Evaluation of Microsatellite Typing, ITS Sequencing, AFLP Fingerprinting, MALDI-TOF MS, and Fourier-Transform Infrared Spectroscopy Analysis of Candida auris.

Candida auris is an emerging opportunistic yeast species causing nosocomial outbreaks at a global scale. A few studies have focused on the C. auris genotypic structure. Here, we compared five epidemiological typing tools using a set of 96 C. auris isolates from 14 geographical areas. Isolates were analyzed by microsatellite typing, ITS sequencing, amplified fragment length polymorphism (AFLP) fingerprint analysis, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and Fourier-transform infrared (FTIR) spectroscopy methods. Microsatellite typing grouped the isolates into four main clusters, corresponding to the four known clades in concordance with whole genome sequencing studies. The other investigated typing tools showed poor performance compared with microsatellite typing. A comparison between the five methods showed the highest agreement between microsatellite typing and ITS sequencing with 45% similarity, followed by microsatellite typing and the FTIR method with 33% similarity. The lowest agreement was observed between FTIR spectroscopy, MALDI-TOF MS, and ITS sequencing. This study indicates that microsatellite typing is the tool of choice for C. auris outbreak investigations. Additionally, FTIR spectroscopy requires further optimization and evaluation before it can be used as an epidemiological typing method, comparable with microsatellite typing, as a rapid method for tracing nosocomial fungal outbreaks.

Anidulafungin Susceptibility Testing of Candida glabrata Isolates from Blood Cultures by the MALDI Biotyper Antibiotic (Antifungal) Susceptibility Test Rapid Assay.

Echinocandins are the recommended first-line antifungals for treatment of invasive candidiasis. The increasing number of Candida glabrata strains resistant against echinocandins is an emerging health care concern. The rapid detection of resistant C. glabrata isolates is an urgent requirement for clinical laboratories. In this study, we developed the MALDI Biotyper antibiotic (antifungal) susceptibility test rapid assay (MBT ASTRA) for the rapid detection of anidulafungin-resistant C. glabrata isolates directly from positive blood cultures. Of 100 C. glabrata strains, MBT ASTRA classified 69 as susceptible and 29 as resistant. Microdilution assays performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, used as a standard reference, identified 65 susceptible, 9 intermediate, and 26 resistant isolates. Sequencing of hot spot 1 and hot spot 2 regions of the FKS1 and FKS2 genes classified 86 susceptible and 14 resistant isolates. The MBT ASTRA had sensitivity and specificity of 80% and 95%, respectively, compared to the microdilution method. Positive and negative agreement of MBT ASTRA was calculated at 100% and 80%, respectively, compared with the molecular sequencing approach. Together, these results revealed a high accuracy of MBT ASTRA compared to microdilution according to the CLSI and PCR analysis, resulting in a categorical agreement of 90% and 83%, respectively. The validity of MBT ASTRA was 98%. Importantly, MBT ASTRA provided antifungal susceptibility testing (AFST) within 6 h that was both accurate and reliable compared to the other two approaches, which require at least 24 h or are costly. Therefore, this method has the potential to facilitate clinical AFST rapidly at low sample costs for clinical labs already equipped with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Candida auris Identification and Rapid Antifungal Susceptibility Testing Against Echinocandins by MALDI-TOF MS.

Candida auris was first reported in an ear swab from Japan in 2009; it then promptly spread over five continents and turned into a global nosocomial problem. The main challenges faced by many researchers are the mis-identification by conventional methods in clinical laboratories and failure in treatment. About 90% of C. auris strains are intrinsically resistant to fluconazole (FLU), and it is developing resistance to multiple classes of available antifungals. Echinocandins are the most potent class of antifungals against C. auris; however, reduced susceptibility to one or many echinocandin drugs has been recently observed. Thus, the main issues addressed in this paper are the fast and accurate identification of C. auris derived from Sabouraud dextrose agar and blood culture bottles as well as the rapid antifungal susceptibility test by MALDI-TOF MS. This study successfully identified all isolates of C. auris (n = 50) by MALDI-TOF MS, with an average log score of ≥ 2. An accuracy of 100% was found on both agar plate and blood culture bottles. MALDI Biotyper antibiotic susceptibility test-rapid assay (MBT ASTRA) was used for rapid antifungal susceptibility testing (AFST). A comparison between MBT ASTRA and the Clinical and Laboratory Standards Institute guidelines (CLSI) detected a sensitivity and specificity of 100% and 98% for anidulafungin, and 100% and 95.5% for micafungin, respectively. A categorical agreement of 98% and 96% was calculated for the two methods. For caspofungin, sensitivity and specificity of 100 and 73% were found, respectively, with a categorical agreement of 82%. MBT ASTRA has the great potential to detect C. auris isolates non-susceptible against echinocandin antifungals within 6 h, which makes it a promising candidate for AFST in clinical laboratories in the future.

Proof of Concept for MBT ASTRA, a Rapid Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)-Based Method To Detect Caspofungin Resistance in Candida albicans and Candida glabrata.

