Evaluation of hospital environment for presence of Mucorales during COVID-19-associated mucormycosis outbreak in India - a multi-centre study.

BACKGROUND: An unprecedented rise in the number of COVID-19-associated mucormycosis (CAM) cases has been reported in India. Myriad hypotheses are proposed for the outbreak. We recently reported uncontrolled diabetes and inappropriate steroid therapy as significant risk factors for the outbreak. However, Mucorales contamination of hospital environment was not studied. AIM: To perform a multi-centre study across India to determine possible Mucorales contamination of hospital environment during the outbreak. METHODS: Eleven hospitals from four zones of India representing high to low incidence for mucormycosis cases were included in the study. Samples from a variety of equipment used by the patients and ambient air were collected during May 19th, 2021 through August 25th, 2021. FINDINGS: None of the hospital equipment sampled was contaminated with Mucorales. However, Mucorales were isolated from 11.1% air-conditioning vents and 1.7% of patients' used masks. Other fungi were isolated from 18% of hospital equipment and surfaces, and 8.1% of used masks. Mucorales grew from 21.7% indoor and 53.8% outdoor air samples. Spore counts of Mucorales in air were significantly higher in the hospitals of North and South zones compared to West and East zones (P < 0.0001). Among Mucorales isolated from the environment, Rhizopus spp. were the most frequent genus. CONCLUSION: Contamination of air-conditioning vents and hospital air by Mucorales was found. Presence of Mucorales in these areas demands regular surveillance and improvement of hospital environment, as contamination may contribute to healthcare-associated mucormycosis outbreaks, especially among immunocompromised patients.

A multicentre observational study on the epidemiology, risk factors, management and outcomes of mucormycosis in India.

OBJECTIVES: To describe the epidemiology, management and outcome of individuals with mucormycosis; and to evaluate the risk factors associated with mortality. METHODS: We conducted a prospective observational study involving consecutive individuals with proven mucormycosis across 12 centres from India. The demographic profile, microbiology, predisposing factors, management and 90-day mortality were recorded; risk factors for mortality were analysed. RESULTS: We included 465 patients. Rhino-orbital mucormycosis was the most common (315/465, 67.7%) presentation followed by pulmonary (62/465, 13.3%), cutaneous (49/465, 10.5%), and others. The predisposing factors included diabetes mellitus (342/465, 73.5%), malignancy (42/465, 9.0%), transplant (36/465, 7.7%), and others. Rhizopus species (231/290, 79.7%) were the most common followed by Apophysomyces variabilis (23/290, 7.9%), and several rare Mucorales. Surgical treatment was performed in 62.2% (289/465) of the participants. Amphotericin B was the primary therapy in 81.9% (381/465), and posaconazole was used as combination therapy in 53 (11.4%) individuals. Antifungal therapy was inappropriate in 7.6% (30/394) of the individuals. The 90-day mortality rate was 52% (242/465). On multivariate analysis, disseminated and rhino-orbital (with cerebral extension) mucormycosis, shorter duration of symptoms, shorter duration of antifungal therapy, and treatment with amphotericin B deoxycholate (versus liposomal) were independent risk factors of mortality. A combined medical and surgical management was associated with a better survival. CONCLUSIONS: Diabetes mellitus was the dominant predisposing factor in all forms of mucormycosis. Combined surgical and medical management was associated with better outcomes. Several gaps surfaced in the management of mucormycosis. The rarer Mucorales identified in the study warrant further evaluation.

Epidemiological profile and spectrum of neglected tropical disease eumycetoma from Delhi, North India.

