In vitro activity of terbinafine against Indian clinical isolates of Candida albicans and non-albicans using a macrodilution method

ObjectiveTerbinafine, an allylamine derivative, is the most representative of this new chemical class of antimycotic compounds. The aim of this study was to determine the anti Candida activity of terbinafine compared with amphotericin, fluconazole, ketoconazole and itraconazole.MethodThe drugs were tested against 25 fresh clinical isolates of Candida recovered from blood, urine, sputum and oral and vaginal wash of patients with underlying diseases like cancer, tuberculosis, diabetes, post-operative patients (immunocompromised) and oropharyngeal candidiasis (OPC) or vulvovaginal candidiasis (VVC). The macrodilution method was performed according to the recommendation of National Committee for Clinical Laboratory Standards (NCCLS) M 27-A using RPMI 1640 medium in MOPS buffered to pH 7.1: twofold drug dilution scheme was adapted with the concentration range of 0.03125–64 μg/ml. Yeast inoculum was adjusted to 0.5 McFarland standard with working suspension at 1:100 dilution. The end point for terbinafine was defined as 80% inhibition compared with growth in control wells.ResultUnder in vitro conditions, terbinafine proved to be highly active against Candida albicans, C. parapsilosis, C. guilliermondii, C. glabrata and C. tropicalis (minimum inhibitory concentration, MIC range, 0.03125–4 μg/ml) and showed potentially useful activity against C. krusei (MIC range, 1–4 μg/ml). Statistically significant difference was observed for terbinafine between C. parapsilosis, C. glabrata and C. guilliermondii and C. albicans.ConclusionTo the knowledge of the authors, the in vitro data presented in this study is the first from India on terbinafine activity against clinical strains of C. albicans and non-albicans species with such lower MICs (MIC range 0.03125–4 μg/ml). Thus, in addition to its potent activity against dermatophytes, terbinafine is also very effective against Candida species. It could be interesting for clinical laboratories and physician for making appropriate drug choices and patient management decision. In vivo investigations of its antifungal activity against Candida should consequently be pursued.     

Thymus vulgaris essential oil and thymol inhibit biofilms and interact synergistically with antifungal drugs against drug resistant strains of Candida albicans and Candida tropicalis

Role of biofilm in disease development and enhance tolerance to antifungal drugs among Candida species has necessitated search for new anti-fungal treatment strategy. Interference in pathogenic biofilm development by new antifungal compounds is considered as an attractive anti-infective strategy. Therefore, the objective of this study was to evaluate Thymus vulgaris essential oil and its major active compound, thymol for their potential to inhibit and eradicate biofilms alone and in combination with antifungal drugs against Candida spp. with especial reference to Candida tropicalis. Anti-candidal efficacy of T. vulgaris and thymol in terms of minimum inhibitory concentration (MIC) was first determined to select the sub-MICs against C. albicans and C. tropicalis. Biofilm formation in the presence and absence of test agents was determined in 96-well microtiter plate by XTT reduction assay and effect of essential oils at sub-MICs of the test agents on biofilm development on glass surface was analysed by light and scanning electron microscopy. Synergistic interaction between essential oils and antifungal drugs were studied by checkerboard method. Effect of sub-MIC of T. vulgaris (0.5 × MIC) and thymol (0.5 × MIC) on biofilm formation showed a significant reduction (P < 0.05) in biofilms. Light microscopy and SEM studies revealed disaggregation and deformed shape of C. albicans biofilm cells and reduced hyphae formation in C. tropicalis biofilm cells at sub-MICs of thymol. Significant effect of T. vulgaris and thymol was also recorded on pre-formed biofilms of both C. albicans and C. tropicalis. T. vulgaris and thymol also showed synergy with fluconazole against both in planktonic and biofilm mode of growth of C. albicans and C. tropicalis. However, synergy with amphotericin B is clearly evident only in planktonic Candida cells. Thyme oil and thymol alone or in combination with antifungal drugs can act as promising antibiofilm agent against drug resistant strains of Candida species and needs further in vivo study to synergise its therapeutic efficacy.     

