TechLab and Alexon Giardia Enzyme-Linked Immunosorbent Assay Kits Detect Cyst Wall Protein 1

A Giardia lamblia antigen detected by the TechLab Giardia Test (TechLab, Inc., Blacksburg, Va.) and the Alexon ProSpecT Giardia microplate assay (Alexon, Inc., Sunnyvale, Calif.) was purified by immunoaffinity chromatography from supernatant fluids of encystment cultures. Two major proteins (M_r 22,000 and 26,000) were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining that did not resemble the GSA65 antigen reportedly detected by the Alexon test. These proteins reacted intensely with the monoclonal antibodies used in both commercial enzyme-linked immunosorbent assays (ELISAs). Both proteins had identical N-terminal amino acid sequences and were identified as cyst wall protein 1 (CWP1). The 26-kDa form appeared early during encystment followed by the appearance of the 22-kDa form. Recombinant CWP1 (M_r 26,000) was strongly positive in both commercial tests. CWP1 was stable in human stool specimens, resistant to degradation by proteases and N- and O-glycanases, and unaffected by oxidation with sodium periodate. Two minor proteins with M_rs of 32,000 and 39,000 were detected in CWP1 preparations by using a sensitive fluorescent protein stain. Both were identified as CWP2, and neither reacted with the monoclonal antibodies from the commercial tests. We analyzed 535 stool specimens for CWP1 by using both commercial ELISAs and resolved discrepant results by using routine ova and parasite examination (O&P) and on immunofluorescence antibody assay. The presence of CWP1 correlated well between both ELISAs (98.7% correlation). Our results demonstrate that both commercial ELISAs detect CWP1, which is a useful diagnostic marker because it is highly stable, is secreted in large amounts by encysting trophozoites, and correlates well with O&P.     

In vitro activity of anidulafungin,caspofungin, fluconazole and amphotericin B against biofilms and planktonic forms of Candida species isolated from blood culture in patients with hematological malignancies

ObjectiveThe aim of the present study was to evaluate the in vitro susceptibility of anidulafungin, caspofungin, fluconazole and conventional amphotericin B against biofilms and planktonic forms of Candida species isolated from blood culture in patients with hematological malignancies.Materials and methodsAntifungal susceptibility for planktonic forms and biofilms of Candida was determined by broth microdilution method as described by Clinical and Laboratory Standards Institute M27 methodology and metabolic XTT-based [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay, respectively.ResultsA total of 75 Candida isolates were evaluated between 2006–2018 yy at the National Research Center for Hematology, Russia, Moscow. Biofilm production was detected in 34 (45.3%) Candida species. Antifungal susceptibility was tested for 27 common species of Candida forming biofilms (8 C.krusei, 7 C.tropicalis, 7 C.albicans, 5 C.parapsilosis).MICs below the susceptibility breakpoints were found for 100% of planktonic forms of Candida species for anidulafungin, 85.2% for caspofungin, and 66.7% for fluconazole. Amphotericin B MIC₉₀ for Candida species were less than or equal to 1 μg/ml. Candida biofilms were susceptible in vitro for both tested echinocandins, but MIC₈₀ of anidulafungin were lower compared to caspofungin. The highest MIC₈₀ against Candida biofilms was found for fluconazole (>1,024 μg/ml for all tested isolates) and for conventional amphotericin B (range 4–16 μg/ml).ConclusionThe majority of Candida isolates grown as planktonic forms were susceptible to anidulafungin, caspofungin, conventional amphotericin B and fluconazole. Anidulafungin displayed higher activity against Candida biofilms than caspofungin. All Candida biofilms were resistant to fluconazole and conventional amphotericin B.     

