortality rate (P=0.03). Seven patients (9.0%) with isolates exhibiting an MIC ≥1.5 µg/mL according to the E-test method died, and nine patients (11.6%) survived (P=0.76). Of the patients for whom MICs were determined using the broth microdilution method, 11 (14.1%) patients with MICs of 1.0 µg/mL died, and 16 (20.5%) survived (P=0.92). The median APACHE II score of survivors was 22.5, and the median score of nonsurvivors was 25.0 (P=0.03). The presence of the agrII gene was not related to the 30-day mortality rate. CONCLUSIONS: Patients with severe hospital-acquired pneumonia presented with MRSA isolates with low to intermediate vancomycin MICs in the ICU setting. At the Hospital de Clínicas de Porto Alegre (Porto Alegre, Brazil), the 30-day mortality rate was high, and was similar among patients with severe hospital-acquired pneumonia infected with MRSA isolates that exhibited MICs of ≤1.5 µg/mL determined using the E-test method and ≤1.0 µg/mL determined using the broth microdilution method in those who achieved optimal serum vancomycin levels. The APACHE II scores which provides an overall estimate of ICU mortality were independently associated with mortality in the present study, regardless of the MICs determined. Molecular markers, such as the agrII gene, were not associated with higher mortality in the present study.

HISTORIQUE: La vancomycine est le traitement de première intention des infections par le Staphylococcus aureus résistant à la méthicilline (SARM), mais les échecs thérapeutiques ne sont pas rares, même lorsque la concentration minimale inhibitrice (CMI) de la souche de SARM se situe dans la plage susceptible de vancomycine. OBJECTIF: Décrire le lien entre les marqueurs moléculaires comme les gènes mecA et agrII, le taux de vancomycine sérique et la CMI de vancomycine, et le taux de mortalité au bout de 30 jours des patients atteints d'une pneumonie à SARM d'origine nosocomiale à l'unité de soins intensifs (USI). MÉTHODOLOGIE: La présente étude prospective de cohorte incluait tous les patients ayant une pneumonie à SARM d'origine nosocomiale ou d'une pneumonie acquise sous ventilation mécanique qui ont été hospitalisés à l'USI d'un hôpital de soins tertiaires entre juin 2009 et décembre 2011. Les chercheurs ont déterminé la CMI de la vancomycine au moyen des méthodes d'E-test et de microdilution en bouillon. Les variables qu'ils ont analysées sont l'âge, le sexe, les états comorbides, la concentration minimale de vancomycine sérique, le score APACHE (évaluation de physiologie aiguë et de maladie chronique II) et la présence du gène agrII. La mortalité au bout de 30 jours était l'issue primaire. RÉSULTATS: Trente-six patients (42,4 %)sont décédés dans les 30 jours suivant la culture de référence du SARM. Une analyse de régression multiple qui incluait les variables de la CMI (déterminée au moyen des méthodes d'E-test ou de microdilution en bouillon, le score APACHE II, le taux de vancomycine sérique et la présence du gène f agrII a révélé que seul le score APACHE II était lié au taux de mortalité au bout de 30 jours (P=0,03). Sept patients (9,0 %) dont les isolats présentaient une CMI d'au moins 1,5 µg/mL d'après la méthode d'E-test sont décédés, et neuf patients (11,6 %) ont survécu (P=0,76). Chez les patients dont la CMI a été déterminée au moyen de la méthode de microdilution en bouillon, 11 (14,1 %) ayant une CMI de 1,0 µg/mL sont décédés et 16 (20,5 %) ont survécu (P=0,92). Les survivants avaient un score APACHE II médian de 22,5, et les non-survivants, de 25,0 (P=0,03). La présence du gène agrII n'était pas liée au taux de décès au bout de 30 jours. CONCLUSIONS: Les patients ayant une grave pneumonie d'origine nosocomiale présentaient des isolats de SARM à la CMI faible à intermédiaire à la vancomycine à l'USI. Au Hospital de Clínicas de Porto Alegre (Porto Alegre, Brésil), le taux de mortalité au bout de 30 jours était élevé, tout comme chez les patients atteints d'une grave pneumonie d'origine nosocomiale infectés par des isolats du SARM dont la CMI était égale ou inférieure à 1,5 µg/mL d'après par la méthode d'E-test (ou égale ou inférieure à 1,0 µg/mL d'après la méthode de microdilution en bouillon) qui ont atteint des taux optimaux de vancomycine sérique. Les scores APACHE II qui procurent une évaluation globale de la mortalité à l'USI s'associaient de manière indépendante avec la mortalité dans la présente étude, quelle que soit la CMI établie. De plus, les marqueurs moléculaires, tels que le gène agrII, n'étaient pas liés à un taux de mortalité plus élevé y.