Antibacterial activity of antagonistic bacterium Bacillus subtilis DJM-51 against phytopathogenic Clavibacter michiganense subsp. michiganense ATCC 7429 in vitro

To investigate antibacterial activity against the tomato pathogen Clavibacter michiganense subsp. michiganense ATCC 7429 (Cmm ATCC 7429), Bacillus subtilis DJM-51 was isolated from rhizosphere soil. For isolation of bacteria, samples were taken from rhizosphere soil. The isolate, DJA-51, had strong antagonistic ability against Tomato pathogen Cmm ATCC 7429 on nutrient-broth yeast extract agar (NBYA) as indicated by inhibition zones around colonies. On the basis of the nucleotide sequence of a conserved segment of the 16S rRNA gene, the bacterium has been identified as B. subtilis DJM-51. The growth of Cmm ATCC 7429 on NBYA plates was inhibited by culture broth of B. subtilis DJM-51 including cells, by the supernatant of culture broth of B. subtilis DJM-51, and by the liquid material resulting from butanol extract of bacterial cultures. The OD value in co-culture mixture was lower than the control throughout the entire incubation period. Antibiotics obtained from B. subtilis DJM-51 inhibited the growth of Tomato pathogen Cmm ATCC 7429. These results provide potentially information about the protection of tomato from pathogen Cmm ATCC 7429 under greenhouse conditions in Quebec.     

Rapid antifungal susceptibility testing of fluconazole and amphotericin B by flow cytometry using FUN-1®: a preliminary study

ObjectiveThe increasing incidence of severe fungal infections and the difficulties encountered with the existing antifungal susceptibility testing methods led us to develop a new technique based on flow cytometry, using the fluorochrome FUN -1^®.Materials and methodsWe tested the susceptibility of 14 Candida spp. strains (5 reference and 9 clinical strains) to amphotericin B and fluconazole by flow cytometry versus M27-A2 CLSI reference method. Fluorescence was measured with a FACScalibur^® flow cytometer (Becton Dickinson). Amphotericin B susceptibility was studied on the 14 strains whereas fluconazole one was only performed on the five reference strains.ResultsResults were obtained after 30 min incubation for amphotericin B and 6 h for fluconazole. Reproducibility and repeatability tests have been performed on C. albicans ATCC 90028 and were > 95% and > 98%, respectively. The flow cytometry method resulted in clear-cut endpoints, which abolishes the source of variability of the M27-A2 reading. The results obtained showed that the patterns of susceptibility to amphotericin B were the same with the 2 methods used for all the strains. Our preliminary study with fluconazole gave the same results with the 5 control strains.ConclusionFUN-1^® staining seems to be a rapid alternative to conventional methods to assess susceptibility of Candida spp. clinical isolates.     

Evolution of fungemia in an Italian region

BackgroundFungemia represents a public health concern. Knowing aetiology and activity of the antifungals is critical for the management of bloodstream infections. Therefore, surveillance on local/international levels is desirable for a prompt administration of appropriate therapy.MethodsData on fungi responsible for fungemia and antifungal susceptibility profiles were collected from a laboratory-based surveillance over 2016–2017 in 12 hospitals located in Lombardia, Italy. The trend of this infection in twenty years was analysed.ResultsA total of 1024 episodes were evaluated. Rate of candiaemia progressively increased up to 1.46/1000 admissions. C.albicans was the most common species (52%), followed by C. parapsilosis (15%) and C glabrata (13%). As in the previous surveys the antifungal resistance is rare (echinocandins < 2%, fluconazole 6%, amphotericin B 0.6%). Fungi other than Candida were responsible for 18 episodes: Cryptococcus neoformans (5 cases), Fusarium spp. (4), Magnusiomyces clavatus (3), Saccharomyces cerevisiae (3), Rhodotorula spp. (2), Exophiala dermatitidis (1). All fungi, except S.cerevisiae, were intrinsically resistant to echinocandins. Some isolates showed also elevated azole MIC.ConclusionsNo particular changes in terms of species distribution and antifungal susceptibility patterns was noted. However, surveillance programs are needed to monitor trends in antifungal resistance, steer stewardship activities, orient empirical treatment.     

