AIM: To perform site-based comparative analysis for samples collected from the nasal region and Oral cavity subjected to microscopic detection of fungal hyphae in KOH mount in a group of patients with rhinomaxillary mucormycosis.
Methodology- 40 patients full-filled eligibility criteria. The diagnostic outcome of detection of fungal hyphae from the KOH samples obtained was the primary endpoint of the study. Based on this, the samples were grouped into 3 groups viz- Oral, Nasal and Both. The secondary outcome was to check if there was any diagnostic delay in these three groups of patients
Results: The mean number of days for delayed diagnosis for oral site involvement was 56.33 ± 37.53, for Nasal involvement was 32.86 ± 19.53 and for both oral & nasal involvement was 22.00 ± 12.94. This difference was statistically significant at p=0.03. The mean delay in diagnosis was significantly less when both oral & nasal regions are involved as compared to the only oral region involved at P=0.01.
Conclusion- To avoid the chance of delayed diagnosis or false-negative results its best to collect samples from both nasal tissues and the most representative site in the dentoalveolar segment depending on the extensiveness of the disease.
Mucor mycosis (previously called as zygomycosis) is a rare but serious angio-invasive infection caused by a group of fungi called mucormycetes. When inhaled, spores of these ubiquitous fungi can infect lungs, paranasal air sinuses and may even involve the brain and eyes. Mucormycosis mainly affects people who are immunocompromised and in patients already infected with other diseases. The incidence rate of mucormycosis globally varies from 0.005 to 1.7 per million population. The prevealence of the disease has risen in the past several decades, possibly because of the increased use of steroids, cytotoxic drugs, and other immunosuppressive medical therapies (such as bone marrow transplant) or simply because of the increased recognition lately . The current trend indicates a surge in the disease burden in those with pre-existing diabetes, systemic corticosteroids, and patients infected and/or recovered from SARS-CoV-2 (COVID-19) infection. India had reported a recent surge in mucormycosis cases with an estimated prevalence of 140 per million population, which is about 80 times higher than the prevalence in developed countries. Being an aggressive, life-threatening infection it requires prompt diagnosis and early treatment for successful management.
The diagnosis of mucormycosis itself is challenging as the clinical approach to diagnosis lacks sensitivity and specificity . Definitive diagnosis is performed by histopathological examination, making microscopy (direct and on histopathology) and culture the cornerstone of diagnosis .
Various specimens can be considered for microscopy depending upon the clinical manifestations and the site of infection. Direct microscopy of a fresh tissue sample with a few drops of potassium hydroxide (KOH) helps to identify the typically broad, hyaline, ribbon-like, irregular fungal hyphae with wide-angle branching. This is accompanied by tissue necrosis and angioinvasion of the fungi giving us a presumptive diagnosis. An early treatment with specific antifungal drugs is of utmost importance to reduce the morbid state.
In our clinical setting, many patients suspected to have rhinomaxillary mucormycosis were found KOH negative on multiple nasal pus swabs and nasal biopsies despite being clinically symptomatic and with radiographically subtle changes not suggestive of evident necrosis where debridement or surgical exploration is not abosultely indicated. This led to delayed diagnosis and thereby leading to a more destructive lesion at later stage. However when subjected to alveolar bone biopsies and sinus biopsies, samples detected fungal hyphae.
The aim of this study was to perform a site based comparative analysis for samples collected from nasal region (swabs, biopsy) and Oral cavity (sinus lining, alveolar bone from extracted socket) subjected to microscopic detection of fungal hyphae in KOH mount for early diagnostic screening in a group of patients suspected for rhinomaxillary mucormycosis based on the history in the endemic.
This study was approved by Instituitional Ethics Committee at All India Institute of Medical Sciences- Raipur (AIIMSRPR/RC(P)/2021/385). Data of all patients admitted to the Department of Dentistry at were retrospectively studied for a duration of 5 months. Only patients subjected to the institutional KOH mount from both nasal swab/nasal endoscopic biopsies and biopsies from alveolar bone/dental extraction sockets/ maxillary sinus lining either through Caldwell Luc approach were eligible to be included in the study. These cases in India surged shortly as a sequale to COVID- 19 infection, so the study participants included had a history of COVID-19 infection. The diagnostic outcome of detection of fungal hyphae from the KOH samples were the primary endpoint of the study. Based on this, the samples were grouped into three groups viz- Group I: Oral being patients positive for biopsies from necrotic alveolar bone, sinus lining; Group II: Nasal being patients positive for biopsies from nasal tissues and pus swabs and Group III: Both being patients positive for biopsies from nasal and oral cavity.
