MALDI-TOF-MS for Rapid Detection of Fungi causing Mucormycosis during COVID-19 Pandemic from a Tertiary Care Hospital in Southern India: A Retrospective Cohort Study

1. Associate Professor, Department of Microbiology, PSG Institute of Medical Sciences and Research, Coimbatore, Tamil Nadu, India. 2. Professor and Head, Department of Microbiology, PSG Institute of Medical Sciences and Research, Coimbatore, Tamil Nadu, India.

Abstract

Introduction: Mucormycosis is an opportunistic fungal infection that became a public health emergency during the second wave of Coronavirus Disease-2019 (COVID-19). It is an acute, angioinvasive, opportunistic, and emerging mycosis caused by mucormycetes, resulting in a significantly fatal fungal infection among immunocompromised and/or immunosuppressed individuals. Mucormycosis has a specific predilection for blood vessels (angioinvasive) and tissues, leading to extensive necrosis and thromboembolic events. Globally, the incidence rate of mucormycosis varies from 0.005 to 1.7 per million people. Early diagnosis and appropriate therapy are crucial for clinical outcomes.

Aim: To evaluate the mycological profile of mucormycosis during the COVID-19 pandemic using automated Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectroscopy (MALDI-ToF-MS) in comparison with conventional fungal identification methods.

Materials and Methods: A retrospective cohort study was conducted, collecting data from July 2020 to June 2022, involving 1,176 patients from various clinical departments who attended PSG Hospitals affiliated with PSG Institute of Medical Sciences and Research, Coimbatore, Tamil Nadu, India. Data analysis was performed from July 2022 to August 2022. Clinical specimens, including Bronchoalveolar Lavage (BAL), Sputum, Cerebrospinal Fluid (CSF), nasal swabs, tracheal aspirates, pus, blood, corneal scraping, eye discharge, and necrotised tissues, were subjected to Potassium Hydroxide (KOH) mount, conventional culture coupled with micrometry-aided microscopy, and MALDI-ToF-MS among suspected mucormycosis cases. The results were collected in Microsoft Excel, and data analysis was conducted using the International Business Machines Statistical Package for the Social Sciences statistical software (IBM SPSS) version 24.0. The study variables were expressed as descriptive statistics: frequency (N) and percentage (%). The incidence of mucormycosis, mycological profile, and diagnostic utility of conventional micrometry-aided microscopy-assisted culture were compared with the automated MALDI-ToF-MS protein signature method of testing.

Results: Fungal elements were positive in 130 (11.05%) out of a total of 1,176 clinical samples, such as necrotised tissue, biopsy, and sputum, in the KOH mount. Fungal culture was positive in 258 (21.94%) out of 1176 samples, with mucormycosis detected in 73 (28.29%) of the culture-positive samples. The incidence of mucormycosis in this study was 73 (6.21%) out of 1176. A total of 101 (8.59%) samples showed positive results for mucormycetes through KOH, culture, and MALDI-ToF-MS. MALDI-ToF-MS could rapidly and specifically identify and confirm the causative agents of mucormycosis at their genus and species levels compared to KOH or culture.

Conclusion: Simple, low-cost, and less laborious conventional culture coupled with micrometry-aided microscopy can detect Mucorales at the species level, where KOH microscopy could not differentiate between species. MALDI-ToF-MS can be employed for the rapid detection and confirmation of mucormycosis agents at the genus or species level.

Aseptate hyphae, Early diagnosis, Fungal culture, Matrix assisted laser desorption ionisation time of flight mass spectrometry, Microscopy, Mucorales

Mucormycosis is an acute, angioinvasive, opportunistic, and emerging mycosis caused by mucormycetes, resulting in a significantly fatal fungal infection among immunocompromised and/or immunosuppressed individuals (1),(2). Factors such as age, uncontrolled Diabetes Mellitus (DM), neutropenia, severe burns, prolonged steroid therapy, intravenous drug abuse, malnutrition, iron overload conditions, and organ transplantation are considered significant risk factors for mucormycosis (1),(2),(3). Other high-risk groups include those living with the Human Immunodeficiency Virus (HIV) and those on immunomodulating drugs such as the antifungal voriconazole (4). Mucormycosis mainly presents as sino-orbital, rhino-cerebral, pulmonary, cutaneous, gastrointestinal, and disseminated infections (1),(2),(3). Mucormycosis has a specific predilection for blood vessels (angioinvasive) and tissues, resulting in extensive necrosis and thromboembolic events. Globally, the incidence rate of mucormycosis varies from 0.005 to 1.7 per million people. The estimated prevalence of mucormycosis in India, derived by computational-model-based simulation, was found to be 140 per million people as of December 2020 (4),(6),(7). This estimate is about 80 times higher than the prevalence rates of mucormycosis reported in developed countries (8). The mortality rate of mucormycosis ranges between 20-96%, depending on the site and extent of involvement. The case fatality rate of patients with mucormycosis was 46%, with the highest rates noted in disseminated forms (68%) and the lowest rates found in cutaneous presentations (31%) of mucormycosis (8),(9).