Misidentification of Candida dubliniensis isolates with the VITEK MS

The phylogenetic relatedness of Candida dubliniensis and C. albicans may lead to misidentification of C. dubliniensis and underestimation of its clinical significance. We evaluated the performance of VITEK-MS in identifying C. dubliniensis isolates following growth on different culture media. Correct identification was documented in 98% of the isolates grown on blood agar media whereas only 44% were correctly identified from SDA or CHROMagar. The use of non-manufacturer validated media for identifying C. dubliniensis with VITEK-MS, may result in misidentification of these isolates as C. albicans. This finding calls for reassessing the accuracy of fungal isolates identification in local workflows using non-validated culture media.     

Étude d’activité in vitro et de stabilité de suspensions antifongiques pour bain de bouche : vers une remise en question de pratiques empiriques ?

Establishment of an effective prophylaxis against oral candidiasis by local treatment is essential for immunocompromised patients. The aim of the study is to assess effectiveness and stability of antifungal suspensions for mouthrinses. The assessed suspensions are compounded by one solvent among sterile water, spring water or sodium bicarbonate associated with amphotericin B (Fungizone^®) or nystatine (Mycostatine^®). Two others mixes are assessed: Mycostatine^®-bicarbonate and Mycostatine^®-Hextril^®-bicarbonate as well as the two straight antifungal. In vitro activity is tested on five Candida species (C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis) after a five minutes contact between yeasts and the assessed suspension. A galenic study is realized during 3 days. Mixes associating a polyene with sodium bicarbonate have no effectiveness on Candida albicans, others mixes shows intermediate effectiveness (the percentage of yeast growth inhibition lies between 35% and 68%). Effectiveness results of Hextril^®-based mixes are not explainable because of alcohol in its composition. Spring water-based mixes must be evicted due to microbiologic contaminations after 48 hours. Mycostatine^®-Hextril^®-bicarbonate mix is not stable during 3 days. All those mouthrinses, poorly effective, excepted on C. glabrata, should be avoided. Straight Mycostatine^® shows a good antifungal effectiveness excepted on C. krusei and its use should be recommended.     

Epidemiology,risk factors and antimicrobial profile of Vulvovaginal Candidiasis (VVC): A study among women in the central region of Saudi Arabia

BackgroundCandida spp. is a fungal resident of the normal microbiota of gastrointestinal tract, reproductive mucosa and oral cavity. Hence, a majority of the healthy population may be prone to the most common fungal infection such as candidiasis that can be caused by any species of Candida. In women, vaginitis or vulvovaginal candidiasis (VVC) forms a significant part of urogenital infections with a high recurrence rate thus posing a public health issue worldwide.ObjectivesThe aim of this study was to determine the prevalence of VVC, its possible risk factors and the antifungal susceptibility of the species isolated from women attending a hospital in the central region of Saudi Arabia.MethodsSamples of high vaginal swabs (HVS) were obtained from 208 women aged 15–64 years with signs and symptoms of VVC. The samples were cultured on Sabouraud agar plates (SDA) and incubated at 30 °C for 10 days. Candida spp. were initially identified using morphologic characteristics, wet mount, germ tube test and finally confirmed with Vitek 2.ResultsAmong the samples, 34% were culture positive. Out of the positive samples, 68% were Candida albicans, followed by C. tropicalis (27%) and C. glabrata (2.7%). Majority of the C. albicans (16%) were observed in women between ages of 21–30 years. All the isolates were sensitive to the antifungals tested. Also, the presence of Candida spp. did not correlate to risk factors such as pregnancy, diabetes and use of antibiotics.ConclusionsPrevalence of vulvovaginal candidiasis was observed in the central region of Saudi Arabia with the predominant organism as Candida albicans.     