Eleven-year retrospective survey of candidaemia in a university hospital in southwestern Greece

The aim of this study was to investigate the isolation and distribution rate of Candida spp. in blood cultures and evaluate antifungal susceptibility during an 11-year period (1998–2008) at a tertiary-care hospital. The causative species were as follows: Candida albicans, 163 strains (64%); Candida parapsilosis, 35 strains (13.7%); Candida glabrata, 25 strains (9.8%); Candida tropicalis, 19 strains (7.4%); and other Candida spp., 13 strains (5.1%). Candidaemia is predominantly caused by C. albicans. C. parapsilosis is the most common non-albicans Candida isolated in neonatal intensive-care units. All Candida isolates remain susceptible to amphotericin B, whereas the highest degree of resistance was observed for azoles.     

Species identification and antifungal susceptibility of uncommon blood yeast isolates

Background/PurposeAccurate identification of Candida species is increasingly important in the era of emergence of Candida auris. We aimed to compare the identification performance of two matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems (Vitek MS and Bruker biotyper MS) and an oligonucleotide array for uncommon blood yeast isolates and demonstrate the susceptibilities among those isolates.MethodCandida species isolates from blood culture other than Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, and Candida krusei identified by biochemical methods were collected from multiple hospitals and further identified by an oligonucleotide array based on the internal transcribed spacer-1 (ITS-1) and ITS-2 sequences of the rRNA genes, Vitek MS and Bruker biotyper MS. The minimal inhibitory concentrations (MICs) of these clinical isolates were determined by the Sensititre YeastOne (SYO) system.ResultsAmong 136 isolates, Candida guilliermondii was most common (52, 38.2%), followed by C. lusitaniae (13, 9.6%) and C. haemulonii (12, 8.8%). The oligonucleotide array, Vitek MS and Bruker biotyper MS correctly identified 89.7% (122), 90.4% (123), and 92.6% (126) of these isolates, respectively. Elevated minimal inhibitory concentrations (MICs) of fluconazole were observed for C. haemulonii (MIC₉₀: 256 mg/L), and C. guilliermondii (MIC₉₀: 16 mg/L) with 28.4% of uncommon Candida isolates with MIC ≧ 8 mg/L.ConclusionsFor uncommon Candida species, the unmet need for current databases of two commercial MALDI-TOF MS systems is highlighted, and the oligonucleotide array may serve as a supplement.     

Role of glutathione in the oxidative stress response in the fungal pathogen Candida glabrata

Candida glabrata, an opportunistic fungal pathogen, accounts for 18–26 % of all Candida systemic infections in the US. C. glabrata has a robust oxidative stress response (OSR) and in this work we characterized the role of glutathione (GSH), an essential tripeptide-like thiol-containing molecule required to keep the redox homeostasis and in the detoxification of metal ions. GSH is synthesized from glutamate, cysteine, and glycine by the sequential action of Gsh1 (γ-glutamyl-cysteine synthetase) and Gsh2 (glutathione synthetase) enzymes. We first screened for suppressor mutations that would allow growth in the absence of GSH1 (gsh1? background) and found a single point mutation in PRO2 (pro2-4), a gene that encodes a γ-glutamyl phosphate reductase and catalyzes the second step in the biosynthesis of proline. We demonstrate that GSH is important in the OSR since the gsh1? pro2-4 and gsh2? mutant strains are more sensitive to oxidative stress generated by H₂O₂ and menadione. GSH is also required for Cadmium tolerance. In the absence of Gsh1 and Gsh2, cells show decreased viability in stationary phase. Furthermore, C. glabrata does not contain Saccharomyces cerevisiae high affinity GSH transporter ortholog, ScOpt1/Hgt1, however, our genetic and biochemical experiments show that the gsh1? pro2-4 and gsh2? mutant strains are able to incorporate GSH from the medium. Finally, GSH and thioredoxin, which is a second redox system in the cell, are not essential for the catalase-independent adaptation response to H₂O₂.     