The incidence of candidemia caused by Candida albicans and Candida glabrata is constantly increasing and is accompanied by the rising use of the few available antifungals. The widespread use of echinocandins and azoles for the treatment of invasive candidemia has enhanced the development of antifungal resistance, resulting in an increasing health care problem. Hence, the rapid detection of resistant strains is required. This study aimed to evaluate the detection of C. albicans and C. glabrata strains resistant to caspofungin by the matrix-assisted laser desorption ionization Biotyper antibiotic susceptibility test rapid assay (MBT ASTRA). This novel semiquantitative technique facilitates the detection of caspofungin-resistant strains within 6 h. MBT ASTRA results were compared to the data obtained by the use of Clinical and Laboratory Standards Institute (CLSI) guidelines. Clinical isolates of C. albicans (n = 58) and C. glabrata (n = 57) were analyzed by MBT ASTRA and the CLSI microdilution method. Antifungal susceptibility testing against caspofungin by the CLSI microdilution method classified the C. albicans isolates into 36 susceptible and 22 resistant strains and the C. glabrata isolates into 5 susceptible, 33 resistant, and 19 intermediate strains. For C. albicans, the comparison of MBT ASTRA and the CLSI method revealed an excellent categorical agreement of 100%. A sensitivity and a specificity between MBT ASTRA and the CLSI microdilution method of 94% and 80%, respectively, were detected for C. glabrata strains, based on categorical agreement. In conclusion, the results obtained by MBT ASTRA indicate that this is a very promising approach for the rapid detection of Candida isolates resistant to caspofungin.

Prospective evaluation of the chromogenic medium CandiSelect 4 for differentiation and presumptive identification of non-Candida albicans Candida species.

Rapid identification of pathogenic yeasts is a crucial step in timely and appropriate antifungal therapy. For diagnostics in the clinical laboratory, simplified alternatives to barcoding are needed. CandiSelect 4 (CS4) medium, a chromogenic medium for isolation of clinical yeasts, allows routine recognition of Candida albicans and presumptive identification of Candida tropicalis, Candida glabrata, and Candida krusei. We evaluated an extension of this method with 46 non-Candida albicans Candida (NCAC) and 7 Malassezia species. The medium supported growth of all species tested and a wide diversity of cultural types were observed. Colony colours were in violet, turquoise (including green and blue), or white tinges. Eight NCAC species produced violet pigmentation similar to that of C. albicans. Most NCAC species, including C. glabrata and C. tropicalis were distributed in the turquoise group. Malassezia species were invariably blue.

Safety, Immunogenicity, and Protective Efficacy of Intradermal Immunization with Aseptic, Purified, Cryopreserved Plasmodium falciparum Sporozoites in Volunteers Under Chloroquine Prophylaxis: A Randomized Controlled Trial.

Immunization of volunteers under chloroquine prophylaxis by bites of Plasmodium falciparum sporozoite (PfSPZ)-infected mosquitoes induces > 90% protection against controlled human malaria infection (CHMI). We studied intradermal immunization with cryopreserved, infectious PfSPZ in volunteers taking chloroquine (PfSPZ chemoprophylaxis vaccine [CVac]). Vaccine groups 1 and 3 received 3× monthly immunizations with 7.5 × 10(4) PfSPZ. Control groups 2 and 4 received normal saline. Groups 1 and 2 underwent CHMI (#1) by mosquito bite 60 days after the third immunization. Groups 3 and 4 were boosted 168 days after the third immunization and underwent CHMI (#2) 137 days later. Vaccinees (11/20, 55%) and controls (6/10, 60%) had the same percentage of mild to moderate solicited adverse events. After CHMI #1, 8/10 vaccinees (group 1) and 5/5 controls (group 2) became parasitemic by microscopy; the two negatives were positive by quantitative real-time polymerase chain reaction (qPCR). After CHMI #2, all vaccinees in group 3 and controls in group 4 were parasitemic by qPCR. Vaccinees showed weak antibody and no detectable cellular immune responses. Intradermal immunization with up to 3 × 10(5) PfSPZ-CVac was safe, but induced only minimal immune responses and no sterile protection against Pf CHMI.

Trichomonas vaginalis: investigation of a novel diagnostic method in urine samples using cysteine proteinase 4 gene and PCR technique.

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted disease that leads to vaginitis, urethritis, ectocervicitis and has been associated with human immunodeficiency virus (HIV). Detection of T. vaginalis based on wet-mount microscopy and culture methods is insensitive and time consuming, respectively. Thus the quest for reliable PCR techniques of T. vaginalis in vaginal discharge and urine sample is more importance. In this study, 500 urine and vaginal-discharge samples were collected from women referred to Sexual Transmitted Disease Clinic of Mirzakuchakkhan Hospital in Tehran, Iran between May 2008 and March 2009. Wet-mount and culture methods were done on the vaginal discharges, and PCR assay targeting cysteine proteinase 4 (CP4) was performed on the urine samples. The present study demonstrated 16 (3.2%) of patients were infected with T. vaginalis using culture and wet-mount, whereas PCR assay using CP4 could detect 12 (2.4%) positivity. Sensitivity and specificity of urine PCR assay compared to culture were 80% (95% CI, 54-96) and 99.6% (95% CI, 98.96-100), respectively. These results indicate that using urine-based detection method for T. vaginalis may not be appropriate in women.