Proposal for a screening protocol for Candida auris colonization.

BACKGROUND: Candida auris is an emerging multidrug-resistant yeast which can cause severe infection in hospitalized patients. Since its first detection in 2009, C. auris has spread globally. The control and elimination of this pathogen in a hospital setting is particularly challenging because of its ability to form biofilms, allowing for long-term patient colonization and persistence in the environment. Identification of C. auris from cultures is difficult due to the morphologic similarities to other yeasts, its slow growth, and the low culture sensitivity when using standard agars and temperatures. AIM: We have developed a screening protocol for C. auris colonization using an in-house-developed polymerase chain reaction (PCR), combined with confirmatory culture in optimized conditions. METHODS: C. auris-specific primers and probe were developed, targeting the internal transcribed spacer (ITS) region, and specificity was confirmed in silico using the BLAST tool. The PCR was validated using a panel of 12 C. auris isolates and 103 isolates from 22 other Candida species and was shown to be 100% accurate. The limit of detection of the assay was determined at approximately four cells per PCR. FINDINGS: C. auris screening was introduced on February 15th, 2023, and was used for patients who had been admitted to a healthcare facility abroad in the two months prior to admission to our hospital. The screening protocol included swabs from nose, throat, rectum, axilla, and groin. In the first eight months, 199 patients were screened and seven were found positive (4%). CONCLUSION: Our proposed screening protocol may contribute to control C. auris in hospitals.

Interspecies discrimination of A. fumigatus and siblings A. lentulus and A. felis of the Aspergillus section Fumigati using the AsperGenius® assay.

The AsperGenius® assay detects several Aspergillus species and the A. fumigatus Cyp51A mutations TR34/L98H/T289A/Y121F that are associated with azole resistance. We evaluated its contribution in identifying A. lentulus and A. felis, 2 rare but intrinsically azole-resistant sibling species within the Aspergillus section Fumigati. Identification of these species with conventional culture techniques is difficult and time-consuming. The assay was tested on (i) 2 A. lentulus and A. felis strains obtained from biopsy proven invasive aspergillosis and (ii) control A. fumigatus (n=3), A. lentulus (n=6) and A. felis species complex (n=12) strains. The AsperGenius® resistance PCR did not detect the TR34 target in A. lentulus and A. felis in contrast to A. fumigatus. Melting peaks for L98H and Y121F markers differed and those of the Y121F marker were particularly suitable to discriminate the 3 species. In conclusion, the assay can be used to rapidly discriminate A. fumigatus, A. lentulus and A. felis.

PCR-based detection of Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multicentre validation of the AsperGenius assay® in 201 patients with haematological disease suspected for invasive aspergillosis.

OBJECTIVES: In patients with invasive aspergillosis (IA), fungal cultures are mostly negative. Consequently, azole resistance often remains undetected. The AsperGenius® multiplex real-time PCR assay identifies clinically relevant Aspergillus species and four resistance-associated mutations (RAMs; TR34/L98H/T289A/Y121F) in the Cyp51A gene. This multicentre study evaluated the diagnostic performance of this assay on bronchoalveolar lavage (BAL) fluid and correlated the presence of RAMs with azole treatment failure and mortality. METHODS: Stored BAL samples from patients with haematological diseases with suspected IA were used. BAL samples that were galactomannan/culture positive were considered positive controls for the presence of Aspergillus. Azole treatment failure and 6 week mortality were compared in patients with and without RAMs that had received ≥5 days of voriconazole monotherapy. RESULTS: Two hundred and one patients each contributed one BAL sample, of which 88 were positive controls and 113 were negative controls. The optimal cycle threshold cut-off value for the Aspergillus species PCR was <38. With this cut-off, the PCR was positive in 74/88 positive controls. The sensitivity, specificity, positive predictive value and negative predictive value were 84%, 80%, 76% and 87%, respectively. 32/74 BAL samples were culture negative. Azole treatment failure was observed in 6/8 patients with a RAM compared with 12/45 patients without RAMs (P = 0.01). Six week mortality was 2.7 times higher in patients with RAMs (50.0% versus 18.6%; P = 0.07). CONCLUSIONS: The AsperGenius® assay had a good diagnostic performance on BAL and differentiated WT from Aspergillus fumigatus with RAMs, including in culture-negative BAL samples. Most importantly, detection of RAMs was associated with azole treatment failure.

Diagnostic Performance of Galactomannan Antigen Testing in Cerebrospinal Fluid.

Testing cerebrospinal fluid (CSF) for the presence of galactomannan (GM) antigen may help in diagnosing cerebral aspergillosis (CA). However, the use of the CSF GM test as a diagnostic test has been little studied. We evaluated its diagnostic performance by comparing the CSF GM optical density indexes (ODI) at different cutoffs in patients with probable and proven CA to those in patients without CA. Patients from 2 tertiary referral hospitals with suspected CA between 2004 and 2014 and in whom CSF GM ODI had been determined were selected. European Organization for Research and Treatment of Cancer/Invasive Infectious Diseases Study Mycoses Group (EORTC/MSG) definitions of invasive aspergillosis and CA were used, but with the exclusion of the test to be validated (i.e., the CSF GM test) as a microbiological EORTC/MSG criterion. The study population consisted of 44 patients (4 with proven CA, 13 with probable CA, and 27 with no CA). Of the 17 patients with CA, 15 had a CSF GM ODI of ≥2.0. Of 27 patients without CA, 26 had a CSF GM ODI of <0.5 and 1 had a CSF GM ODI of 8.2. When a GM CSF ODI cutoff of 1.0 was used, the sensitivity, specificity, and positive and negative predictive values were 88.2%, 96.3%, 93.8%, and 92.9%, respectively. The same results were found when a CSF GM ODI cutoff of 0.5 or 2.0 was used. Testing GM in CSF has a high diagnostic performance for diagnosing CA and may be useful to diagnose or virtually rule out the infection without the need for a cerebral biopsy.