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ORIGINAL ARTICLE Table of Contents  
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Relevance of conventional microscopy in the diagnosis of mucormycosis during COVID-19 pandemic


1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Otorhinolaryngology and Head and Neck Surgery, All India Institute of Medical Sciences, New Delhi, India
3 Department of Onco-Anaesthesia, Pain and Palliative Care, All India Institute of Medical Sciences, New Delhi, India

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Date of Submission30-Oct-2021
Date of Decision30-Dec-2021
Date of Acceptance21-Jan-2022
Date of Web Publication14-Nov-2022
 

  Abstract 


Introduction: Mucormycosis is a lethal disease which bewildered the health-care community of India during the ongoing second wave of the COVID-19 pandemic. The diagnosis is challenging considering the poor isolation in culture. Aims: The aim of the study was to emphasize the utility of potassium hydroxide (KOH) mount examination using conventional light microscopy for early diagnosis of mucormycosis in resource-limited settings. Materials and Methods: A retrospective analysis of results for all the samples including tissue biopsies, swabs, and pus received in the laboratory for KOH microscopy was done, and results were recorded. The clinical and demographic details of the patients were collected from the hospital information system. Results: A total of 75 samples from 50 patients were received in the laboratory. Out of these, 43 samples from 35 patients showed fungal hyphae (38 patients with only nonseptate hyaline hyphae, 2 with septate hyaline hyphae, and 3 samples with mixed infections). All patients except one were positive for severe acute respiratory syndrome coronavirus 2 infection. The most common age group was 45–59 years (40%), followed by 30–44 years (34.28%) with a male predominance. There was a significant difference in hemoglobin A1C (P = 0.005) and ferritin (P = 0.017) levels between laboratory-confirmed mucormycosis patients and clinically suspected mucormycosis patients without confirmation. Conclusion: Early diagnosis and initiation of targeted therapy is the cornerstone for treating mucormycosis patients. Hence, a rapid and reliable mode of diagnosis is the need of the hour. Conventional microscopy is such a tool that may be used, especially in resource-limited settings.

Keywords: COVID-19, diagnosis, microscopy, mucormycosis, potassium hydroxide


How to cite this URL:
Vidyarthi AJ, Das A, Khan S, Panda S, Singh G, Thakar A, Bhatnagar S, Xess I, Chaudhry R. Relevance of conventional microscopy in the diagnosis of mucormycosis during COVID-19 pandemic. J Microsc Ultrastruct [Epub ahead of print] [cited 2022 Nov 30]. Available from: https://www.jmau.org/preprintarticle.asp?id=361129





  Introduction Top


While the world was still engrossed in battling the COVID-19 disease, the recent surge of another deadly infection called mucormycosis gripped the population of India. Mucormycosis is caused by saprophytic fungi from the order Mucorales, which are found in the environment and release spores that are easily dispersed.[1] Whereas the exact incidence of mucormycosis in India is unknown due to the lack of population-based studies, the estimated prevalence in India is around 70–80 times higher than the developed world corresponding to around 0.14 cases/1000 population.[2],[3] Manifestations are varied with rhino-orbital-cerebral mucormycosis (ROCM) being the most common form (45% to 74%), followed by cutaneous (10%–31%), pulmonary (3%–22%), renal (0.5%–9%), gastrointestinal (GI) (2%–8%), and disseminated infections (0.5%–9%). While ROCM and pulmonary infections are caused by inhalation of spores, cutaneous infections have been reported to occur due to the nonintact/damaged skin getting exposed to the spores. GI mucormycosis has been associated with contaminated foods or products being introduced to the GI tract. These are angioinvasive fungi and may hematogenously spread to the brain and other organs leading to disseminated infections.[2] Uncontrolled diabetics and immunosuppressed patients including those with stem cell or solid organ transplant, hematologic malignancies, and those on prolonged treatment with corticosteroids are predisposed for this infection.[4] The most common genera that cause infections in humans are Rhizopus and Mucor species, but others include Apophysomyces, Rhizomucor, Cunninghamella, Lichtheimia, Cokeromyces, and Saksenaea.[5]

