Development of single-tube real-time PCR assay for the rapid detection of Aspergillus and Fusarium-The two most common causative agents in fungal keratitis.

BACKGROUND: To compare the performance of conventional, semi-nested and real-time panfungal ITS PCRs for diagnosing fungal keratitis (FK) and develop genus-specific real-time PCR for the most common aetiology of FK. METHODS: This multicentric study includes 232 corneal samples from suspected FK patients from four centres across India between November 2019 through August 2021. A total of 87 corneal buttons were included for the comparison of conventional, semi-nested and real-time ITS PCRs, of which 68 were from confirmed FK patients. Of these 87 samples, 44 (microscopy and culture positive for Aspergillus sp. and/or Fusarium sp.) were used for the standardisation of genus-specific real-time primers/probes. Subsequently, the best method showing highest sensitivity and specificity was validated in 188 samples. RESULTS: On Bayesian comparison, conventional ITS2 PCR showed best performance (sensitivity and specificity of 55.88% and 100%, respectively). Since, real-time ITS2 PCR was also considerably efficient (sensitivity and specificity of 51.47% and 84.21%, respectively) in comparison with the conventional PCR but faster, cost-effective, and less labor-intensive, ITS-2 real-time PCR is a suitable method that can be applied along with culture and microscopy. During validation, real-time PCR with genus-specific primers showed 61.76% and 91.18% sensitivity with specificity of 98.05% and 79.22%, respectively, for Aspergillus sp. and Fusarium sp. Aspergillus probe, Fusarium probe and duplex PCR showed sensitivity of 52.94%, 50% and 54.41% with specificity of 92.86%, 82.47% and 75%, respectively. No cross-reactivity of genus-specific PCRs was observed during standardisation. CONCLUSIONS: ITS-2 real-time PCR can be applied as an adjunct with conventional methods for the diagnosis of FK. The genus-specific duplex real-time PCRs are rapid which reduces the turnaround time (TAT) avoiding the need for sequencing.

Fusarium Keratitis From a Comprehensive Eye Health Care Facility in South India: Molecular Characterization by MALDI-TOF Versus Polymerase Chain Reaction Sequencing, Species Complex Distribution, and Clinical Correlation.

PURPOSE: Fusarium keratitis possesses significant diagnostic and therapeutic challenges. Medically relevant Fusaria belong to various species complexes and show prominent differences in their antifungal susceptibility profile which may influence the clinical outcome. Rapid diagnostic methods are warranted for precise identification of species complexes for prompt initiation of correct antifungals. The aim of the study was to compare between matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and polymerase chain reaction sequencing for correct species-level identification and to analyze the clinical outcome among different Fusarium species complexes. METHODS: Twenty-nine culture-proven Fusarium keratitis cases were included in this study. A phylogenetic tree was constructed after TEF1α gene sequencing and isolates were subjected to MALDI-TOF MS, followed by database expansion and identification. Clinical outcome and risk association among species complexes were analyzed retrospectively. RESULTS: Maximum likelihood phylogeny categorized 68.9% isolates as Fusarium solani species complex (FSSC), 17.2% as Fusarium dimerum species complex (FDSC), followed by 13.7% as Fusarium fujikuroi species complex (FFSC). With extended database, MALDI-TOF MS could correctly speciate 96.5% (28/29) isolates. Previous antibiotic usage ( P = 0.034) and preoperative antifungal treatment with natamycin, voriconazole, or ketoconazole ( P = 0.025) were significantly higher in the FSSC group. The patients in the FFSC group had a significantly longer duration of symptoms at the time of clinical presentation to the clinic (15 days vs. 5 days, P = 0.030). Among 11 patients with a clinically poor outcome, 9 (31%) had FSSC infection. CONCLUSIONS: Patients infected with the FSSC had more aggressive infection with poor prognosis. MALDI-TOF MS can serve as the best alternative method to conventional molecular identification with reduced turnaround time, which may help the ophthalmologists to consider the appropriate antifungals or early surgical intervention for improved outcome.

Clinical and mycological profile of fungal keratitis from North and North-East India.