The secondary outcome was to check if there was any diagnostic delay in these three groups of patients. Diagnostic delay was defined as the combined patient delay (time from first symptoms until presentation to a health practitioner) and professional delay (time from first presentation to microscopic detection of fungal elements) in days. Any delay over 30 days was considered to be significant adding to the disease burden.
The characteristics studied were extent of clinical disease by assessing extraoral, intraoral and nasal endoscopic findings; radiographic evidence of disease involving rhinomaxillary complex, duration for establishing diagnosis after first clinical symptom (diagnostic delay), host factors like uncontrolled random blood glucose, postoperative evidence of recurrent infection and incidence of wound dehiscence after total surgical debridement. Patients with missing data ailing the study outcomes were excluded from the study.
Two data abstractors were appointed to review data retrospectively. First investigator determined the procedure or location of biopsy site for each patient along with clinical and radiographic data in the preset case record format. Second investigator recorded the KOH lab results (outcome) for the patients satisying the eligibility criteria. Names were redacted out of the reports by a third person before outcome data abstraction to avoid any form of bias. Thus possibilitiy of selection and reporting bias was eliminated. All the demographic and quantitative data were presented as degrees of distribution and graphs.
Association among variables like diagnostic delay were examined using Kruskal Wallis test followed by Mann Whitney Post hoc test based on the site involved. All cateogorical data were assessed by Chi square test for each group. A p value of < 0.01 was considered to be statistically significant.
Over the study period, 85 patients were admitted under the Department of Dentistry for treatment of rhinomaxillary mucormycosis. Of these, 40 patients fulfilled the eligibility criteria and data of these patients were recorded. Any missing data essential to establish primary outcome were excluded.
Patients were divided into three groups based on KOH mount results as Oral positive, nasal positive and both positive groups. The characteristics of patients is given in Table 1. The mean age of patients for Group I was 47.33 ± 13.34 years, for Group II was 46.71 ± 8.44 years and for both sites were 49.00 ± 12.00 years. Males were more predominantly distributed [57.1 – 71.4%] in all three KOH specific sites as compared to females [14.3 – 42.9%]. However, there was no significant difference observed in the mean age (p=0.93) as well as gender distribution (p=0.50) of the patients with respect to KOH specific sites (Table 1). Hence each study group was homogenous.
Correlation between Intra oral findings and the KOH specific sites :
The test results revealed that the gingival lesions and dental mobility showed a near equal correlation with all three KOH specific sites, ranging from 42.9 – 57.1% and 66.7 – 85.7%, respectively. However, no significant correlation was observed between gingival lesions, dental mobility & KOH specific sites. Incidentally, a few cases showed a positive expression of maxillary mobility in oral site as compared to other sites, but there was no significant correlation found between the three KOH specific sites (Table 2).
Correlation between Radiographic findings of Bony Erosions and the KOH specific sites using Chi Square Test:
The test results revealed that the bony erosions in the radiographic findings were predominantly found to be confined to the maxillary bone without alveolar bone involvement [42.9 – 47.6%] in all three KOH specific sites. The bony erosions localization in maxillary bone with alveolar bone involvement and those that are confined to maxillary and zygomatic bone showed an equal distribution [28.6%] in group II and group III patients as compared to 42.9% and 9.5% in group 1, respectively. However, no significant correlation was observed between bony erosions in radiographic findings and the three group sites (Table 3).
Correlation between Endoscopic findings and the KOH specific sites using Chi Square Test:
The test revealed that only nasal crusting was predominant in the nasal site [28.6%], whereas nasal crusting & pus discharge were more predominantly encountered [71.4%] when both oral & nasal KOH specific sites were involved. A relative proportion of patients were negative for endoscopic findings for group I (19.3%) and group II (14%). The data on the endoscopic findings were not available with respect to all sites [14.3% to 42.9%]. However, no significant correlation was observed between endoscopic findings and KOH specific sites (Table 4).
Comparison of mean duration of delay in diagnosis based on the site involved using Kruskal Wallis Test followed by Mann Whitney Post hoc Test:
The test results showed that the mean number of days for delayed diagnosis for oral site involvement was 56.33 ± 37.53, for nasal involvement was 32.86 ± 19.53 and for both oral and nasal involvement was 22.00 ± 12.94 days.. Multiple comparisons between groups revealed that the mean delay in diagnosis was significantly less when both oral & nasal regions were involved as compared to only oral region is involved at p=0.01. However, no significant differences were observed between other combinations (Table 5).