Aspergillus peritonitis in peritoneal dialysis patients: A systematic review

Fungal peritonitis in patients undergoing peritoneal dialysis (PD) is very difficult to treat and is associated with significant morbidity and mortality. Among fungal pathogens, Aspergillus peritonitis presents a higher mortality rate when compared to Candida peritonitis and its identification as well as appropriate treatment remains a challenge for the physicians. We critical reviewed all published cases in literature of Aspergillus peritonitis in PD patients. The results showed that a total of 55 cases (51% males) of Aspergillus peritonitis in PD patients were reported from 1968 to 2019. Mean patient age was 49.54 ± 19.63 years and mean PD duration prior to fungal infection was 33.31 ± 32.45 months. Aspergillus fumigatus was isolated in 17/55 patients, Aspergillus niger in 15, Aspergillus terreus in 9, unidentified Aspergillus spp. in 6, Aspergillus flavus in 4, whereas sporadic cases of other Aspergillus spp. were reported. As far as predisposing factors are concerned, 75% of patients suffered from prior bacterial peritonitis receiving antimicrobial therapy. Initial antifungal treatment was intravenous and/or intraperitoneal administration of amphotericin B formulations monotherapy in 47.2% of patients or in combination with fluconazole in 13.2%, or with itraconazole in 13.2%, or with caspofungin in 3.8%, or with ketoconazole or with 5-FC in 1.9%, each. Peritoneal catheter removal was performed in 85.5% of cases. Mortality rate was 38.2%, while 81.8% of the survived patients switched to hemodialysis. Conclusively, Aspergillus peritonitis diagnosis can be difficult, due to unspecific symptoms. Early treatment with appropriate antifungal agents can be determinant for patient prognosis. Despite appropriate treatment, reported mortality remains high.     

Mode of action and anti-Candida activity of Artemisia annua mediated-synthesized silver nanoparticles

Candida albicans is a polymorphic opportunistic commensal that causes both superficial and systemic fungal infections especially in immunocompromised patients. Biologically synthesized silver nanoparticles (AgNPs) have emerged as potential antifungal agents. The present work evaluates the antifungal activity of Artemisia annua synthesized AgNPs against three Candida species (C. albicans ATCC 90028, C. tropicalis ATCC 750 and C. glabrata ATCC 90030). The in vitro effect of AgNPs was investigated for fungal growth, sterol content, secretion of hydrolytic enzymes and yeast-to-hyphal transition. The green synthesized AgNPs were effective against all the three species with minimum inhibitory concentration (MIC) in the range 80–120 μgml⁻¹. Candida glabrata showed greater sensitivity for AgNPs followed by Candida tropicalis and C. albicans. AgNPs at 4MIC were as effective as fluconazole (FLC) and caused only 5% haemolysis while FLC caused 50% haemolysis at the same concentration. The secretion of hydrolytic enzymes was the lowest in case of AgNP exposed C. glabrata. Yeast-to-hyphal transition was significantly reduced in treated C. albicans cells and showed disfigured morphology in SEM images. The decrease in ergosterol content was slightly higher (94%) in both C. glabrata and C. tropicalis in comparison to C. albicans (69%). Green synthesized AgNPs thus have immense potential as an antifungal and can play a crucial role in the management of Candida infections especially those caused by C. glabrata.     

