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Comparative evaluation of histopathological analysis, KOH wet mount and fungal culture to diagnose fungal infections in post-COVID patients


 Department of Pathology, Government Medical College, Bhavnagar, Gujarat, India

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Date of Submission27-Jun-2021
Date of Decision13-Dec-2021
Date of Acceptance30-Dec-2021
Date of Web Publication25-Nov-2022
 

   Abstract 


Context and Aim: There is increasing prevalence of post-COVID fungal infection of rhinoorbitocerebral region especially mucormycosis and aspergillosis in India.[1] Early diagnosis of these fungal infections are of utmost importance, since it may improve outcome and survival.[2],[3],[4],[5],[6],[7],[8] The objective of this study was to evaluate and compare routine laboratory diagnostic methods, that is, histopathological examination, KOH wet mount and fungal culture in the diagnosis of post-COVID fungal infections. Method: A total of 106 specimens of clinically suspected patients of post-COVID fungal infection of rhinoorbitocerebral region received in histopathology department were included in this study. The data of KOH wet mount and culture were acquired from the microbiology department after histopathological examination. Result: Approximately 88.68% of patients were diagnosed having fungal infections by one of the laboratory methods. The sensitivity of histopathological examination was highest (79.78%), followed by KOH wet mount (58.51%) and fungal culture (35.10%). Rhizopus species of zygomycetes group were the most common isolate (24.24%) on SDA culture. Overall 76% concordance was found between histopathological examination and fungal culture report for morphological identification of fungi. Conclusion: For the diagnosis of post-COVID fungal infection of Rhino-orbito-cerebral region, histopathological examination is was found to be more sensitive and rapid method to detect fungal hyphae. It leads to early treatment, prevents morbidity and mortality.

Keywords: Aspergillosis, culture, fungal infections, histopathology, KOH wet mount, mucormycosis, post COVID, rhino-orbito-cerebral infection


How to cite this URL:
Baxi SN, Gohil MR, Navadiya AJ, Bapodra MK, Patel HR. Comparative evaluation of histopathological analysis, KOH wet mount and fungal culture to diagnose fungal infections in post-COVID patients. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Feb 5]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=361986





   Introduction Top


A wide range of opportunistic bacterial and fungal infections in post-COVID patients have been reported in India amongst which mucormycosis and aspergillosis fungal infections are reported in high numbers.[1] Both these fungi target the rhino-orbital-cerebral regions and hence have high mortality and morbidity. Early diagnosis and timely initiation of treatment are crucial for the successful management of the disease.[2],[3],[4],[5],[6],[7],[8]

Presumptive diagnosis of fungal infections is based on signs and symptoms and radiology whereas definitive diagnosis is by KOH mount, culture, and histopathological examination.[1],[4],[9],[10] According to the revised EORTC/MSG (European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group) criteria, the diagnosis of fungal infection of rhinoorbitocerebral region can only be made by cultures obtained by sterile procedure or by histopathologic, cytopathologic evaluation of tissue specimens[11] as serological tests such as aspergillus galactomannan antigen and RT-PCR for mucor are available, but are very costly and/or not easily available, and radiological methods are not specific to detect specific fungal infection.

In this study, we analyzed the three common routinely used lab diagnostic methods to diagnose fungal infections in post COVID patients and compare efficacy of all in establishing a reliable diagnosis of fungal infection that may be crucial for early treatment.


   Material and Methods Top


A total of 106 specimens of suspected post-COVID fungal infection of rhino-cerebral-orbital region, received in histopathological department, were included in this study. The data of KOH wet mount and Culture were acquired from the Microbiology Department after histopathological examination. The specimens which were not sent for KOH wet mount and culture were excluded from the study.

Specimens received in 10% buffered formalin were processed as per routine histopathological specimen, stained with hematoxylin and eosin stain, and examined by two of the authors. Broad, aseptate, wide-angled hyphae were reported as mucorale [Figure 1]b and slender, septate, acute-angled branched hypahe were reported as aspergillus [Figure 1]c, and few cases having both typed hyphae were reported as mixed infection [Figure 1]d. Whenever fungal hyphae were not detected by routine hematoxylin and eosin stain, Grocott's methenamine silver (GMS) stain was done for detection of fungal hyphae.
Figure 1: Microscopic images of fungus KOH wet mount and histopathology in H&E stained smears, 40×.jpg

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At the same time, the microbiology department received a part of the same specimen for microbiological assessment. KOH preparation was made by incubation for 1–2 h with 10% KOH. Slides were microscopically evaluated within 4 h by competent microbiologists for the presence of branching thread-like structures (hyphae) or beaded spherical structures (spores). When they were present, it was considered to be a positive test [Figure 1]a and genus identification was done if it was possible.