Vulvovaginal candidiasis among symptomatic women of childbearing age attended at a Medical Analysis Laboratory in Franceville,Gabon

BackgroundVulvovaginal candidiasis (VVC) is one of the most common lower genital tract infections in women; this unpleasant and extremely embarrassing pathology is one of the main reasons for gynaecological consultation. In Gabon, the prevalence of VVC remains poorly described even though VVC is known to be the leading gynaecological condition in several countries. This retrospective cross-sectional study sought to assess the prevalence of VVC among symptomatic women in southeastern Gabon.MethodsClinical samples were collected from patients suspected to have VVC during a 2-year period (from January 2016 to December 2017). Gram staining of vaginal smears provided indications of vaginal flora and confirmed the presence of yeast. Sabouraud-chloramphenicol and chromID Candida media were used to isolate yeast, and species identification was performed using morphological tests and the Vitek 2 Compact automated system.ResultsFor the 873 patients included in this study, the prevalence of VVC was 28.52%. Eleven Candida species were identified, with greater representation of Candida albicans (82.73%) than of Non C. albicans candida (NCAC) (17.27%), which were distributed as follows: Candida famata (4.02%), Candida spp. (3.61%), Candida rugosa (3.21%), Candida lipolytica (1.61%), Candida parapsilosis (1.61%), Candida glabrata (1.21%), Candida tropicalis (0.80%), Candida krusei (0.40%), Candida dubliniensis (0.40%), and Candida sphaerica (0.40%).ConclusionThis study offers the first estimation of VVC among Gabonese women in childbearing age with the symptoms. It showed that VVC is very common in Gabon. C. albicans as the most commonly represented species.     

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.     

Recurrent vulvo-vaginal candidiasis in Abidjan (Côte d’Ivoire): Aetiology and associated factors

Recurrent vulvovaginal candidiasis (RVVC) is a major health problem for sexually active women because of its severe effect on their quality of life. A thorough knowledge of their epidemiology leads to their efficient management. Therefore, a cross-sectional study was conducted in 2014 in women with leucorrhoea associated or not with other clinical signs. Recurrence was based on the occurrence of at least four annual episodes of Candida vulvo-vaginitis. An individual interview based on a questionnaire was conducted to identify the socio-demographic parameters that could be associated with the RVVC. Vaginal samples were collected at the obstetrical gynaecology department of the University Hospital of Cocody and at the Pasteur Institute of Côte d’Ivoire. On each sample, a direct examination and culture on Sabouraud-chloramphenicol medium with or without actidione were performed. Yeast identification was performed using chromogenic media (CandiSelect^®4 [Bio-Rad]) and the study of sugar assimilation using the Auxacolor^® 2 gallery (Bio-Rad). A total of 400 patients were included. The average age was 29.2 years (SD = 7.2 years). Of these, 94 had recurrent vulvovaginal candidiasis, with a prevalence of 23.5% (CI_95%: 19.49–28.02). Five species of the genus Candida have been identified: Candida albicans (59.6%), Candida glabrata (19.1%), Candida tropicalis (16%), Candida krusei (4.2%) and Candida inconspicua (1.1%). Some factors such as education level, history of sexually transmitted infection, type of underwear used, frequency of personal hygiene and type of product used for these hygiene have been associated with the occurrence of RVVCs. The occurrence of RVVCs is relatively high in our study population. Non-albicans Candida species occupy a significant place in this disease epidemiology. By addressing the factors associated with the occurrence and/or persistence of RVVCs, it will be possible to reduce their incidence in sexually active women.     

A Comprehensive Evaluation of the Bruker Biotyper MS and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Yeasts,Part of the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Study, 2012 to 2013

Among the 2,683 yeast isolates representing 41 different species (25 Candida and Candida-related species and 16 non-Candida yeast species) collected in the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program (2012 to 2013), the Bruker Biotyper MS matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system exhibited significantly higher accuracy rates than the Vitek MS system for identification of all yeast isolates (98.8% versus 95.4%, P <0.001 by Pearson''s chi-square test) and for all Candida and Candida-related species isolates (99.4% versus 95.5%, P < 0.001).     