Mucormycosis species being ubiquitous and an uncommon commensal in human paranasal sinuses and oral cavity, turns pathogenic only if the host immunity is compromised. This can occur due to various reasons including prolonged steroidal therapy, immunocompromised patients, uncontrolled diabetes, malignancies such as lymphomas and leukemias, renal failure and in patients infected with COVID-19 infection. In the present study, 70% participants tested Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) positive, 10% were symptomatic but tested negative, 12.5% had no COVID-19 association. Immune dysregulation caused by the SARS-CoV-2 virus and the use of broad-spectrum antibiotics and corticosteroids particularly in patients with poorly controlled diabetes with ketoacidosis were proposed to be the cause of infection .
Factors attributable to COVID-19 related mucormycosis includes hyperglycemia, sera response to infection, altered adipose tissue sensitivity and β-cell destruction, altered mucosal clearance and local immunity, use of immune-mediated therapies (Corticosteroids, Tocilizumab, etc). Additionally, mucosal erosion secondary to aggressive use of steam inhalation or the use of high flow oxygen have also been considered as factors promoting the fungus colonisation.
Uncontrolled diabetic patients and in patients under corticosteroid usage normally present with reduced capability of the host in combating fungal infections. It can lead to impaired ciliary motility of the nasal mucosa thereby leading to ineffective phagocytosis of the invading organisms, instead providing them with an excellent substrate for proliferation. Presence of ketone reductase in the fungi help them thrive through critical situations when there is ketoacidosis and metabolic acidosis .
Infection is caused by asexual spore formation. In an immunocompromised host, when airborne spores settle on the oral or nasal mucosa germination will follow and hyphae will develop as polymorphonuclear leukocytes are less effective in removing the invading hyphae. Hyphae then begin to invade arteries, wherein they propagate within the vessel walls and lumens causing thrombosis, ischemia, and infarction with dry gangrene of the affected tissues. Later hematogenous spread to other organs can occur leading to spead of the infection . Due to their relatively bigger dimensions as compared to other species, they are easily retained in the paranasal sinuses.
The time lapse between the onset of symptoms and diagnostic procedures was proved to be associated with mortality . Chamilos et al., demonstrated that delayed antifungal therapy increases mortality in patients with haematologic malignancy that have invasive mucormycosis infection. They emphasised the importance of not just timely administration of antifungal agents but also aggressive diagnostic strategies . According to Jeong et al., timely debridement of necrotic tissue can reduce the extent of infecting moulds and improve the penetration of antifungal agents to the site of infection . In the absence of reliable diagnostic tools except direct examination and culture of infected tissues, molecular approaches are currently just in phase of trial and economical feasibility is difficult in developing countries like India with overburdened health management system.
Succesful management of this deadly disease is early diagnosis and early treatment which includes a combination of medical and surgical debridement. Knowing the importance of early diagnosis, which is the most challenging aspect of mucormycosis management, we were left with the option of microscopic examination of tissue speciments in patients who were clinically suspected to have the fungal infection. Commonly patients with rhinomaxillary infection presented with mobile tooth (70%), numbness in the infraorbital region (40%), palatal ulceration (25%), blurred vision (7.5%), nasal discharge (20%), intraoral pus draining sinus tract or fistula (65%), exposed bone- alveolar (20%), palatal (5%), both (7.5%) and absent (67.5%) with common history of COVID-19 infection. Most of the patients were hospitalised under oxgyen and were usually given steroids to combat the deadly viral infection. Computed tomography and magnetic resonance imaging done revealed thickening of maxillary sinus (right- 5%, left- 10%), any two sinuses (47.5%), three sinuses (32.5%) and all paranasal sinuses in 5% of the cases. Sublte erosive changes of the maxillary bone (80%) with alveolar bone involvement and without alveolar erosion were seen in 35% and 45% of the cases, respectively. Changes extending to zygomatic bone were seen in 15% of the cases.
Infection usually starts in the nasal cavity and spreads to the paranasal sinuses. Bhandari et al., in 2021 stated that the humid environment of the nose and paranasal sinuses favors the growth and invasion of fungi. Early implantation of fungi is common in the maxillary sinus with a mass of fungal growth called a fungal ball and with no evidence of bone erosion. The most common site involved in mucor is middle turbinate, followed by middle meatus and septum. In undiagnosed or untreated cases, the infiltration of the bone is common .
When the infection extends through the nasal turbinates, the orbit frequently becomes involved, and orbital cellulitis, extraocular muscle paresis, proptosis, and chemosis are commonly found. Extension of the infection posteriorly into the brain results in the formation of abscesses and necrosis of the frontal lobes. When the disease invades inferiorly into the mouth, a black, necrotic eschar is often found on the palate, this finding is highly suggestive of the presence of invasive mucormycosis .