A part of the material was also inoculated on Sabouraud's dextrose agar (SDA) incubated at 37°C for isolation of filamentous fungi. Cultures plates were examined for growth daily during the first week and twice a week during the next 3 weeks. Primary fungal isolates were subcultured onto SDA media for identification of species. The fungal species were identified based on their gross colony characteristics and microscopic morphology [Figure 2]a, [Figure 2]B, [Figure 2]c by a competent microbiologist.
Figure 2: Microscopy of gross fungal colony, 100×.jpg

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The data of KOH and culture were collected after completion of the histopatho report to check for concordance. In case of nonconcordance, the histopathology slides were reviewed by a third pathologist. Results so obtained were tabulated and statistically analyzed.


   Results Top


Of 106 suspected patients, total 12 patients (11.3%) were found to be negative for fungal infection by all three lab tests. Histopathological examination, KOH wet mount, and Fungal culture on SDA agar showed positive results in 75 (70.75%), 55 (51.88%), and 33 cases (31.13%), respectively [Table 1].
Table 1: Sensitivity of different diagnostic methods to diagnose fungal infections of rhino-orbito-cerebral region

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Hence, in our study, histopathological examination had the best sensitivity (79.78%), KOH had a sensitivity of 58.51%, and culture surprisingly was the least sensitive test with 35.11% sensitivity.

Of 33 SDA fungal culture positive, Rhizopus species of Zygomycetes group was the most common 8 cases (24.24%), and Absidia species of Zygomycetes group was the least common -1 case only (3.03%) [Table 2]. One case (3.03%) had cultural characteristics of Mucorales as well as Aspergillus niger and was labeled as mixed infection.
Table 2: Genus and species isolated From SDA culture

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Of 94 positive cases, only 40 cases had positivity by any single test, amongst which histopathological examination had highest positivity. Fifteen cases (16%) fungal infections were found to be positive by all 3 methods and 39 cases (41.5%) were positive by two methods. 12 cases were negative for fungal infection by all 3 lab tests [Table 3].
Table 3: Details of fungal detection by different methods

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There was correlation of results of histopathological examination and fungal culture in 76% of cases [Table 4]. Of seven patients which were diagnosed having mixed fungal infection histopathologically, two had aseptate fungal isolates and 5 had septate fungal isolates on SDA culture. They have been counted as concordant because at least one of the organisms was identified on culture.
Table 4: Comparison of results of histopathology with fungal culture for identification of fungal morphology

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   Discussion Top


Post-COVID fungal infection of Rhino-orbito-cerebral region was increasingly reported in India during second wave.[1] Among which Mucormycosis and Aspergillosis were most common. Mucormycosis is disease caused by mold fungi of the genus Rhizopus, Mucor, Rhizomucor, Cunninghamella and Absidia of Order- Mucorales, Class- Zygomycetes. Aspergillosis is caused by mold fungi genus-Aspergillus, Order- Eurotiales, Class- Eurotiomycetes. Early diagnosis is of utmost important, since both fungi are angioinvasive and cause tissue necrosis which leads to high mortality and morbidity.[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12]

According to the revised EORTC/MSG criteria, the diagnosis of proven invasive fungal infection can only be made by cultures obtained by sterile procedure or histopathologic, cytopathologic or direct microscopic evaluation of tissue specimens.[11]

We evaluated three diagnostic methods and found that histopathology examination was most sensitive in detecting fungus (79.8%) followed by KOH wet mount (58.51%) and fungal culture (35.11%). These findings are comparable with other studies which evaluated different diagnostic methods for diagnosis of other fungal infection.[13],[14],[15] Due to urgent need of reporting the fungal infection within 24 hours, we had released reports of presence/absence of fungi and its type purely on H and E stain, GMS stain was done in only few cases. Despite of it, Histopathological examination detected all the cases of fungus, detected in our hospital. It is difficult to compare any two tests in our study further because, although culture is considered as gold standard test, in this study it had very poor sensitivity, so we did not consider any one of the three tests as gold standard and we were not able to calculate specificity.