In Vitro Activities of Isavuconazole and Comparator Antifungal Agents Tested against a Global Collection of Opportunistic Yeasts and Molds

Isavuconazole is a new broad-spectrum triazole with a favorable pharmacokinetic and safety profile. We report the MIC distributions for isavuconazole and 111 isolates of Candida (42 Candida albicans, 25 Candida glabrata, 22 Candida parapsilosis, 14 Candida tropicalis, and 8 Candida krusei isolates), as determined by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution (BMD) methods. Also, the relative activities of isavuconazole, itraconazole, fluconazole, posaconazole, voriconazole, and the three echinocandins were assessed against a recent (2011) global collection of 1,358 isolates of Candida spp., 101 of Aspergillus spp., 54 of non-Candida yeasts, and 21 of non-Aspergillus molds using CLSI BMD methods. The overall essential agreement (EA) (±2 log₂ dilutions) between the CLSI and EUCAST methods was 99.1% (EA at ±1 log₂ dilution, 90.1% [range, 80.0 to 100.0%]). The activities of isavuconazole against the larger collection of Candida spp. and Aspergillus spp. were comparable to those of posaconazole and voriconazole; the MIC₉₀ values for isavuconazole, posaconazole, and voriconazole against Candida spp. were 0.5, 1, and 0.25 μg/ml and against Aspergillus spp. were 2, 1, and 1 μg/ml, respectively. Isavuconazole showed good activities against Cryptococcus neoformans (MIC₉₀, 0.12 μg/ml) and other non-Candida yeasts (MIC₉₀, 1 μg/ml) but was less potent against non-Aspergillus molds (MIC₉₀, >8 μg/ml). Isavuconazole MIC values for three mucormycete isolates were 4, 1, and 2 μg/ml, whereas all three were inhibited by 1 μg/ml posaconazole. Isavuconazole demonstrates broad-spectrum activity against this global collection of opportunistic fungi, and the CLSI and EUCAST methods can be used to test this agent against Candida, with highly comparable results.     

Validation of 24-hour fluconazole MIC readings versus the CLSI 48-hour broth microdilution reference method: results from a global Candida antifungal surveillance program

We performed 24- and 48-h MIC determinations and disk diffusion testing of fluconazole against more than 11,000 clinical isolates of Candida species. By using the reference MIC breakpoints, the categorical agreement between the 24-h and reference 48-h broth microdilution results ranged from 93.8% (all Candida species) to 94.9% (all Candida species minus Candida krusei), with only 0.1% very major errors (VME). The essential agreement (within 2 log₂ dilutions) between the 24-h and 48-h results was 99.6%. The categorical agreement between the 24-h disk diffusion results and the 24-h MIC results, using the previously established breakpoints, was 94.4%, with 0.1% VME. Both the MIC and the disk diffusion results obtained for fluconazole after only 24 h of incubation may be used to determine the susceptibilities of Candida spp. to this widely used antifungal agent.     

In vitro susceptibility of invasive isolates of Candida spp. to anidulafungin, caspofungin, and micafungin: six years of global surveillance