Isolation of <Emphasis Type="BoldItalic">Acanthamoeba</Emphasis> species in surface waters of Gilan province-north of Iran

We analyzed water samples to determine the prevalence of free-living Acanthamoeba in water sources from Gilan, greater area, Iran. A total of 27 surface water samples were collected from environmental sources, including natural (rivers, lakes, springs, and lagoon) and freshwater source. The samples were filtrated and transferred to non-nutrient agar plates seeded with Escherichia coli and incubated for 2 to 7 days at 30°C or 42°C. The plates were examined by microscopy to morphologically identify Acanthamoeba species. Following DNA extraction, PCR was used to confirm the microscopically identification. A total of 19 out of 27 samples (70.3%) were positive for Acanthamoeba species based on the morphological criteria, and 14 (73.7%) were confirmed by PCR method. The high frequency of Acanthamoeba spp. in different environmental water sources of Gilan is an alert for the public health related to water sources in Iran.     

Thinner tuberosity osteotomy is more resistant to axial load in medial open-wedge distal tuberosity proximal tibial osteotomy: A biomechanical study

BackgroundThe purpose of this study was to investigate axial load resistance of the tibia depending on the thickness of tibial tuberosity osteotomy in medial open-wedge distal tuberosity proximal tibial osteotomy (OWDTO). The hypothesis is that a thin tibial tuberosity osteotomy shows high axial load resistance of the tibia.MethodsThe OWDTO model was constructed from imitation bones of the tibia. Distal tibial tuberosity osteotomy was performed with thicknesses of 7, 14, and 21 mm (n = 5 for each group). Cyclic axial-load fatigue tests were performed to investigate the strain at five measurement points on the OWDTO model. An axial-load failure test was also performed to investigate the maximum strain for failure.ResultsThe 7-mm OWDTO model showed a significantly lower stain range than the 14-mm model at the middle part of the lateral hinge (P = 0.0263, mean difference: −852.6 με), posterior part (P = 0.0465, mean difference: −1040.0 με), posterior tibial cortex (P < 0.0001, mean difference: −583.4 με), and plate (P = 0.0029, mean difference: −121.6 με). There were no significant differences in the strain at the tibial tuberosity between the groups. The axial load for complete failure was significantly higher in the 7-mm model than in the 21-mm model (P = 0.0010, mean difference: 2577.0 N). The failure points were at the lateral hinges.ConclusionsThinner distal tibial tuberosity osteotomy is more resistant to axial load and may be recommended for the prevention of tibial and lateral hinge fractures after OWDTO.     

Bacillus subtilis isolated from the human gastrointestinal tract

As part of an ongoing study to determine the true habitat of Bacillus species, we report here the isolation and characterisation of Bacillus subtilis from the human gastrointestinal tract (GIT). Strains were obtained from ileum biopsies as well as from faecal samples and their biotypes defined. 16S rRNA analysis revealed that most isolates of B. subtilis were highly conserved, in contrast to RAPD-PCR fingerprinting that showed greater diversity with 23 distinct RAPD types. The majority of B. subtilis strains examined possessed features that could be advantageous to survival within the GIT. This included the ability to form biofilms, to sporulate anaerobically and secretion of antimicrobials. At least one isolate was shown to form spores that carried an exosporium, a loosely attached outer layer to the mature endospore, this being the first report of B. subtilis spores carrying an exosporium. This study reinforces a growing view that B. subtilis and probably other species have adapted to life within the GIT and should be considered gut commensals rather than solely soil microorganisms.     

In vitro Azole antifungals susceptibility of Candida spp. isolates from HIV-infected patients with periodontitis