Considering the fulminant nature and high mortality rates of this disease, early diagnosis and initiation of treatment is crucial and necessary.[6] As fungal culture is time-consuming and molecular tests require sophisticated equipment and expertise, potassium hydroxide (KOH) microscopy with or without fluorescent staining may be instrumental in providing early diagnosis, especially in resource-limited settings. Here, we present the data of patients admitted at our hospital during the recent mucormycosis outbreak with complaints suggestive of ROCM.


  Materials and Methods Top


This study was performed at National Cancer Institute (NCI), an allied tertiary care center of All India Institute of Medical Sciences, which served as a dedicated COVID-19 care facility during the second wave of the pandemic. The analysis was performed in accordance with the ethical standards of the Institute Ethics Committee (IEC-780/12.11.2021, RP-16/2021).

A retrospective analysis of the laboratory results for various samples (tissue biopsies, nasal swabs, crusts and secretions) received for KOH microscopy between May 19th 2021 and July 15th 2021 was performed. All samples were taken from patients clinically suspected for mucormycosis infection on the basis of symptoms such as nasal or sinus congestion, facial swelling, headache, or black lesions on the nasal bridge/mouth. The samples were incubated in KOH at 37°C, and a wet mount was later made with a centrifuged deposit. The clinical and demographic details of the patient were also collected from the hospital information system.

Samples showing broad, nonseptate or pauciseptate, hyaline fungal hyphae were taken as being suggestive of mucormycosis.[6]


  Results Top


A total of 75 samples were received in the Microbiology Laboratory of NCI from 50 suspected sinonasal mucormycosis patients from the ward and the emergency department [Figure 1].
Figure 1: Flow diagram showing number of patients and their laboratory testing status by Potassium hydroxide microscopy

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Out of the 75 samples, most were tissue biopsies (n = 63), followed by nasal crusts and secretions (n = 7), nasal swabs (n = 3), pus (n = 1), and bronchoalveolar lavage (n = 1).

On performing KOH microscopy, fungal elements were seen in 43 samples, whereas 32 samples were reported negative for fungal elements. Among the positive samples, 38 samples showed broad, nonseptate/pauciseptate hyaline hyphae, and 3 samples showed mixed infection with both nonseptate and septate hyphae. However, two samples showed only septate hyphae. Out of the 41 samples reported positive for nonseptate hyphae [Figure 2], 40 were tissue biopsies while 1 was a pus discharge. Interestingly, none of the nasal swabs, secretions, or nasal crust samples (n = 10) showed positive results. For three patients, the initial nasal swab/secretions received were reported negative for fungal elements. However, they too were later found to be positive on examination of the tissue samples taken during debridement. A total of 35 patients were found to be positive for mucormycosis. The demographic details of these patients are as per the table [Table 1].
Figure 2: Potassium hydroxide microscopy showing broad, nonseptate, hyaline hyphae (Magnification × 400)

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Table 1: Demographic details of the patients