Purpose: To study the clinical presentation, mycological profile, and risk factors of fungal keratitis (FK) cases presenting at two tertiary-care centers, one each at North (Chandigarh) and Northeast (Assam) India, and to compare the spectrum of fungi recovered from the clinical and environmental samples at both locations. Methods: All patients with suspected FK were enrolled from both the centers between January 2018 and December 2019. Corneal samples were collected and processed as per standard laboratory protocols. Demographic details and clinical and mycological profiles were noted in all patients. Environmental sampling from the soil, air, and the vegetative matter was performed from both locations and neighboring districts. Results: Of the 475 suspected cases, 337 (71%) were diagnosed as FK (median age: 50 years; 77.2% males). The presence of diabetes, hypertension, blurred vision, and corneal discoloration was significantly higher in patients with FK compared to those without FK. Aspergillus sp. (52.1%) and Fusarium sp. (47.61%) were the predominant etiological agents isolated from cases in North and Northeast India, respectively. FK due to melanized fungi was associated with diabetes, trauma with animal tail, and corneal discoloration. A similar spectrum of fungi was seen in environmental and clinical samples in both the regions. Conclusion: The difference in etiological agents of FK and environmental fungal isolates in North and Northeast India highlights the need to identify the ecological niche of potential fungal pathogens. Prospective, multicenter studies, systematic environmental sampling, and the evaluation of the differences in causative agents and clinical presentation of FK from different parts of the country can substantially improve our understanding of its region-specific clinico-epidemiological profile.

A Selective Medium for Isolation and Detection of Candida auris, an Emerging Pathogen.

Identification of Candida auris is challenging and requires molecular or protein profiling-based approaches, availability of which is limited in many routine diagnostic laboratories, necessitating the development of a cost-effective, rapid, and reliable method of identification. The objective of this study was to develop a selective medium for C. auris identification. Eighteen C. auris and 30 non-C. auris yeasts were used for the standardization of the selective medium. Sodium chloride (10% to 13% concentration) and ferrous sulfate (8 mM to 15 mM) were added to yeast extract-peptone-dextrose (YPD) agar in various combinations followed by incubation at 37°C, 40°C, or 42°C for 2 to 3 days. For validation, 579 yeast isolates and 40 signal-positive Bactec blood culture (BC) broths were used. YPD agar comprising 12.5% NaCl and 9 mM ferrous sulfate incubated at 42°C for 48 h, named Selective Auris Medium (SAM), allowed selective growth of C. auris A total of 95% (127/133) of C. auris isolates tested grew on the standardized media within 48 h, and the remaining 6 isolates grew after 72 h, whereas the growth of 446 non-C. auris yeast isolates was completely inhibited. The specificity and sensitivity of the test medium were both 100% after 72 h of incubation. The positive and negative predictive values were also noted to be 100% after 72 h of incubation. The formulated selective medium can be used for the detection and identification of C. auris The SAM is inexpensive, can easily be prepared, and can be used as an alternative to molecular diagnostic tools in the clinical microbiology laboratory.

Identification and broth-microdilution antifungal susceptibility testing of yeast directly from automated blood cultures.

Aim: To standardize MALDI-TOF-MS based identification and antifungal susceptibility (AFST) for yeasts directly from automated blood cultures to reduce turnaround time. Materials & methods: Direct-ID after lysis-centrifugation (0.5% SDS) standardized in 40 and validated in 250 yeast positive samples. Direct-AFST was standardized with fluconazole (28 samples) and evaluated (70 samples) for seven antifungals. Results: Direct-ID had a high sensitivity (97.2%) and specificity (94.3%). Correct species-level identification showed 100% in C. tropicalis, C. krusei, C. parapsilosis. Direct-AFST had a 100% categorical agreement with culture-AFST for posaconazole, anidulafungin and >90% categorical agreement for amphotericin B, voriconazole and fluconazole. Conclusion: Direct-ID and subsequent direct-AFST is a rapid and robust method to reduce the turnaround time for the diagnosis of invasive candidiasis.