The echinocandins are being used increasingly as therapy for invasive candidiasis. Prospective sentinel surveillance for the emergence of in vitro resistance to the echinocandins among invasive Candida sp. isolates is indicated. We determined the in vitro activities of anidulafungin, caspofungin, and micafungin against 5,346 invasive (bloodstream or sterile-site) isolates of Candida spp. collected from over 90 medical centers worldwide from 1 January 2001 to 31 December 2006. We performed susceptibility testing according to the CLSI M27-A2 method and used RPMI 1640 broth, 24-h incubation, and a prominent inhibition endpoint for determination of the MICs. Of 5,346 invasive Candida sp. isolates, species distribution was 54% C. albicans, 14% C. parapsilosis, 14% C. glabrata, 12% C. tropicalis, 3% C. krusei, 1% C. guilliermondii, and 2% other Candida spp. Overall, all three echinocandins were very active against Candida: anidulafungin (MIC₅₀, 0.06 μg/ml; MIC₉₀, 2 μg/ml), caspofungin (MIC₅₀, 0.03 μg/ml; MIC₉₀, 0.25 μg/ml), micafungin (MIC₅₀, 0.015 μg/ml; MIC₉₀, 1 μg/ml). More than 99% of isolates were inhibited by ≤2 μg/ml of all three agents. Results by species (expressed as the percentages of isolates inhibited by ≤2 μg/ml of anidulafungin, caspofungin, and micafungin, respectively) were as follows: for C. albicans, 99.6%, 100%, and 100%; for C. parapsilosis, 92.5%, 99.9%, and 100%; for C. glabrata, 99.9%, 99.9%, and 100%; for C. tropicalis, 100%, 99.8%, and 100%; for C. krusei, 100%, 100%, and 100%; and for C. guilliermondii, 90.2%, 95.1%, and 100%. There was no significant change in the activities of the three echinocandins over the 6-year study period and no difference in activity by geographic region. All three echinocandins have excellent in vitro activities against invasive strains of Candida isolated from centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging echinocandin resistance among invasive clinical isolates of Candida spp.     

Involvement of amyloid proteins in the formation of biofilms in the pathogenic yeast Candida albicans

Candida species represent a major fungal threat for human health. Within the Candida genus, the yeast Candida albicans is the most frequently incriminated species during episodes of candidiasis or candidemia. Biofilm formation is used by C. albicans to produce a microbial community that is important in an infectious context. The cell wall, the most superficial cellular compartment, is of paramount importance regarding the establishment of biofilms. C. albicans cell wall contains proteins with amyloid properties that are necessary for biofilm formation due to their adhesion properties. This review focuses on these amyloid proteins during biofilm formation in the yeast C. albicans.     

Echinocandin and Triazole Antifungal Susceptibility Profiles for Clinical Opportunistic Yeast and Mold Isolates Collected from 2010 to 2011: Application of New CLSI Clinical Breakpoints and Epidemiological Cutoff Values for Characterization of Geographic and Temporal Trends of Antifungal Resistance

The SENTRY Antimicrobial Surveillance Program monitors global susceptibility and resistance rates of newer and established antifungal agents. We report the echinocandin and triazole antifungal susceptibility patterns for 3,418 contemporary clinical isolates of yeasts and molds. The isolates were obtained from 98 laboratories in 34 countries during 2010 and 2011. Yeasts not presumptively identified by CHROMagar, the trehalose test, or growth at 42°C and all molds were sequence identified using internal transcribed spacer (ITS) and 28S (yeasts) or ITS, translation elongation factor (TEF), and 28S (molds) genes. Susceptibility testing was performed against 7 antifungals (anidulafungin, caspofungin, micafungin, fluconazole, itraconazole, posaconazole, and voriconazole) using CLSI methods. Rates of resistance to all agents were determined using the new CLSI clinical breakpoints and epidemiological cutoff value criteria, as appropriate. Sequencing of fks hot spots was performed for echinocandin non-wild-type (WT) strains. Isolates included 3,107 from 21 Candida spp., 146 from 9 Aspergillus spp., 84 from Cryptococcus neoformans, 40 from 23 other mold species, and 41 from 9 other yeast species. Among Candida spp., resistance to the echinocandins was low (0.0 to 1.7%). Candida albicans and Candida glabrata that were resistant to anidulafungin, caspofungin, or micafungin were shown to have fks mutations. Resistance to fluconazole was low among the isolates of C. albicans (0.4%), Candida tropicalis (1.3%), and Candida parapsilosis (2.1%); however, 8.8% of C. glabrata isolates were resistant to fluconazole. Among echinocandin-resistant C. glabrata isolates from 2011, 38% were fluconazole resistant. Voriconazole was active against all Candida spp. except C. glabrata (10.5% non-WT), whereas posaconazole showed decreased activity against C. albicans (4.4%) and Candida krusei (15.2% non-WT). All agents except for the echinocandins were active against C. neoformans, and the triazoles were active against other yeasts (MIC₉₀, 2 μg/ml). The echinocandins and triazoles were active against Aspergillus spp. (MIC₉₀/minimum effective concentration [MEC₉₀] range, 0.015 to 2 μg/ml), but the echinocandins were not active against other molds (MEC₉₀ range, 4 to >16 μg/ml). Overall, echinocandin and triazole resistance rates were low; however, the fluconazole and echinocandin coresistance among C. glabrata strains warrants continued close surveillance.     