Mycetoma is a chronic granulomatous, suppurative and progressive inflammatory disease that usually involves the subcutaneous tissue and bones after traumatic inoculation of the causative organism. In India, actinomycotic mycetoma is prevalent in south India, south-east Rajasthan and Chandigarh, while eumycetoma, which constitutes one third of the total cases, is mainly reported from north India and central Rajasthan. The objective was to determine the epidemiological profile and spectrum of eumycetoma from a tertiary care hospital in Delhi, North India. Thirty cases of eumycetoma were diagnosed by conventional methods of direct microscopy, culture and species-specific sequencing as per standard protocol. The spectrum of fungal pathogens included Exophiala jeanselmei, Madurella mycetomatis, Fusarium solani, Sarocladium kiliense, Acremonium blochii, Aspergillus nidulans, Fusarium incarnatum, Scedosporium apiospermum complex, Curvularia lunata and Medicopsis romeroi. Eumycetoma can be treated with antifungal therapy and needs to be combined with surgery. It has good prognosis if it is timely diagnosed and the correct species identified by culture for targeted therapy of these patients. Black moulds required prolonged therapy. Its low reporting and lack of familiarity may predispose patients to misdiagnosis and consequently delayed treatment. Hence health education and awareness campaign on the national and international level in the mycetoma belt is crucial.

Cladosporium sphaerospermum causing brain abscess, a saprophyte turning pathogen: Case and review of published reports.

Emergence of saprophytic fungi thriving in dead plant material and soil as opportunistic human pathogens is of great concern. Cladosporium species are environmental saprophytes reported to cause various superficial and invasive fungal infections worldwide. C. sphaerospermum, a predominantly indoor fungus has been reported from cases of meningitis, subcutaneous and pulmonary fungal infections in the past. Herein we report the first case of cerebral abscess due to C. sphaerospermum in an immunocompetent host who was successfully managed by combined medical and surgical therapy.

Ocular infection caused by Hormographiella aspergillata: A case report and review of literature.

Hormographiella aspergillata, a basidiomycete is a rare cause of human infection. We report a case of 70-year-old female with corneal ulcer and endophthalmitis caused by this agent. The patient had an intraocular implantation of lens following a cataract surgery. Corneal tissue obtained during therapeutic penetrating keratoplasty showed presence of septate hyphae on microscopy and culture grew H. aspergillata which was confirmed by sequencing of ITS region. Patient was started on systemic voriconazole and topical natamycin, however the eye could not be salvaged. To our knowledge, this is the first report of ocular infection caused Hormographiella aspergillata in an immunocompetent patient.

Identification of Malassezia species by MALDI-TOF MS after expansion of database.

The taxonomy of Malassezia species is evolving with introduction of molecular techniques, and difficulty is faced to identify the species by phenotypic methods. Among 15 known Malassezia species, the present Bruker database could identify only 2 species. The present study was aimed to improve Matrix -assisted laser desorption ionization time-flight mass spectrometry (MALDI-TOF MS) based identification of Malassezia species. A total of 88 isolates (DNA sequencing confirmed) for database preparation and, for the validation of database, 190 isolates confirmed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) were used. The main spectrum profile dendrogram showed the sufficient discrimination between all the species by MALDI-TOF MS. The updated Malassezia database could identify 94.7% and 5.3% strains to the species and genus level, respectively. MALDI-TOF MS is a significantly reliable technique, and results were comparable with PCR-RFLP with kappa value 0.9. In conclusion, MALDI-TOF MS could be a possible alternative tool to other molecular methods for rapid and accurate identification of Malassezia species.

Epidemiology and clinical characteristics of invasive mould infections: A multicenter, retrospective analysis in five Asian countries.

Formal, large-scale, multicenter studies of invasive mould infection (IMI) in Asia are rare. This 1-year, retrospective study was designed to assess the incidence and clinical determinants of IMI in centers in five countries (Thailand, Taiwan, Singapore, China, India). Patients treated in a single year (2012) were identified through discharge diagnoses, microbiology, and histopathology logs, and entered based on published definitions of IMI. A total of 155 cases were included (median age 54 years; 47.7% male). Of these, 47.7% had proven disease; the remainder had probable IMI. The most frequent host factors were prolonged steroid use (39.4%) and recent neutropenia (38.7%). Common underlying conditions included diabetes mellitus (DM; 30.9%), acute myeloid leukemia (19.4%), and rheumatologic conditions (11.6%). DM was more common in patients with no recent history of neutropenia or prolonged steroid use (P = .006). The lung was the most frequently involved site (78.7%), demonstrating a range of features on computed tomography (CT). Aspergillus was the most common mould cultured (71.6%), primarily A. fumigatus and A. flavus, although proportions varied in different centers. The most often used antifungal for empiric therapy was conventional amphotericin. Ninety-day mortality was 32.9%. This is the first multicenter Asian study of IMI not limited to specific patient groups or diagnostic methods. It suggests that DM and rheumatologic conditions be considered as risk factors for IMI and demonstrates that IMI should not be ruled out in patients whose chest features on CT do not fit the conventional criteria.