In present setup, various tissue specimens were sent for microscopic examination (using KOH stain) and fungal culture. Swabs were sent from nasal mucosa, sinus tract, or tissue specimen from necrotic bone or suspected tissue from paranasal sinus with the aid of endoscopy. Few of these showed fungal elements giving way for hospitalisation and definitive treatment while some turned negative despite clinically evident disease leading to delayed medical management. Multimodal therapy is key to reduce the disease burden which involves both surgical debridement and systemic antifungal therapy. Without antifungal therapy the disease burden and recurrence increases even after performing surgical debridement.
Harrill et al., stated that the median time from the symptom onset to diagnosis was 7 months . In the present study, the mean number of days for delayed diagnosis amongst various study groups were 56.33 ± 37.53, 32.86 ± 19.53 and 22.00 ± 12.94 days, respectively and which was statistically significant.
Because of the nonspecific clinical signs, a high index of suspicion should be made in the existence of risk factors. Nasal scrapings and fine-needle aspiration cytology were performed to give the diagnostic results which showed fungal hyphae. Swabs taken and cultures from sinuses were negative in most of the cases (25%). Thus in the rhinomaxillary group which present with common disease presentations, we found 52.5% of participants to show fungal hyphae from the oral tissue (necrotic bone or sinus lining via Caldwell Luc), 12.5% from nasal tissue/ pus as well as from both tissue specimens with extensive involvement.
Mucorales appear in the tissue as irregularly shaped, broad hyphae with right-angle branching. The hyphae are easily visualized in routine hematoxylin-eosin stained sections as well as in tissue stained by the periodic acid– Schiff reaction or by Grocott-Gomori methenamine-silver nitrate stains. Hyphae are usually found in the midst of an acute neutrophilic infiltrate along with hyphal invasion of the blood vessels .
In a report to check the diagnostic values of KOH examination, histological examination, culture for onychomycosis and Periodic acid Schiff stain (PAS)were highly specific but poorly sensitive. KOH was highly sensitive but poorly specific and Grocott’s Methenamine ilver Stain (GMS) were both highly sensitive and specific .
Various other techniques for diagnosis include Immunohistochemistry, polymerase chain reaction (PCR) − based methods and matrix-assisted laser desorption or ionization time-of-flight mass spectrometry (MALDI-TOF MS). In a study done in combining a semiquantitative method using high-resolution melt analysis and real-time quantitative PCR (RQ PCR) assays showed sensitivity and specificity of 100% and 93%, respectively and high negative predictive value (99%) in detecting mucormycetes. Despite its higher sensitivity and specificity these diagnostic aids are not available in clinical set ups of India for it to be used commonly giving way to conventional techniques. Thus KOH mount setup is feasible for Indian scenario however the sites should be carefully selected in order to collect suitable sample showing hyphae infiltration in the necrotic tissue helping to predicate the diagnosis at early stage rendering them to medical management with systemic antifungals.
The limitation of the study is its retrospective temporality with limited sample size. The present study concurs that in rhinomaxillary mucormycosis group, the fungal spores have two different pattern of invasion evident with oro-nasal signs and symptoms. To avoid the chance of delayed diagnosis or false negative results, its best to collect samples from both nasal tissues as well as most representative site in dentoalveolar segment depending on the extensiveness of the disease.
In rhinomaxillary mucormycosis, treatment is surgical debridement followed by systemic antifungals. In order to start antifungal therapy, it is important to establish a diagnosis prior because of the financical burden and nephrotoxicity it imposes. The microscopic examination and its reliability depends on the viablility and infiltration of fungal hyphae in the samples obtained. Based on the evidence in our retrospective study, we imply that both nasal swabs and invasive biopsies from oral representative sites are required to negate the possibility of delayed diagnosis.
Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by Instituitional Ethics Committee at All India Institute of Medical Sciences- Raipur (AIIMSRPR/RC(P)/2021/385).
Consent to participate: A written informed consent was obtained from all individual participants included in the study.
Consent for Publication: Consent is obtained for publication of this data from the patients.
Conflict of Interest Statement: None of the authors have any financial or personal interest associated with this article. No funding has been received by any author in relation to this study.
Author contributions: SSA and SR are the operating surgeons/attending physicians, SSA and SR collected the data, PA and VG analysed the data, SSA and PA prepared the manuscript, SR and VG proof read and edited the manuscript. All the authours read the final version of this manuscript and agreed upon the writeup.
Funding: Non funded study.
Availability of Data nd Material: All the research related data is available with the principal investigator.
Tables 1 to 5 are available in the Supplementary Files section.