A multicentre study of antifungal strategies and outcome of Candida spp. peritonitis in intensive-care units

Information on the species causing Candida peritonitis, their in vitro susceptibility, antifungal strategies in this setting and patient outcome is still scarce. AmarCand was a prospective, non-interventional study in 271 adult intensive-care unit (ICU) patients with proven invasive Candida infection who received systemic antifungal therapy (France, 2005–2006). Of these ICU patients, 93 (median age 65 years, simplified acute physiology score II 52) had Candida peritonitis, including 73 nosocomial peritonitis, 53 concomitant bacterial peritoneal infections and 26 candidaemias. Candida species were C. albicans (n = 63/108 isolates, 58%), C. glabrata (n = 22, 20%), C. krusei (n = 9), C. kefyr (n = 5), C. parapsilosis (n = 3), C. tropicalis (n = 3), C. ciferii (n = 2) and C. lusitaniae (n = 1). Of tested isolates, 28% were fluconazole-resistant or susceptible dose-dependent (C. albicans 3/32, C. glabrata 9/14, C. krusei 4/4). Empiric antifungal treatment was started 1 day (median) after peritonitis diagnosis, with fluconazole (n = 72 patients), caspofungin (n = 12), voriconazole (n = 3), amphotericin B (n = 2), or a combination (n = 4). Following susceptibility testing, empiric antifungal treatment was judged inadequate in 9/45 (20%) patients and modified in 30 patients (fluconazole was replaced by caspofungin (n = 14) or voriconazole (n = 4)). Mortality in ICU was 38% (35/93) and was not influenced by type of Candida species, fluconazole susceptibility, time to treatment, candidaemia, nosocomial acquisition, or concomitant bacterial infection. No specific factors for death were identified. In summary, a high proportion of fluconazole-resistant or susceptible dose-dependent strains was cultured. These results confirm the high mortality rates of Candida peritonitis and plead for additional investigation in this population. Antifungal treatment for severe cases of Candida peritonitis in ICU patients remains the standard care.     

Changing Virulence Factors among Vaginal Non-albicans Candida species

Background: Vulvovaginal candidiasis (VVC) is caused by overgrowth of Candida species in the female lower genital tract and most commonly caused by Candida albicans. The production of various virulence factors may attribute to their pathogenicity. Hence, this study was aimed to determine the production of various virulence factors of Candida spp. causing VVC. Materials and Methods: A total of 51 Candida spp. were isolated prospectively from 50 patients among 211 clinically suspected cases of VVC. The haemolytic activity, biofilm production, proteinase activity, phospholipase activity and esterase activity were detected by standard methods. Statistical analysis was performed using OpenEpi version 3.01. Results: Haemolytic activity was observed in 42 Candida isolates (82.4%), biofilm activity in 21 Candida isolates (41.2%), proteinase and esterase activity in 19 Candida isolates (37.3%) each and phospholipase activity in 15 Candida isolates (29.4%). Phospholipase activity was observed in all of the C. albicans strains, whereas all strains of Candida krusei were able to produce biofilm. All strains of Candida parapsilosis and 87% strains of Candida glabrata were haemolytic. Five of the eight C. glabrata strains were found to produce strong proteinase (Pr_z score ≤0.63). About 30.4% strains of C. glabrata and 20% strains of C. krusei were found to be positive for esterase activity. This is one of the few studies which revealed esterase activity among C. glabrata and C. krusei strains. Conclusions: This study highlighted that there is a change in the virulence factors among the non-albicans Candida species, especially C. glabrata strains which were haemolytic and produce strong proteinase activity and esterase activity. It may be one of the explanation of the most common causative agent of VVC in our study. Multicentric studies from this area might be required to get a more generalised conclusion.     