ObjectiveThe objective of the present study was to determine the in vitro Azole antifungals susceptibility of Candida spp. strains isolated from HIV-positive patients with periodontitis.MethodsOral examination was performed in 500 HIV-positive patients, of which 228 were included in the study for having periodontitis which and separated in two groups based on their TCD4⁺ T-cells: (A) n = 110 (≤200 CD4⁺); (B) n = 118 (>200 CD4⁺). Candida spp. were isolated from the subgingival biofilm and crevicular fluid by seeding on CHROMagar plates and confirmed by endpoint PCR and MALDI-TOF. The susceptibility test in vitro for five antifungals was performed using the disc diffusion method.ResultsFrom the 228 HIV-positive patients with periodontitis, 174 were positive to Candida spp., and 204 isolations were obtained. 138 (67.64%) were C. albicans, and 66 (32.35%) were Candida non-albicans species. The most frequent Candida non-albicans species in order of frequency were C. glabrata with 48 (23.52%), C. tropicalis with 10 (4.9%), C. krusei with 7 (3.43%), and C. dubliniensis with 1 (0.49%). All species presented resistance to any antifungal: 149 to 5-fluorocytosine (73.0%), 149 to fluconazole (73.0%), and 144 to voriconazole (70.7%). Miconazole and econazole presented the highest susceptibility rates with 129 (63.2%) and 130 (63.7%) isolations, respectively.ConclusionThe Candida spp. involved in periodontitis of HIV-positive patients have a multi-resistant feature. It is necessary to implement recurrent research regarding the antifungal resistance of the Candida spp. that take part in periodontitis pathogenesis to promote an effective treatment in HIV patients.     

In vitro activity of ibrexafungerp against Candida species isolated from blood cultures. Determination of wild-type populations using the EUCAST method

ObjectivesIbrexafungerp is a new oral glucan synthase inhibitor with in vivo and in vitro activity against Candida spp., including echinocandin- and azole-resistant isolates. We studied the in vitro activity of ibrexafungerp against Candida species isolated from blood cultures and assessed wild-type upper limits against the five Candida species most frequently associated to candidaemia.MethodsIsolates (n = 958) causing incident episodes of candidaemia in patients admitted to Gregorio Marañón hospital (Madrid, Spain) between January 2007 and April 2021 were studied. Antifungal susceptibility to ibrexafungerp, fluconazole, micafungin and anidulafungin was tested (EUCAST E.Def 7.3.2) and wild-type upper limits determined against C. albicans (n = 462), C. glabrata (n = 120), C. parapsilosis (n = 249), C. tropicalis (n = 73) and C. krusei (n = 24). fksgene sequencing was carried out in non-wild-type isolates.ResultsIbrexafungerp showed antifungal in vitro activity against the studied isolates. Wild-type upper limits for ibrexafungerp were >0.25 mg/L against C. albicans, >1 mg/L against C. parapsilosis, C. glabrata, and C. tropicalis, and >2 mg/L against C. krusei. Percentages of ibrexafungerp non-wild-type isolates were low (C. parapsilosis and C. krusei, 0%; C. albicans, 0.22% (1/462); C. glabrata, 0.83% (1/120); and C. tropicalis, 1.37% (1/73)). Ibrexafungerp proved in vitro activity against fluconazole- or echinocandin-resistant isolates.DiscussionWe show in vitro activity of ibrexafungerp against the tested Candida species. Furthermore, we provide ibrexafungerp wild-type upper limits, which allows defining the wild-type populations of the five most relevant Candida species.     

How to fix a tibial tubercle osteotomy with distalisation: A finite element analysis

BackgroundAntero-medialisation osteotomy combined with a distalisation procedure may require a more stable fixation as the osteotomy fragment loses both proximal and distal support. This finite element analysis aimed to compare the mechanical behaviour of different fixation techniques in tibial tubercle antero-medialisation osteotomy combined with distalisation procedure.MethodsTibial tubercle osteotomy combined with distalisation was modelled based on computerised tomography data, which were acquired from a patient with patellar instability requiring this procedure. Six different fixation configurations with two 3.5-mm cortical screws (1), two 4.5-mm cortical screws (2), three 3.5-mm cortical screws (3), three 4.5-mm cortical screws (4), three 3.5-mm screws with 1/3 tubular plate (5), and four 3.5-mm screws with 1/3 tubular plate (6) were created. A total of 1654 N of force was applied to the patellar tendon footprint on the tibial tubercle. Sliding, gap formation, and total deformation between the osteotomy components were analyzed.ResultsMaximum sliding (0.660 mm), gap formation (0.661 mm), and displacement (1.267 mm) were seen with two 3.5-mm screw fixation, followed by two 4.5-mm screws, three 3.5-mm screws, and three 4.5-mm screws, respectively, in the screw-only group. Overall, the minimum displacement was observed with the four 3.5-mm screws with 1/3 tubular plate fixation model.ConclusionsPlate fixation might be recommended for tibial tubercle antero-medialisation osteotomy combined with distalisation procedure because it might allow early active range of motion exercises and weight-bearing.     

