| Abstract|| |
Context and Aim: There is increasing prevalence of post-COVID fungal infection of rhinoorbitocerebral region especially mucormycosis and aspergillosis in India. Early diagnosis of these fungal infections are of utmost importance, since it may improve outcome and survival.,,,,,, 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. Materials and Methods: 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 article:|
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 2023;66:540-4
|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 [serial online] 2023 [cited 2023 Sep 27];66:540-4. Available from: https://www.ijpmonline.org/text.asp?2023/66/3/540/361986
| Introduction|| |
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. 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.,,,,,,
Presumptive diagnosis of fungal infections is based on signs and symptoms and radiology whereas definitive diagnosis is by KOH mount, culture, and histopathological examination.,,, 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 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.
[TAG:2]Materials and Methods[/TAG:2]
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.
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|| |
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.
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].
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|| |
Post-COVID fungal infection of Rhino-orbito-cerebral region was increasingly reported in India during second wave. 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.,,,,,,,,,,
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.
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.,, 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.,, 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. 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., Walsh et al., Lackner et al. 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. 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., 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.,
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. 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.,
| Conclusion|| |
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.
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| References|| |
Kubin CJ, McConville TH, Dietz D, Zucker J, May M, Nelson B, et al
. Characterization of bacterial and fungal infections in hospitalized patients with COVID-19 and factors associated with healthcare-associated infections. Open Forum Infect Dis 2021;8:ofab201. doi: 10.1093/ofid/ofab201.
Pfaller MA, Pappas PG, Wingard JR. Invasive fungal pathogens: Current epidemiological trends. Clin Infect Dis. 2006;43(Suppl 1):S3-14.
Neblett Fanfair R, Benedict K, Bos J, Bennett SD, Lo YC, Adebanjo T, et al
. Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri, in 2011. N Engl J Med 2012;367:2214-25.
Roden MM, Zaoutis TE, Buchanan WL, Knudsen TA, Sarkisova TA, Schaufele RL, et al
. Epidemiology and outcome of zygomycosis: A review of 929 reported cases. Clin Infect Dis 2005;41:634-53.
Vitrat-Hincky V, Lebeau B, Bozonnet E, Falcon D, Pradel P, Faure O, et al
. Severe filamentous fungal infections after widespread tissue damage due to traumatic injury: Six cases and review of the literature. Scand J Infect Dis 2009;41:491-500.
Hajdu S, Obradovic A, Presterl E, Vecsei V. Invasive mycoses following trauma. Injury 2009;40:548-54.
Lanternier F, Dannaoui E, Morizot G, Elie C, Garcia-Hemoso D, Huerre M, et al
. A global analysis of mucormycosis in France: The RetroZygo study (2005- 2007). Clin Infect Dis 2012;54(Suppl 1):S35-43.
Warkentien T, Rodriguez C, Lloyd B, Wells J, Weintrob A, Dunne J, et al
. Invasive mold infections following combat-related injuries. Clin Infect Dis 2012;55:1441-9.
Badiee P, Alborzi A, Karimi M, Pourabbas B, Haddadi P, Mardaneh J, et al
. Diagnostic potential of nested PCR, galactomannan EIA, and beta-D-glucan for invasive aspergillosis in pediatric patients. J Infect Dev Ctries 2012;13:352-7.
Badiee P, Alborzi A. Detection of Aspergillus species in bone marrow transplant patients. J Infect Dev Ctries 2010;4:511-6.
De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, et al
. Revised definitions of invasive fungal disease from the 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 (EORTC/MSG) Consensus Group. Clin Infect Dis 2008;46:1813-21.
Soni A. Paranasal mucormycosis in an immunocompetent individual: Importance of early diagnosis. Int J Oral Health Med Res 2017;4:52-6.
Shenoy MM, Teerthanath S, Karnaker VK, Girisha BS, Krishnaprasad MS, Pinto J. Comparison of potassium hydroxide mount and mycological culture with histopathologic examination using periodiic aciid-Schiifff sttaiiniing of the nail clippings in the diagnosis of onychomycosis. Indian J Dermatol Venereol Leprol 2008;74:226-9.
] [Full text]
Sangoi AR, Rogers WM, Longacre TA, Montoya JG, Baron EJ, Banaei N. Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: A ten-year retrospective review at a single institution. Am J Clin Pathol 2009;131:364-75.
Begari V, Pathakumari P, Takalkar AA. Comparative evaluation of KOH mount, fungal culture and PAS staining in onychomycosis. Int J Res Dematol 2019;5:554-8.
Walsh TJ, Gamaletsou MN, McGinnis MR, Hayden RT, Kontoyiannis DP. Early clinical and laboratory diagnosis of invasive pulmonary, extrapulmonary, and disseminated mucormycosis (zygomycosis). Clin Infect Dis 2012;54:S55-60.
Lackner M, Caramalho R, Lass-Flörl C. Laboratory diagnosis of mucormycosis: Current status and future perspectives. Future Microbiol 2014;9:683-95.
Cornely OA, Alastruey-Izquierdo A, Arenz D, Chen SCA, Dannaoui E, Hochhegger B, et al
. Global guideline for the diagnosis and management of mucormycosis: An initiative of the European confederation of medical mycology in cooperation with the mycoses study group education and research consortium. Lancet Infect Dis 2019;19:e405-421.
Ribes JA, Vanover-Sams CL, Baker DJ. Zygomycetes in human disease. Clin Microbiol Rev 2000;13:236-301.
Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st
century. Clin Microbiol Rev2011;24:247-80.
Heaton SM, Weintrob AC, Downing K, Keenan B, Aggarwal D, Shaikh F, et al
. Histopathological techniques for the diagnosis of combat-related invasive fungal wound infection. BMC Clin Pathol 2016;16:11.
Mayuri R Gohil
404/A Wing, Shetrunjay Residency, Opp. Sir T. Hospital, Bhavnagar - 364 001, Gujarat
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]