Multicenter Evaluation of the New Vitek 2 Yeast Susceptibility Test Using New CLSI Clinical Breakpoints for Fluconazole

A fully automated antifungal susceptibility test system recently updated to reflect the new species-specific clinical breakpoints (CBPs) of fluconazole for Candida (Vitek 2 AF03 yeast susceptibility test; bioMérieux, Inc., Durham, NC) was compared in three different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) method by testing 2 quality control strains, 10 reproducibility strains (4 Candida species and 6 Cryptococcus neoformans strains), and 746 isolates of Candida species (702 isolates, 13 species) and 44 isolates of C. neoformans against fluconazole. Excellent essential agreement (EA) (within 2 dilutions) between the reference and Vitek 2 MICs was observed for fluconazole and Candida species (94.0%). The EA was lower for fluconazole and C. neoformans at 86.4%. The mean times to a result with the Vitek 2 test were 9.1 h for Candida species and 12.1 h for C. neoformans. Categorical agreement (CA) between the two methods was assessed by using the new species-specific CBPs. For less common species without fluconazole CBPs, the epidemiological cutoff values (ECVs) were used to differentiate wild-type (WT; MIC, ≤ECV) from non-WT (MIC, >ECV) strains. The CAs between the two methods were 92.0% for Candida species (0.3% very major errors [VME] and 2.6% major errors [ME]) and 84.1% for C. neoformans (4.5% VME and 11.4% ME). The updated Vitek 2 AF03 IUO yeast susceptibility system is comparable to the CLSI BMD reference method for testing the susceptibility of clinically important yeasts to fluconazole when using the new (lower) CBPs and ECVs.     

Role for the fibrinogen-binding proteins Coagulase and Efb in the Staphylococcus aureus–Candida interaction

Staphylococcus aureus and Candida species are increasingly coisolated from implant-associated polymicrobial infections creating an incremental health care problem. Synergistic effects between both genera seem to facilitate the formation of mixed S. aureus–Candida biofilms, which is thought to play a critical role in coinfections with these microorganisms. To identify and characterize S. aureus factors involved in the interaction with Candida species, we affinity-panned an S. aureus phage display library against Candida biofilms in the presence or absence of fibrinogen. Repeatedly isolated clones contained DNA fragments encoding portions of the S. aureus fibrinogen-binding proteins coagulase or Efb. The coagulase binds to prothrombin in a 1:1 ratio thereby inducing a conformational change and non-proteolytic activation of prothrombin, which in turn cleaves fibrinogen to fibrin. Efb has been known to inhibit opsonization. To study the role of coagulase and Efb in the S. aureus–Candida cross-kingdom interaction, we performed flow-cytometric phagocytosis assays. Preincubation with coagulase reduced the phagocytosis of Candida yeasts by granulocytes significantly and dose-dependently. By using confocal laser scanning microscopy, we demonstrated that the coagulase mediated the formation of fibrin surrounding the candidal cells. Furthermore, the addition of Efb significantly protected the yeasts against phagocytosis by granulocytes in a dose-dependent and saturable fashion. In conclusion, the inhibition of phagocytosis of Candida cells by coagulase and Efb via two distinct mechanisms suggests that S. aureus might be beneficial for Candida to persist as it helps Candida to circumvent the host immune system.     

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.