The timeline of the first wave of the COVID-19 pandemic extended from April 1, 2020, to January 31, 2021, for a period of 10 months, followed by the second wave, which lasted from March 1, 2021, to June 30, 2021, for a period of three months. India accounted for 71% of global mucormycosis cases from December 2019 to April 2021 (7). Mucormycosis cases emerged rampantly during the second wave of COVID-19, with pre-existing diabetes mellitus as the predominant risk factor, making it a notifiable disease causing an incipient mucormycosis epidemic. The Government of India declared an “epidemic of mucormycosis” in many states of India during the second wave of COVID-19 pandemic. The condition of mucormycosis was known as black fungus and considered a “notifiable disease” or “alert” due to its immediate threat to life, declared as a public health emergency (10). The main cause of the high incidence of mucormycosis was the excessive use of steroids for treating COVID-19 and the immunosuppressive properties of the virus (9),(11). Early diagnosis and appropriate early surgical and/or medical therapy played a significant role in clinical outcomes. Though the culture identification of Mucorales species is not of much importance in terms of therapeutic management, it still provides us with details about individual species. Conventional micrometry-aided microscopy-assisted culture of mucormycosis is an epidemiological tool and is much cheaper, rapid, and does not require very costly equipment, unlike MALDI-ToF-MS, which is the novelty and importance of this study. The conventional techniques for identifying fungi are based on morphological, biochemical, and/or immunological characteristics and can take 2-5 days or longer (12). For definitive interpretations, it is frequently necessary to combine multiple phenotypic techniques (13). Due to the biological complexity of fungal agents, research into them is generally challenging, mainly due to the co-existence of multiple fungal phenotypes (hyphae and/or conidia) in the same organism (14). In these situations, KOH mount is a technique that helps directly examine the ribbon-like structures called hyphae in fungi (15).

The clinical course and outcome can vary among different members and according to the involved anatomical location. Late diagnosis, misdiagnosis, or untreated mucormycosis without necessary surgical debridement of affected parts, along with specific intravenous antifungals, can result in potentially significant and fatal fungal infections. Since mucormycosis is an aggressive and life-threatening infection, it requires quick and accurate identification of the infecting fungus species, followed by treatments such as antifungal medications and surgery (8),(15). The diagnosis of mucormycosis through the KOH mount examination has limitations as it cannot identify fungi at the genus and species level (16),(17). Another emerging technique is MALDI-ToF-MS, which allows for early, more reliable, and rapid laboratory diagnosis of mucormycosis, making it a potential tool compared to other conventional methods (16). Conventional micrometry-aided Lactophenol Cotton Blue (LPCB) microscopy-assisted culture of mucormycosis seems to be a simple and more precise diagnosis of clinically suspected mucormycosis, though Polymerase Chain Reaction (PCR) is considered the gold standard. MALDI-ToF-MS is also considered an alternative gold standard in cases where infrastructure is available. Histopathological Examinations (HPE) can be very helpful in the direct demonstration of fungi.

The purpose of this study was to evaluate the mycological profile of mucormycosis during the COVID-19 pandemic using automated MALDI-ToF-MS in comparison with conventional fungal identification methods which was the primary objective of the study. The secondary objectives of the study were to identify the agents causing mucormycosis from specimens received for KOH and/or LPCB stain, along with micrometry-aided microscopy of mucormycosis agents from positive mucormycosis cultures during the study period from the mycology lab register, Hospital Information System (HIS), and Medical Records Department (MRD).

Material and Methods

A retrospective cohort study was conducted from July 2020 to June 2022, involving a total of 1,176 samples received from 71,176 patients. Analysis of the data was done from July 2022 to August 2022. Ethical approval for the study was obtained from the Institutional Human Ethics Committee (IHEC) with reference number PSG/IHEC/2022/Appr/Exp/163; Project No. 22/164 dated 30-06-2022. A waiver of consent was applied for as there was no active involvement of patients in the study.