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Viral markers (HIV, HBV, and HCV) were negative in all these patients. Interestingly, all these patients except one were positive for severe acute respiratory syndrome coronavirus 2 infection at the time of admission, as reported on the basis of cartridge-based nucleic acid amplification test done on GeneXpert platform (Cepheid, USA). History of steroid use was present in 19 out of 33 patients while history was not available for 2 patients. Out of the 35 laboratory-confirmed cases of mucormycosis, 32 (91%) had diabetes. Out of these 32 patients, 27 (84.3%) had a hemoglobin A1C (HbA1C) value of > 8% corresponding to poor sugar control. Unfortunately, the history of diabetes could not be retrieved for two patients. Despite having history of diabetes, HbA1C values was not available for another two patients. Furthermore, except for 5 patients for whom the data were not available, the ferritin levels were found to be high (>200 ng/ml) in 28 out of 30 (93.3%) laboratory-confirmed cases of mucormycosis, as evident in [Figure 3]. The mean (+standard deviation [SD]) of the HbA1C values was 10.051 (+2.391) and 7.84 (+2.493) among the laboratory-confirmed mucormycosis patients and patients without laboratory confirmation, respectively, whereas the mean (+ SD) of the serum ferritin values was 985.34 (+504.71) ng/ml and 612 (+451.35) ng/ml among those two groups of patients, respectively. Independent samples t-test performed revealed that significant differences of both HbA1C (P = 0.005) and serum ferritin levels (P = 0.017) existed between laboratory-confirmed cases of mucormycosis and clinically suspected cases of mucormycosis without laboratory confirmation.
Figure 3: Scatter plot showing relationship between hemoglobin A1C and ferritin values among laboratory-confirmed cases

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  Discussion Top


Owing to the angioinvasive and fulminant nature of the disease, mucormycosis is a lethal infection, especially if the diagnosis and treatment is delayed. Fungal culture, although the gold standard for diagnosis, is time-consuming and technically demanding. Moreover, the yield from clinical samples is poor, being approximately 50%.[7] While matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a brilliant tool for identification of bacteria, it is still undergoing validation for Mucorales. Serological methods including enzyme-linked immunosorbent assays and immunoblots too are still under research. Furthermore, in spite of being a rapid and reliable diagnostic method, polymerase chain reaction (PCR) requires sophisticated infrastructure in addition to a high degree of technical expertise and precision. Interestingly, the interpretation of PCR for Mucorales is also a challenge considering the presence of spores in the environment. In addition, these assays are still under research and are associated with poor sensitivity and specificity for being used as stand-alone tests for diagnosis. Some molecular tests provided encouraging results from serum/blood samples.[8],[9],[10] However, at present, it is recommended to use them only in conjunction with the conventional diagnostic methods.[7],[11] Hence, it is imperative that we rely for diagnosis on a method that can give us rapid results even in resource-limited settings to facilitate early diagnosis and treatment. Hence, microscopy may be the investigation of choice. During the recent outbreak, early diagnosis was of foremost importance as the decision-making regarding the appropriate antifungals and surgical debridement was dependent on it.[6] As is evident from the data, we could diagnose 70% of the patients only on the basis of microscopy and high degree of clinical suspicion. The only precondition is a good and appropriate sample, which is true for any test modality to be effective. Our results were congruent with another study from India which reported the positivity of KOH microscopy as 84%.[12] KOH microscopy was thus a reliable, inexpensive, and valuable modality for presumptive diagnosis of these cases. Considering the fact that culture is the gold standard for diagnosis and histopathology showing evidence of angioinvasion may probably be the diagnostic modalities of choice, we thus acknowledge that their absence is a limitation of this study. However, considering the criticality of early diagnosis and desperation for starting the treatment, these time-consuming modalities may not be as helpful as KOH microscopy, especially in resource-limited settings.

The most common predisposing conditions for mucormycosis infection reported are diabetes mellitus (DM), malignancies, transplantation, neutropenia, prolonged corticosteroid use, iron overload, illicit intravenous drug use, trauma, malnourishment, and immunosuppression due to any other cause. The same was evident in our study. Approximately 80% of the patients diagnosed with the infection were uncontrolled diabetics. This correlates with another multicenter study which reported that 77% of the ROCM cases occurred in diabetics.[13] Severe COVID-19 itself is a high ferritinemic state and whether high ferritin can predispose to opportunistic mycoses including mucormycosis remains a contentious topic.[14] Three carrier protein molecules, namely transferrin, ferritin, and lactoferrin, were found to bind with iron in mammalian hosts, and this sequestration helps to avoid the effects of iron toxicity besides providing defense against the microbes.[15] On the other hand, a high ferritin level may also lead to tissue damage by the reactive oxygen species generated as an effect of increased intracellular iron concentration in response to high ferritinemia. As a consequence of the tissue injury, intracellular iron is released in the circulation posing a unique risk to mucormycosis.[14] Majority of our patients (both laboratory-confirmed cases of mucormycosis and others) had increased serum ferritin values. Interestingly, we observed that the ferritin values among the laboratory-confirmed cases of mucormycosis were significantly much higher than the cases without laboratory confirmation.