KOH wet mount of fresh tissue specimen is economical, minimal invasive and very rapid method. It gives results within 2-4 hours, is easy to perform test and requires minimal infrastructure.[13],[14],[15] But on the other hand it has low sensitivity whenever hyphae are sparse, so a negative KOH report cannot rule out fungal infection. Artifacts may give a false-positive result in unexperienced hands; moreover, KOH preparations are unable to identify the species. We found 58.51% sensitivity of KOH smears in detecting fungi which was higher than the fungal culture. Shenoy et al.[13] also found KOH wet mount to have a sensitivity of 64% and fungal culture to have a sensitivity of 42% in diagnosis of onycomycosis.

Although culture is considered as the gold standard test and is necessary to identify the genus and species and eventual antifungal susceptibility, it is more expensive than KOH preparations and takes 2-3 weeks for the report to be available. We found only 34.7% sensitivity of culture to detect fungus in our study which is comparable with studies of Shenoy et al.,[13] Walsh et al.,[16] Lackner et al.[17] Low sensitivity of culture may be due to the fact that under normal laboratory condition sporulation fails and some genera require special culture condition. Grinding or homogenization of tissue specimen or previously administered antifungal drugs destroys the delicate fungal hyphae. Some fungi especially mucorale species do not survive at refrigerated temperature, so if the stored sample is inoculated, that may give false-negative results. The reason for such a poor sensitivity of a gold standard method in detection of the fungi in rhino-orbito-cerebral fungi could be the highly necrotic tissue which has few viable organisms, caused by angioinvasive Mucorales and Aspergillous species.

A positive culture from a sterile site confirms diagnosis while a positive culture from a non-sterile site could be due to contaminant and must be combined with clinical, radiological, and histopathological examination to establish a probable diagnosis.[18] Hence, when fungal hyphae is not seen on histopathology, positive culture may be a false positive result. We found 2.8% cases in which culture isolated aspergillus while histopathology and KOH both were negative which may be due to contamination.

Histopathologic examination remains the major diagnostic tool in mycology because it gives rapid results usually within 24–48 h and presumptive identification of fungal infection can be done and coinfection can be identified. Mucorales genera produce typically non-pigmented, wide, ribbon-like hyphae with no or few septation (pauciseptate) and right-angle branching.[19],[20] Aspergillus species are typically thin, septate, and form acute angle branching. These features are easily seen on H and E stain slides. Moreover, tissue invasion and blood vessels invasion causing thrombosis and tissue necrosis or inflammatory reaction can only be seen histopathologically which also help to determine whether an organism represents contamination, colonization or true infection.[20],[21]

We have encountered few cases with discrepant histopathology and culture results. Of 18 aseptate hyphae diagnosed by histopathology 6 cases had growth for septate mold that is Aspergillus. Histopathological slides of these cases were reviewed by authors but didn't show classical morphology of aspergillosis. Swollen and distorted hyphae seen in extensive necrotic areas may lead to morphological misdiagnosis of mucorales. Sometimes hyphae become crinkled, folded, and fragmented in appearance, so accurate assessment of septation and type of branching may be extremely difficult. In such cases, test for aspergillus galactomannan antigen should be recommended although it is an expensive test.

We found overall 76% concordance between histopathological examination and fungal culture report. Heaton et al.[21] found 85% concordance. In general, accuracy of microscopic identification of fungal species using either histopathological or cytological specimen has been estimated to range from 20% to 80% depending upon expertise of the consultant pathologist and other factors like the species of fungal infection and whether special stains were used along with H and E stain.[14],[17]


   Conclusion Top


For the diagnosis of post-COVID fungal infection of Rhino-orbito-cerebral region, histopathological examination by H and E stain is the most sensitive and rapid test to detect fungal hyphae compared to KOH preparations and fungal culture. and it should be confidently relied upon by the treating clinicians to initiate prompt treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Mayuri R Gohil,
404/A wing, Shetrunjay Residency, Opp. Sir T. Hospital, Bhavnagar - 364 001, Gujarat
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_663_21



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