Inclusion criteria: Patients who were tested positive for SARS-CoV-2/COVID-19, had type 2 diabetes mellitus, and/or were under prolonged steroid use and were suspected or diagnosed with mucormycosis were included in the study.

Exclusion criteria: Patients who had been previously diagnosed or treated for mucormycosis within the last six months were excluded from the study.

Sample size calculation and justification:

Z=1.96 for 95% confidence interval and d=5%. Prevalence: 20% (18).

Desired sample size calculation: Probability.

=1.96²×20×(100-20)/5×5=3.84×1600/25=512.

Therefore, the sample size to be included in this study was determined to be 1000 specimens.
Sample size included: 1176.

These patients, including both inpatients and outpatients, were clinically suspected or diagnosed with mucormycosis and were from various clinical departments such as General Medicine, Ophthalmology, Ear Nose Throat (ENT), and Dermatology at PSG Hospitals affiliated with PSG Institute of Medical Sciences and Research in Coimbatore, Tamil Nadu, India (as shown in (Table/Fig 1)).

Procedure

The clinical specimens, such as BAL, endo-tracheal aspirate, sputum, CSF, nasal swabs, tracheal aspirates, pus, blood, urine, pleural fluid, peritoneal fluid, drain fluid, corneal scraping, eye discharge, and biopsied tissue or necrotised tissues from affected areas (sinuses, orbit, face, nasal mucosa, nasal cavity, middle meatus, skin, mucosal debris, etc.), were subjected to diagnostic KOH mount, fungal culture and LPCB stain along with micrometry-aided microscopy of mucormycosis agents from positive mucormycosis culture from July 2020 to June 2022. The data collection was conducted during this period. The results of LPCB stain, along with micrometry-aided microscopy of mucormycosis agents from positive mucormycosis cultures, were noted in Microsoft Excel as part of the data collection process. LPCB microscopy of cultures positive for mucorales showed coenocytic broad aseptate hyaline hyphae or sparsely septate hyphae with right-angle branching (19). A total of 36 mucormycosis agents, such as Rhizopus spp. and Rhizomucor spp., isolated from mucorales culture-positive specimens, were further subjected to automated bioMérieux Vitek® MS, a MALDI-ToF-MS technique, for identification according to the manufacturer’s instructions. The reports of the MALDI-ToF-MS identification were compared with the culture and KOH reports [20,21]. The bioMérieux Vitek® MALDI-ToF-MS identification had already been performed in the Department of Microbiology, Jawaharlal Nehru Postgraduate Medical Education and Research, Puducherry, India.

Data collection: The KOH mount, culture, and MALDI-ToF-MS results, along with clinical and demographic data of the study population, were collected from the Mycology lab register and HIS for further analysis. Variables such as age, sex, disease diagnosis, and presence of co-morbidity were included in the analysis during the baseline survey. The incidence of mucormycosis, mycological profile, and the diagnostic utility of conventional micrometry-aided LPCB microscopy-assisted culture were compared with automated MALDI-ToF-MS, which is a protein signature method for testing and identifying fungi.

Statistical Analysis

Data analysis was performed using the IBM Statistical Package for the Social Sciences (SPSS) software version 28.0. Descriptive statistics were used to calculate the frequency and percentage of the variables. All analyses were performed using Microsoft Excel 2021 (Office 365, Microsoft Corporation).

Results

Out of a total of 1,176 participants included in the analysis, 720 (61.22%) were males and 456 (38.78%) were females, resulting in a male-to-female ratio of 1.57. The mean age of the participants was 49.28±16.74 years, with a minimum age of 0 years and a maximum age of 90 years. The age-wise distribution of the patients is described in (Table/Fig 2).

Preliminary findings showed that 368 (31.29%) participants had fungal pneumonia, 104 (8.84%) were suspected cases of mucormycosis, 93 (7.91%) had fungal meningitis, and 56 (4.76%) had sepsis, as shown in (Table/Fig 3).

Among the participants, 951 (80.87%) reported no co-morbid conditions. However, some patients had co-morbidities, including 76 (6.46%) post-COVID patients, 27 (2.30%) with Diabetes Mellitus (DM), and 14 (1.19%) with Pulmonary Tuberculosis (PTB), as depicted in (Table/Fig 4).