Although there are various hypotheses, what still remains a mystery is the cause for the sudden upsurge in the number of mucormycosis during the second wave of COVID-19 disease in India. This has not only left the scientists perturbed but also disconcerted the entire medical fraternity. The possible hypotheses as proposed are as follows: first, the high prevalence of DM in the Indian population which is made worse due to illiteracy and lack of awareness regarding the health checkups and treatment for the same. With an estimated 65.1 million cases of diabetes in 2013, expected to reach 109.0 million by 2035, India has the second-highest number of the disease globally, only next to China.[16] It has been reported that around 43% of diabetes cases were unmasked during the diagnostic workup for mucormycosis, especially in North India.[2],[17] Second, the unsupervised and imprudent use of corticosteroids due to over-the-counter sale and self-prescription predisposed the patients to the infection by immune suppression as well as hyperglycemia.[18] Extensive use of antibiotics such as doxycycline, azithromycin, and ivermectin and immune modulators such as tocilizumab worsened the situation on account of immunosuppression. Third, the administration of minerals such as iron and zinc to boost the immunity might have possibly been instrumental in creating the appropriate environment for growth of the fungus and thus counterproductive.[18],[19],[20] Fourth, the potential of the hospital environment being the source for the infection was intriguing. The persistence of fungal spores in the environment, especially moist and humid environment colonizing the surfaces such as bed rails, tables, and taps, has always been a concern.[20] However, since these spores have always been omnipresent, it is unlikely that they would be responsible for the recent outbreak. Furthermore, the possible use of unsterile water instead of sterile distilled water in humidifiers being used for oxygen therapy as recommended could have contributed to the infection. In addition, the continued use/reuse of oxygen masks without cleaning and disinfection is also a concern to be investigated as the food particles stuck in the oxygen masks would have been nidus for infection.[18],[20] However, studies need to be planned to confirm this hypothesis. Another thought-provoking but ignored hypothesis was the industrial oxygen being diverted for use for medical purposes due to the intensively augmented demand. According to the World Health organization, the industrial oxygen along with its processing and transport is very distinct from medical grade oxygen.[21] For instance, the cleanliness and sterility of industrial-grade oxygen cylinders is far inferior to medical-grade cylinders considering the probable presence of impurities and micro leaks along with contaminated equipment.[18] The recommended deep cleaning and disinfection requirements might not have been followed taking into account the urgency and demand. However, all these are just speculations and detailed studies would be required to assess the right hypothesis out of these.


  Conclusion Top


The sudden outbreak of mucormycosis together with the ongoing pandemic of COVID-19 was nothing less than a disaster for the already overwhelmed and trembling health-care system of the country. Being a lethal disease in itself, the criticality of early diagnosis and treatment initiation could not have been emphasized more. The conventional microscopy was a savior in terms of providing rapid and reliable diagnosis even in a resource-limited setting. Further, the scientific community needs to discern the exact cause for the upsurge of mucormycosis cases during the second wave of COVID-19 which may consequently help us in averting another such catastrophe.

Acknowledgment

We are thankful to the Department of Hospital Administration, AIIMS, New Delhi, for their immense support. Furthermore, we would like to thank our technical staff for their relentless efforts to expedite the processing and report generation during the critical time of the outbreak.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Rama Chaudhry,
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmau.jmau_86_21



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    -  Vidyarthi AJ
    -  Das A
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    -  Singh G
    -  Thakar A
    -  Bhatnagar S
    -  Xess I
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