Emergence of cryptic species and clades of Meyerozyma guilliermondii species complex exhibiting limited in vitro susceptibility to antifungals in patients with candidemia.

Members of the Meyerozyma guilliermondii species complex are able to cause superficial and life-threatening systemic infections with low susceptibility to azoles and echinocandins. We tested 130 bloodstream M. guilliermondii complex isolates collected from eight Latin American medical centers over 18 years (period 1 = 2000-2008 and period 2 = 2009-2018) to investigate trends in species distribution and antifungal resistance. The isolates were identified by rDNA ITS region sequencing, and antifungal susceptibility tests were performed against fluconazole, voriconazole, anidulafungin, and amphotericin B using the CLSI microbroth method. M. guilliermondii sensu stricto (s.s.; n = 116) was the most prevalent species, followed by Meyerozyma caribbica (n = 12) and Meyerozyma carpophila (n = 2). Based on rDNA ITS identification, three clades within M. guilliermondii sensu stricto were characterized (clade 1 n = 94; clade 2 n = 19; and clade 3 n = 3). In the second period of study, we found a substantial increment in the isolation of M. caribbica (3.4% versus 13.8%; P = 0.06) and clade 2 M. guilliermondii s.s. exhibiting lower susceptibility to one or more triazoles. IMPORTANCE Yeast-invasive infections play a relevant role in human health, and there is a concern with the emergence of non-Candida pathogens causing disease worldwide. There is a lack of studies addressing the prevalence and antifungal susceptibility of different species within the M. guilliermondii complex that cause invasive infections. We evaluated 130 episodes of M. guilliermondii species complex candidemia documented in eight medical centers over 18 years. We detected the emergence of less common species within the Meyerozyma complex causing candidemia and described a new clade of M. guilliermondii with limited susceptibility to triazoles. These results support the relevance of continued global surveillance efforts to early detect, characterize, and report emergent fungal pathogens exhibiting limited susceptibility to antifungals.

Standardization and characterization of antigens for the diagnosis of aspergillosis.

The aim of this study was to develop and characterize antigens for the diagnosis of aspergillosis. Nine strains of Aspergillus species Aspergillus fumigatus , Aspergillus flavus , and Aspergillus niger were grown in Sabouraud and Smith broth to produce exoantigens. The antigens were tested by immunodiffusion against sera from patients with aspergillosis and other systemic mycoses. The protein fraction of the antigens was detected by SDS-PAGE; Western blot and representative bands were assessed by mass spectrometry coupled to a nano Acquity UltraPerformance LC and analyzed by the Mascot search engine. Concurrently, all sera were tested with Platelia Aspergillus EIA. The most reactive antigens to sera from patients infected by A. fumigatus were produced by A. fumigatus MG2 Sabouraud and pooled A. fumigatus Sabouraud samples, both with a sensitivity of 93% and specificity of 100% and 97%, respectively. Aspergillus niger and A. flavus antigens were reactive against A. niger and A. flavus sera, each one with a sensitivity and specificity of 100%. Two proteins, probably responsible for antigenic activity, ß-glucosidase in A. fumigatus and α-amylase in A. niger were attained. The commercial kit had a specificity of 22%, sensitivity of 100%, positive predictive value of 48%, and negative predictive value of 100%. The antigens produced showed high sensitivity and specificity and can be exploited for diagnostics of aspergilloma.

The performance of real-time PCR, galactomannan, and fungal culture in the diagnosis of invasive aspergillosis in ventilated patients with chronic obstructive pulmonary disease (COPD).

Emerging reports have associated chronic pulmonary obstructive disease (COPD) with invasive aspergillosis (IA), particularly in patients treated with mechanical ventilation and/or corticosteroids. This is a multicentre study in which COPD patients demonstrating a new lung infiltrate while being mechanically ventilated were prospectively evaluated for the presence of IA. From the 47 patients studied, Aspergillus fumigatus was recovered in culture in two patients (4.2%). While serum galactomannan (GM) was negative for 94% of patients, GM levels in respiratory samples were >0.5, >1.0 and >1.5 for 74.5, 40.5, and 21.3% of patients, respectively. PCR was positive for 10 patients in the study but did not differentiate Aspergillus colonization from infection. The combination of PCR and GM in respiratory samples may be an interesting alternative to diagnose IA in COPD patients.

Diversity of Fusarium species causing invasive and disseminated infections.

