key: cord-1011069-sfy5mqsl authors: Philip, Ashish C.; Madan, Prarthna; Sharma, Sonal; Das, Shukla title: Utility of MGG and Papanicolaou stained smears in the detection of Mucormycosis in nasal swab/scraping/biopsy samples of COVID 19 patients date: 2021-12-29 journal: Diagn Cytopathol DOI: 10.1002/dc.24924 sha: 429dfe711780f79be182ff5e23401c5b44599209 doc_id: 1011069 cord_uid: sfy5mqsl BACKGROUND: COVID 19 has been rapidly spreading across the globe. As a result of alteration of the immune milieu by COVID 19 and its treatment, there has been a rise in opportunistic fungal infections particularly Mucormycosis in these patients. Delay in diagnosis of these fungal infections can be fatal. The usual diagnostic modalities used to detect Mucor include potassium hydroxide (KOH) mount, fungal culture, and histopathology. Since histopathology and fungal culture have a long turnaround time we are dependent on KOH mount for rapid results. Here we investigate the role of stained cytology smears in the rapid diagnosis of Mucormycosis. METHODS: A prospective observational study was conducted in a tertiary health care hospital on samples of patients clinically suspected to have Mucormycosis. We performed May Grunwald Giemsa (MGG) and Papanicolaou (PAP) stains on the remnant samples of nasal swabs/scrapings/biopsies after KOH test and fungal culture. We took 16 KOH positive and 16 KOH negative samples. We also examined 16 fresh samples from patients whose earlier samples were reported to be negative on KOH test. RESULTS: The 6/16 KOH positive samples were found to be positive on stained cytology smears and 2 were mixed infections wherein both Mucor and Aspergillus were seen. The 4/16 KOH negative samples were positive for Mucor with one sample having both Mucor and Aspergillus. The 3/16 repeat samples which were earlier negative on KOH test were positive for Mucor. CONCLUSION: Stained cytology smears if used in conjunction with KOH test can increase the overall sensitivity of detection of Mucormycosis and mixed infections. of Mucormycosis have been reported in India. 4 These infections are associated with rapid progression and a poor clinical outcome. Thus, early diagnosis and appropriate quick management are of utmost importance in reducing fatality due to severe fungal co infections in COVID 19 patients. For diagnosis, specimens are usually obtained from nasal and paranasal sinuses and include nasal swabs, scrapings, or nasal crust biopsies. Swabs can directly be taken from the fungal lesions present externally on the skin of face (eyes, nose etc.). 5 The usual diagnostic modalities used to examine these specimens include potassium hydroxide (KOH) mount, fungal culture and histopathology. While KOH has a rapid turnaround time, results from histopathology and culture are often delayed. Although KOH has good sensitivity, it may be supplemented with a diagnostic method which can examine these specimens and give fast and reliable results. [6] [7] [8] Traditionally cytology has been used in aspirated materials for fungal diagnosis. Cytological examination of aspirates helps identify most fungi by their morphological characteristics. Zygomycetes including Mucor are characterized by wide (3-25 μm) ribbon like, irregular branching pauciseptate hyphae. [9] [10] [11] In the present study we examined the remnants of samples including nasal swabs/scrapings/ biopsies after KOH test and fungal culture were done. We performed May Grunwald Giemsa (MGG) and Papanicolaou (PAP) staining on smears prepared from the crushed material. We also performed cytospin on the saline solution in which the sample was sent. Further, we did cytological examination on fresh samples from patients who were previously reported as negative for fungus on KOH wet mount (on an earlier sample) but were clinically suspected to have Mucormycosis. Here, we report our findings and discuss the utility of cytological examination using MGG and PAP staining for diagnosis as well as species identification. Samples having scant material were excluded. Remnants from samples sent to Department of Microbiology for KOH test along with samples sent directly to Department of Pathology for cytological examination, were taken. For swabs, the material suspended in the saline was collected using a wire loop followed by spreading it on two separate glass slides in circular motion. The smears prepared were stained with MGG and PAP stain. A 3 mL of saline in which the swab was suspended was collected in a plain test tube and was cyto-centrifuged following which MGG and PAP was done on the smears prepared. Sample collection and smear preparation were done while taking all precautions to prevent cross contamination. For scrapings and biopsies, a small piece was taken using a loop and kept on the slide and another slide was used to spread it with uniform pressure, following which the same procedure as above was repeated. The same procedure was followed for slides which came directly from wards to the Cytology Laboratory, Department of Pathology. After all the smears were stained, the slides were examined for presence of fungus using Dewinters Opticals Microscope at 4Â, 10Â, Out of 16 patients who were reported as negative for fungus on KOH test, 4 were positive on MGG and/or PAP stain. Out of these, one was found to have both Mucor and Aspergillus. Two out of these four samples were culture positive (Table 1) . Out of 16 repeat samples from patients who were earlier reported as negative for fungus on KOH test done on a previous sample, 3 were positive for Mucor on MGG and/or PAP stain done on the repeat sample ( Figure 4 ). Two out of these three were culture positive on the earlier sample on which KOH was done (Table 1) . Coronavirus Disease 19 (COVID 19) caused by SARS CoV 2 has been sweeping across the globe at a rapid pace. A major cause of concern in patients with severe COVID 19 illness is susceptibility to opportunistic bacterial and fungal infections. 12 Increased susceptibility to F I G U R E 1 Basic structure of study conducted 21 A definitive diagnosis is based on demonstration of fungal hyphae in biopsies from affected tissues. 22 Histopathology as a diagnostic tool helps to differentiate between the presence of fungus as a pathogen and a culture contaminant. 23 Histopathology with Grocott-Gomori Methenamine Silver (GMS) stain, Periodic Acid-Schiff (PAS) can help highlight the fungal wall. 9, 24 Mucorales can also be cultured on Sabouraud Dextrose, brain heart infusion or potato dextrose agar at 25-30 C. 9 However, the utility of fungal culture as a method for diagnosis is limited as it does not conclusively indicate infection with the organism identified. 9 It is relatively expensive and takes at least 3 weeks to give reliable results. 21 Reported sensitivity of fungal culture (57%) is lower than that of KOH mount while the specificity is comparable (92.8%). 15 Traditionally, nasal swab specimens have been tested by KOH wet mount for quick diagnosis of fungal infections. 10 conjunction, will definitely increase the overall sensitivity and specificity (species identification) of diagnosis on the same specimen. 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Data available on request from the authors. https://orcid.org/0000-0003-1077-2576