Clinical specimens collected from the participants for KOH mount, culture, and MALDI-ToF-MS included 211 (17.94%) Bronchoalveolar Lavage (BAL) samples, 122 (10.37%) sputum specimens, 108 (9.18%) Cerebrospinal Fluid (CSF) samples, 90 (7.65%) biopsied tissue or necrotised tissues from affected areas, 76 (6.46%) nasal swabs, and 50 (4.25%) tracheal aspirates, among others. A detailed description of the diagnosis, co-morbidities, and specimens collected is provided in (Table/Fig 5).

KOH mount for fungal elements was positive in 130 (11.05%) out of the total 1176 clinical samples, such as necrotised tissue, biopsy, and sputum. Among these positive findings, 47 (36.15%) showed broad aseptate hyaline hyphae with right-angle branching suggestive of Mucorales (Table/Fig 6).

Fungal culture was positive in 258 out of the total 1176 samples (21.94%). Among these positive cultures, mucormycosis was detected in 73 (28.29%) samples, resulting in an overall incidence of mucormycosis of 6.21% in the study population (Table/Fig 7). The remaining 185 (71.70%) positive cultures were identified as non-mucorales fungal agents, as depicted in (Table/Fig 8).

A total of 101 (8.59%) samples out of 1176 showed positive results for mucormycetes by both KOH mount and culture, while 157 (13.35%) samples showed positive culture results despite initial negative KOH examination. Additionally, 29 (2.47%) samples showed positive results in KOH microscopy but were negative in fungal culture. A comparison of KOH microscopy results with culture is presented in (Table/Fig 9).

Out of the 73 mucormycosis-positive cultures, 36 non duplicate mucormycosis culture isolates were selected for MALDI-ToF-MS identification. Among these, 31 were identified as “Rhizopus oryzae complex” using MALDI-ToF-MS. Three isolates were identified as Rhizopus homothallicus by LPCB, and two isolates were identified as Rhizopus microsporus. These isolates were not tested by MALDI-ToF-MS, as shown in (Table/Fig 10). MALDI-ToF-MS demonstrated rapid and specific identification and confirmation of causative agents of mucormycosis at the genus and species level compared to KOH or culture. A comparison of culture with LPCB versus MALDI-ToF-MS results is depicted in (Table/Fig 10).

Discussion

COVID-19 has increased the risk of invasive fungal infections, such as mucormycosis, aspergillosis, and candidiasis, among affected patients. During India’s second wave of the pandemic, there was an epidemic of mucormycosis, leading to delayed diagnosis and significant morbidity and mortality (10),(22). In routine clinical practice, direct HPE of BAL or necrosed tissue is used for definitive lab diagnosis of mucorales. A presumptive diagnosis can be made using KOH wet mount examination. Conventional fungal culture at 37°C is used for isolation, speciation, and susceptibility testing of mucorales (23),(24). Micrometry-aided LPCB microscopy of culture-positive samples is a rapid and cost-effective tool for diagnosis and antifungal stewardship. While molecular detection by PCR is the gold standard, MALDI-ToF-MS identification of cultured Mucorales is an excellent confirmation method (25). Appropriate sampling, careful sample handling, and effective communication between clinicians and lab personnel are crucial for optimal diagnosis. Treating physicians face the challenge of early diagnosis and initiating appropriate therapy, including surgical resection of infected areas and antifungal treatment with drugs like Liposomal Amphotericin, Isavuconazole, or Posaconazole, for COVID-19 Associated Mucormycosis (CAM).

This study aimed to rapidly detect mucormycosis through culture and identify the causative agents using conventional culture and automated MALDI-ToF-MS protein signature detection. Most participants in the study were between 41 and 60 years old, and the male-to-female ratio was 1.57, slightly lower than reported in other studies (26),(27). The common clinical presentations in the study population included fungal pneumonia, fungal meningitis, suspected cases of mucormycosis, fungal rhinosinusitis, and sepsis, which aligned with a previous study conducted in India in 2021 (10). Other studies have reported new risk factors, such as chronic kidney disease and post-PTB, in Asian countries (2). Single-center studies on the incidence of mucormycosis have shown varying numbers of cases over the years, which could be attributed to improved awareness, advancements in diagnosis, and technical expertise (25),(26),(27),(28). Incidence rates range from 9.5 to 129 cases per year (29),(30). A multicentre study in Indian Intensive Care Units (ICUs) reported a 12% prevalence of mucormycosis (31). Among various anatomical locations, rhino-orbital-cerebral mucormycosis (ROCM) and pulmonary mucormycosis were the predominant forms in this study. Previous studies in India have also reported ROCM as the most common form (45-74%), followed by cutaneous (10-31%) and pulmonary (3-22%) cases (2),(27),(28),(32),(33). Out of the culture-positive isolates, a total of 73 cases (28.29%) were identified as mucormycosis using microbiology-aided diagnosis methods, including culture, KOH, and MALDI-ToF-MS. Multiple studies in India have reported varying numbers of mucormycosis cases, ranging from 27 to 382 (26),(27),(28),(34).