Controlling a possible outbreak of Candida auris infection: lessons learnt from multiple interventions.

BACKGROUND: Multidrug-resistant Candida auris infection has been reported from five continents in recent years. The prevalence of C. auris invasive infection has been estimated at 5.3% for intensive-care-acquired candidaemia in India. The transmission of the organism between the patients and from environment to patients is rapid. AIM: To understand the intra-hospital dynamics of C. auris transmission and to determine the possible interventions to prevent its spread. METHODS: Surveillance of intensive care units was carried out to assess patient colonization, environmental contamination and hand carriage of the yeast among healthcare workers. Interventions including chlorhexidine washing of patients and decontamination of environmental surfaces with stabilized hydrogen peroxide disinfectant (Ecoshield) were undertaken. We further evaluated the effectiveness of frequently used disinfectants in the hospital against C. auris on various inanimate surfaces, and its persistence on hospital fabrics. FINDINGS: Three cases of C. auris bloodstream infection were detected over a period of three months. Many patients admitted at the same time, in the same area, were colonized by C. auris. Surveillance detected C. auris contamination of environmental surfaces and hands of healthcare workers. Interventions such as chlorhexidine washing and appropriate use of disinfectants could eradicate C. auris from patients and hospital environment. CONCLUSION: The frequently used disinfectants in our hospital and current hand hygiene practices were efficient against C. auris if proper contact time and procedures were followed. Evaluation of possible persistence of C. auris on dry fabrics showed that they can persist for up to seven days.

High fungal spore burden with predominance of Aspergillus in hospital air of a tertiary care hospital in Chandigarh.

The prevalence of fungal spores in the hospital air is essential to understand the hospital-acquired fungal infections. Air conditioners (ACs) used in hospitals may either reduce spores in air or be colonised by fungi and aid in its dissemination. The present study was conducted to assess the fungal spore burden in AC and non-AC areas. We found a high fungal spore count in air irrespective of whether the area was AC or non-AC. The most predominant species isolated were Aspergillus flavus and Aspergillus fumigatus. Such high concentrations of pathogenic fungi in air may predispose individuals to develop disease.

Distribution and association of Mycobacterium tuberculosis CAS lineage with multidrug resistance in North India.

SETTING: A tertiary care hospital in North India. OBJECTIVES: To determine the prevalence of different genotypes and examine their association with drug resistance among clinical isolates of Mycobacterium tuberculosis from the northern region of India. DESIGN: We analysed 100 clinical isolates of M. tuberculosis using mycobacterial interspersed repetitive units genotyping and TbD1 analysis. RESULTS: The analysis revealed that 34% of strains belonged to the Delhi/CAS (TbD1-) lineage, 32% had unknown patterns (27 TbD1-, 5 TbD1+), 18% were of Beijing genotype (TbD1-) and 11% were of EAI lineages (TbD1+). Twenty-one strains were multidrug-resistant tuberculosis (MDR-TB), 9 of which belonged to the Delhi/CAS lineage, 4 were of Beijing lineage, 6 were of unknown pattern and one was of EAI lineage. Our meta-analysis showed the overall proportion of CAS lineage to be 42.96% (95%CI 33-52); the CAS lineage had no association with MDR-TB (OR 0.89, 95%CI 0.66-1.20). CONCLUSION: The study highlights the high proportion of CAS lineage strains and absence of association with MDR-TB. The distribution and identification of different genotypes of M. tuberculosis could help in better understanding the factors that influence disease transmission and drug resistance.