Evaluation of the inoculum effect of new antibiotics against carbapenem-resistant enterobacterales

ObjectivesNew antibiotics have been developed to treat multidrug-resistant Enterobacterales. We evaluated the impact of the inoculum size on minimal inhibitory concentrations (MICs) of recently commercialized antibiotics.MethodsWe focused on 40 clinical carbapenemase-producing Enterobacterales and evaluated the impact of the inoculum size on the MICs to cefiderocol and to new β-lactams/β-lactamase inhibitors (ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam) at usual and high inocula (10⁵ and 10⁷ CFU/mL, respectively).ResultsAt usual inoculum, 15% were resistant to cefiderocol (n = 6), 30% to meropenem-vaborbactam (n = 12), 42.5% to ceftazidime-avibactam (n = 17), 55% to imipenem-relebactam (n = 22), and 90% to ceftolozane-tazobactam (n = 36). At higher inoculum, a switch from susceptible to resistant category was observed for 88% (n = 30/34; CI, 71.6–96.2), 75% (n = 3/4; CI, 21.9–98.7), 72% (n = 13/18; CI, 46.4–89.3), 50% (n = 14/28; CI, 31.1–68.9), and 8.7% (n = 2/23; CI, 1.5–29.5) isolates regarding cefiderocol, ceftolozane-tazobactam, imipenem-relebactam, meropenem-vaborbactam, and ceftazidime-avibactam, respectively.DiscussionCefiderocol and meropenem-vaborbactam were the most efficient against carbapenemase-producing Enterobacterales at usual inoculum. When increasing inoculum to 10⁷ CFU/mL, all of the molecules were impacted, particularly cefiderocol and imipenem-relebactam, while others, such as ceftazidime-avibactam, remain mildly affected. Our in vitro results deserved to be confirmed in vivo.     

Three Isothermal Amplification Techniques for Rapid Identification of Cladophialophora carrionii,an Agent of Human Chromoblastomycosis

In this study, we developed rapid and sensitive assays for the detection of Cladophialophora carrionii, a common agent of human chromoblastomycosis. The isothermal techniques evaluated were rolling-circle amplification (RCA), multiplex ligation-dependent probe amplification (MLPA), and loop-mediated isothermal amplification (LAMP). The probes for RCA and MLPA were designed with target sequences in the rDNA internal transcribed spacer gene (ITS) region, and LAMP primers were designed using the elongation factor 1α gene (EF1); these probes and primers specifically amplified DNA of isolates of the species. The three techniques were sufficiently specific and sensitive for discriminating target DNA of C. carrionii from that of related Cladophialophora species and other agents of chromoblastomycosis. RCA, MLPA, and LAMP are advantageous in their reliability and ease of operation compared to standard PCR and conventional methods.     