According to the Centers for Disease Control and Prevention (CDC), samples of respiratory fluid are collected from individuals suspected of having pulmonary mucormycosis for examination (35),(36),(37). In this study, the majority of specimens collected were BAL, followed by sputum, CSF, necrotised tissue, nasal swabs, and tracheal aspirates. Utilising KOH wet mounts enhances visualisation through direct microscopy, and the characteristics of fungal hyphae can aid in early diagnosis (1). Diabetes was the predominant risk factor or co-morbidity observed in this study, consistent with findings from other studies conducted in India (8),(9),(11),(26). Mucorales causing mucormycosis were preliminarily identified by the presence of distinctive broad or narrow pauciseptate or aseptate ribbon-like hyphae with acute or right-angled branching (19). Mucorales grew rapidly when cultured at 25°C and 37°C, displaying white, pale gray, or brown cottony-fluffy colony morphology within 24-72 hours (20). The MALDI-ToF-MS method was used to identify the fungal agents of mucormycosis from the culture-positive samples.

In this study, MALDI-TOF/MS was used for the detection of Mucorales. Out of 36 non duplicate mucorales, 31 were identified as “Rhizopus oryzae complex” using MALDI-TOF/MS. LPCB microscopy, on the other hand, was found to be effective in detecting Rhizopus homothallicus and Rhizopus microsporus, making MALDI-TOF identification unnecessary. Another study conducted in India focused on the epidemiology and diagnosis of mucormycosis, revealing that species of Rhizopus, Lichtheimia, Mucor, Apophysomyces, and Saksenaea complex were the main causative factors (8),(26). The study also reported the detection of new emerging species using MALDI-TOF-MS. While MALDI-TOF-MS is considered sophisticated for diagnosing mucormycosis, culture-based identification coupled with micrometry-aided LPCB microscopy was found to be a more feasible and relevant method (2).

A separate study evaluated non mucorales as causative agents of mucormycosis, reporting Aspergillus spp., Fusarium spp., Pseudallescheria spp., Scedosporium spp., and Pseudomonas aeruginosa as the main agents. These findings were consistent with the present study, which also identified other non mucorales fungi in culture. The study on the applications of MALDI-TOF-MS in clinical microbiology demonstrated its rapid, accurate, and easy identification of cultures. Despite this, the study emphasised the importance of combining culture-based identification with micrometry-aided LPCB microscopy for accurate detection of mucormycosis cases and their causative agents, as confirmed by MALDI-TOF-MS.

The study acknowledged the strength of the large sample size, allowing for effective analysis, as well as the comparison between KOH examination, culture, and MALDI-TOF-MS techniques to determine the accuracy of identifying agents causing mucormycosis. However, the study recommended further research, specifically a phase II study focusing on PCR specificity and sequencing, to augment early identification and confirmation of Mucorales.

Limitation(s)

Limitations of the study included the inability of KOH microscopy to identify mucorales at the genus or species level. Positive cultures from non sterile sites required clinical and radiological data to establish a probable diagnosis, while the low sensitivity of culture posed a challenge, potentially leading to false-negative results. The study also highlighted the need to expand commercially available databases of MALDI-TOF-MS and gather more validation data. PCR for mucormycosis was not conducted due to it not being part of routine diagnostic methods or national guidelines.

Conclusion

In conclusion, simple KOH microscopy, conventional culture, and micrometry-aided LPCB microscopy can detect Mucorales. MALDI-TOF-MS can be used for rapid detection, confirmation, and epidemiological purposes, although treatment does not differ among various species causing mucormycosis.

Acknowledgement

The Authors would like to express their gratitude to Dr. Rakesh Singh, Professor and Head, Department of Microbiology, JIPMER, Puducherry, for providing support in identifying Mucormycosis agents using automated MALDI-TOF-MS. Authors would like to thank our postgraduates and lab technicians for helping in data collection.

DOI and Others

DOI: 10.7860/JCDR/2023/64875.18409

Date of Submission: Apr 19, 2023
Date of Peer Review: May 24, 2023
Date of Acceptance: Aug 24, 2023
Date of Publishing: Sep 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. NA

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