A Novel Y319H Substitution in CYP51C Associated with Azole Resistance in Aspergillus flavus.

This study aimed to explore any mutation in the CYP51 gene conferring azole resistance in Aspergillus flavus. Two voriconazole-resistant and 45 voriconazole-susceptible isolates were included in the study. Sequence analysis demonstrated a T1025C nucleotide change in CYP51C, resulting in the Y319H amino acid substitution in one resistant isolate. However, the earlier described T788G mutation in CYP51C conferring voriconazole resistance in A. flavus isolates was present in all isolates, irrespective of their susceptibility status.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the rapid identification of yeasts causing bloodstream infections.

Few studies have systematically standardised and evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of yeasts from bloodstream infections. This is rapidly becoming pertinent for early identification of yeasts and appropriate antifungal therapy. We used 354 yeast strains identified by polymerase chain reaction (PCR) sequencing for standardisation and 367 blind clinical strains for validation of our MALDI-TOF MS protocols. We also evaluated different sample preparation methods and found the on-plate formic acid extraction method as most cost- and time-efficient. The MALDI-TOF assay correctly identified 98.9% of PCR-sequenced yeasts. Novel main spectrum projections (MSP) were developed for Candida auris, C. viswanathii and Kodamaea ohmeri, which were missing from the Bruker MALDI-TOF MS database. Spectral cut-offs computed by receiver operating characteristics (ROC) analysis showed 99.4% to 100% accuracy at a log score of ≥ 1.70 for C. tropicalis, C. parapsilosis, C. pelliculosa, C. orthopsilosis, C. albicans, C. rugosa, C. guilliermondii, C. lipolytica, C. metapsilosis, C. nivariensis. The differences in the species-specific scores of our standardisation and blind validation strains were not statistically significant, implying the optimal performance of our test protocol. The MSPs of the three new species also were validated. We conclude that MALDI-TOF MS is a rapid, accurate and reliable tool for identification of bloodstream yeasts. With proper standardisation, validation and regular database expansion, its efficiency can be further enhanced.

Fatal cryptococcosis involving multiple sites in an immunocompetent child.

Disseminated cryptococcosis is less common in immunocompetent individuals. Herein, we report a fatal case of cryptococcosis in apparently immunocompetent child with multiple site involvement. The yeast isolated from cerebrospinal fluid, blood, endotracheal, gastric and lymph node aspirate was identified by molecular method as Cryptococcus neoformans var. grubii.

Epidemiological study of a large cluster of fungaemia cases due to Kodamaea ohmeri in an Indian tertiary care centre.

While performing molecular confirmation of phenotypically identified Candida tropicalis isolates, we re-identified a few isolates as Kodamaea ohmeri. This led us to the present epidemiological investigation of K. ohmeri fungaemia cases. All phenotypically identified C. tropicalis blood isolates during October 2008 through to December 2009 at our advanced paediatric centre were included for molecular identification by sequencing of the internal transcribed spacer and D1/D2 regions of rDNA. After identifying a large cluster K. ohmeri fungaemia cases, a case-control study was carried out retrospectively to analyse potential risk factors for K. ohmeri fungaemia. Molecular typing of the isolates was performed using a fluorescent amplified fragment length polymorphism (FAFLP) technique. The antifungal susceptibility testing was performed as per the M27-A3 protocol of CLSI. Thirty-eight (25.7%) of 148 phenotypically identified C. tropicalis isolates were confirmed as K. ohmeri by sequencing and FAFLP. By case-control analysis, piperacillin-tazobactam was significantly associated with the K. ohmeri fungaemia. The FAFLP analysis showed that all K. ohmeri isolates had >92% similarity. The azoles and echinocandins had good in vitro activity against K. ohmeri, though 86.8% of the isolates had MIC of 1 mg/L for amphotericin B. The response to antifungal therapy could be evaluated in 27 patients and 70.4% of patients recovered after antifungal therapy. The present study reports the largest cluster of K. ohmeri fungaemia from a single centre. The study also stresses the need for accurate identification of clinical yeast isolates.