Glucose-based dialysis fluids inhibit innate defense against Staphylococcus aureus

BackgroundStaphylococcus aureus peritonitis is a serious complication of Chronic Peritoneal Dialysis (CPD) and associated with a higher risk for severe and recurrent infections compared with other bacteria. We have previously shown that complement-mediated effectors essential for optimal opsonophagocytosis of S. aureus are inhibited by high glucose concentrations. Since most commonly used peritoneal dialysis (PD) fluids are glucose-based, we hypothesized that glucose-based PD fluids likely inhibit complement host defenses against S. aureus.MethodsCommercially available PD fluids were tested: glucose-based (Dianeal), Dianeal supplemented with amino acids, icodextrin-based (Extraneal) and amino acid-based (Nutrineal). Control PD fluid was generated to simulate Dianeal excluding the glucose. Three commercially available glucose concentrations were tested: Dianeal 1.5% (15gm/1000 ml), Dianeal 2.5% (25 gm/1000 ml) and Dianeal 4.25% (42.5 gm/1000 ml). Complement effectors against S. aureus were analyzed including opsonization with C3-fragments, anaphylatoxin generation, and phagocytosis efficiency. We also evaluated clinical strains, including MRSA strains, and specific complement activation pathways.ResultsGlucose-based PD fluids inhibited complement opsonization of S. aureus (≥7-fold reduction) and inhibited S. aureus-induced generation of anaphylatoxins C3a and C5a (>10-fold reduction) compared to non-glucose based PD fluids. Dianeal 1.5%, 2.5% and 4.25%, all similarly inhibited C3-mediated opsonization. Glucose-based PD fluids showed a ≥4-fold reduction in opsonization of clinical strains of S.aureus, including MRSA strains. Decreased opsonization of S.aureus in the glucose-based PD fluid compared with non-glucose based fluids correlated with decreased phagocytosis by neutrophils.ConclusionComplement-mediated opsonophagocytosis of S. aureus and anaphylatoxin generation were severely inhibited in glucose-based PD fluids compared with non-glucose-based PD fluids. By inhibiting complement host defenses, glucose-based PD fluids may increase the risk of and severity of S. aureus peritonitis for CPD patients using these fluids.     

Comparison of the Copan WASPLab incorporating the BioRad expert system against the SIRscan 2000 automatic for routine antimicrobial disc diffusion susceptibility testing

ObjectivesThis study investigated the agreement at the categorical level between the Copan WASPLab incorporating the BioRad expert system against the SIRscan 2000 automatic for antimicrobial disc diffusion susceptibility testing.MethodsThe 338 clinical strains (67 Pseudomonas aeruginosa, 19 methicillin-resistant Staphylococcus aureus, 75 methicillin-sensitive S. aureus and 177 Enterobacterales isolates) analysed in this study were non-duplicate isolates obtained from consecutive clinical samples referred to the clinical bacteriology laboratory at Geneva University Hospitals between June and August 2019. For the WASPLab the inoculum suspension was prepared in strict accordance with the manufacturer's instruction (Copan WASP srl, Brescia, Italy) by adding 2 mL of the 0.5 McFarland primary suspension used for the SIRscan analysis into a sterile tube filled with 4 mL of sterile saline (1:3 dilution). The inoculum (2 × 30 μL loop/spreader) was spread over the entire surface of Mueller–Hinton agar plates according to the AST streaking pattern defined by Copan. The antibiotic discs were dispensed by the WASP and inoculated media were loaded on conveyors for transfer to the automatic incubators. The plates were incubated for 16 h, and several digital images were acquired. Inhibition zone diameters were automatically read by the WASPLab and were adjusted manually whenever necessary. For the SIRscan 2000 automatic, the antimicrobial disc diffusion susceptibility testing was performed according to the EUCAST guidelines. The gradient strip method was used to resolve discrepancies.ResultsThe overall categorical agreement between the compared methods reached 99.1% (797/804; 95% CI 98.2%–99.6%), 99.5% (1029/1034; 95% CI 98.9%–99.8%), and 98.8% (2798/2832; 95% CI 98.3%–99.1%) for P. aeruginosa, S. aureus and the Enterobacterales, respectively.ConclusionsWASPLab incorporating the BioRad expert system provides a fully automated solution for antimicrobial disc diffusion susceptibility testing with equal or better accuracy than other available phenotypic methods.