INTRODUCTION: Invasive fusariosis (IF) is considered an emerging fungal disease and an important problem worldwide that increasingly affects immunocompromised individuals. There is currently concern about establishing the genetic diversity and phylogenetic relationship of the species Fusarium causing invasive fusariosis. MATERIALS AND METHODS: The aim of this study was to characterize the molecular profile and morphological characteristics of Fusarium species isolated from 21 patients with invasive fusariosis. Multilocus sequence typing was performed for molecular identification of the following genes: the second largest subunit of the RNA polymerase gene (RPB2) and elongation factor 1 alpha (EF-1α). The morphological features of different species were carefully described and revised by experienced mycologists. RESULTS: Morphological and molecular analyses revealed that the F. solani species complex (FSSC) and F. oxysporum species complex (FOSC) were the most common species isolated from patients with invasive fusariosis; FSSC-2 h (5), FSSC-1 (2) and FOSC-183 (2) were the most frequent haplotypes. The macroscopic characterization revealed great variation in the tonalities of the FSSC colonies and particularities amongst the species in relation to the macroconidia structures, while the FOSC was more homogeneous and presented shades from white to lilac. CONCLUSIONS: Our study characterized the diversity, haplotypes, and morphological aspects of Fusarium species and the haplotypes prevalent in patients with invasive fusariosis. FSSC and FSSC-2 h were the predominant species and haplotype, respectively. Although we have described interesting morphological aspects in Fusarium species, particularly haplotypes, their identification cannot rely on phenotypical aspects. Molecular biology techniques are necessary and should be introduced for routine use in mycology laboratories.

Coinfection of disseminated cryptococcosis and BK Virus, a casualty of missed diagnosis during the COVID-19 Pandemic: A case report and review of the literature.

Background and Purpose: The COVID-19 pandemic resulted in an overload of health services and healthcare professionals. The result is a setback in health promotion and prevention, delays in diagnosis, and deaths from other diseases that are currently receiving inadequate attention. This article illustrates the risk of this negligence. Case report: This study aimed to report a case of coinfection of disseminated cryptococcosis and BK virus in a patient without a previous diagnosis of human immunodeficiency virus infection and COVID-19 negative in the context of the COVID-19 pandemic. Despite receiving antifungal therapy, the patient died. Conclusion: This fatal case is a warning regarding delay of diagnosis and neglect of other serious illnesses owing to the current pandemic, including fungal diseases and neglected diagnoses.

Evaluation of Identification and Susceptibility for Candida Spp. Isolated Directly from Positive Blood Culture Bottles.

Determination of the susceptibility profile of isolates of Candida from blood culture bottles is extremely important for correctly guiding patient pharmacotherapy. The aim of this study was to compare the results of analysis of Candida isolated directly from blood culture bottles by the VITEK MS MALDI-TOF identification system and the fluconazole disk diffusion assay with those of standard identification methods. Testing directly from the bottle allowed results 24 to 48 hours quicker than the standard method. There was a categorical agreement of 51.64% (47 of 91 samples) between the results of analysis directly from the bottle and analysis by the standard method. Regarding species identification, there was 96.15% agreement for Candida parapsilosis (25 of 26 samples). Categorical agreement between the rapid and standard disk diffusion methods was 95%, and the agreement between the rapid disk diffusion method and the broth microdilution method was 97%. Only minor errors in the rapid method were observed: 3 (5%) in the standard disk diffusion method and 2 (3%) in the broth microdilution method. Our study concluded that the rapid disk diffusion method for fluconazole is a fast, easy, reproducible, and consistent method. Its timely implementation for testing antifungal agents in the clinical microbiology laboratory can help reduce profile release times, thus helping to determine the most appropriate antifungal treatment.

Continuous infusion of amphotericin B deoxycholate in the treatment of cryptococcal meningoencephalitis: analysis of safety and fungicidal activity.

We measured fungicidal activity of continuous infusion of amphotericin B deoxycholate plus 5'flucytosine using quantitative cultures of cerebrospinal fluid (CSF) obtained from lumbar punctures of human immunodeficiency virus (HIV)-infected patients with neurocryptococcosis during 14 days of treatment. Glomerular renal function was preserved in all patients. Mycological efficacy with progressive reduction in CSF cryptococcal colony-forming units was comparable to standard 4-h infusion of amphotericin B.

Rapid antimicrobial susceptibility of Enterobacteriaceae by disk diffusion directly from blood culture bottles using the EUCAST RAST breakpoints.

OBJECTIVES: To evaluate the disk diffusion technique after 4 and 6 h directly from positive blood culture bottles of Enterobacteriaceae using the rapid antimicrobial susceptibility test (RAST) breakpoints established by The European Committee on Antimicrobial Susceptibility Testing (EUCAST). METHODS: A total of 61 isolates of Escherichia coli and Klebsiella spp. were selected. The results were assessed using the RAST breakpoints (4-6h) as well as the standard breakpoints (18h) from EUCAST. RESULTS: The vast majority of the zone diameters of E. coli and Klebsiella spp. were optimally readable after 6 h of incubation. RAST at 6 h presented best results of categorical agreement (CA) and errors (CA = 94.4%, minor errors [mE] = 4.3%, major errors [ME] = 0.8% and very major errors [VME] = 0.4%) compared to RAST at 4 h (CA = 84.3%, mE = 13.0%, ME = 3.2% and VME = 0.4%). The proportion of results in the area of technical uncertainty decreased over time: from 13.8% at 4 h to 6.8% at 6 h. According to the U.S. Food and Drug Administration and ISO criteria, early readings at 6 h using the RAST breakpoints provided acceptable results (CA > 90%), whereas accuracy of results at 4 h was unacceptable (CA < 90%). CONCLUSION: These data indicate that the early readings at 6 h using the RAST breakpoints for all antibiotics tested in this study (except amikacin) may be used in the clinical microbiology laboratory to anticipate the antimicrobial susceptibility test results of blood cultures. Early readings at 4 h (RAST breakpoints) may also be used, but not for all antibiotics.

Performance of polymyxin B Etest in a setting of high prevalence of KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Polymyxin resistance has been increasing in many regions, and appropriate determination of polymyxin susceptibility is now a major challenge worldwide. Many clinical laboratories rely on gradient diffusion methods to assess polymyxin susceptibility, although broth microdilution (BMD) is the only method currently recommended by the CLSI and EUCAST. The aim of this study was to assess the performance of the polymyxin B (PMB) Etest in a setting with a high prevalence of KPC-producing Klebsiella pneumoniae (KPC-KP). METHODS: A commercial Etest susceptibility testing method was evaluated and compared with the reference BMD method, considering isolates with a minimum inhibitory concentration (MIC) ≤2 mg/L for PMB as susceptible to this drug. A total of 310 clinical KPC-KP isolates were evaluated. RESULTS: Susceptibility was significantly higher by Etest compared with BMD (82.6% vs. 75.8%). The MIC50, MIC90 and modal MICs for PMB were 0.25, 32 and 0.25 mg/L (27.1%) by BMD and 0.5, 16 and 0.5 mg/L (49.7%) by Etest, respectively. Although categorical agreement was 90.0%, there was poor essential agreement (50.6%). A high rate (34.7%) of very major errors (VMEs) and a relatively low rate (2.1%) of major errors were found. CONCLUSION: The considerable number of resistant isolates in this study allowed an accurate estimation of VME rates and, consequently, a more comprehensive assessment of susceptibility testing for polymyxins. Etest did not meet fully the acceptance criteria for US FDA requirements. These data do not support the use of this commercial method for determining PMB MICs in carbapenem-resistant Enterobacterales populations.

Epidemiological aspects and characterization of the resistance profile of Fusarium spp. in patients with invasive fusariosis.

Introduction. The remarkable intrinsic resistance of Fusarium species to most antifungal agents results in high mortality rates in the immunocompromised population.Aims. This study aimed to investigate the epidemiology, clinical features and antifungal susceptibility of Fusarium isolates in patients with invasive fusariosis.Methodology. A total of 27 patients admitted to a referral hospital from January 2008 to June 2017 were evaluated. Antifungal susceptibility testing of isolates was performed by broth microdilution according to the Clinical and Laboratory Standards Institute guidelines.Results. Haematological malignancy was the predominant underlying condition, with an incidence of invasive fusariosis of 14.8 cases per 1000 patients with acute lymphoid leukaemia and 13.1 cases per 1000 patients with acute myeloid leukaemia. The Fusarium solani species complex (FSSC) was the most frequent agent group, followed by the Fusarium oxysporum species complex (FOSC). Voriconazole showed the best activity against Fusarium, followed by amphotericin B. Itraconazole showed high minimum inhibitory concentration values, indicating in vitro resistance. Clinical FSSC isolates were significantly (P<0.05) more resistant to amphotericin B and voriconazole than FOSC isolates.Conclusion. The present antifungal susceptibility profiles indicate a high incidence of fusariosis in patients with haematological malignancy. Species- and strain-specific differences in antifungal susceptibility exist within Fusarium in this setting.