Cytomorphometric Changes of Buccal Exfoliated Pap Stained Smears among COVID-19 Recovered Patients; Sudan 2022

Introduction: COVID-19 is known to cause various changes in oral mucosa which can be detected by exfoliative cytology. The present study aimed to determine the cytomorphometric change of oral mucosa exfoliated Pap-stained smears for COVID-19 recovered patients at Khartoum state 2022. Methods: This was a cross-sectional comparative, community-based study conducted at Khartoum State. Eighty (80) participants who recovered from COVID-19 enrolled in the study as cases and twenty (20) healthy participants as the control group. Results: In the present study the ages of the participants ranged between 19-75 years. Mean was significantly different in cytoplasm diameter (CD), nucleus diameter (ND), and nuclear/cytoplasmic ratio (N/CR) between the patients who recovered from COVID-19 versus the healthy group (69.71μm versus 37.45μm, P. value= 0.001; 10.02μm versus 6.05μm, P. value= 001; and 28.9 μm versus 18.0μm, P. value= 0.016) respectively. Conclusion: The marked changes in cytomorphometric parameters caused by infection with COVID-19 prove that infection with the virus causes changes in the buccal mucosa. These findings will help in the screening and diagnosis of the disease.


INTRODUCTION
Coronaviruses are a large family of viruses are known to cause respiratory infections such as Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and Severe Acute Respiratory Syndrome corona-virus2 (SARS-CoV-2) (1,2).World Health Organization (WHO) officially named (SARS-CoV-2) as coronavirus disease 2019 (COVID-19) (3).The disease spreads through close contact with symptomatic or asymptomatic infected people via droplets and aerosols generated by talking, breathing, sneezing, and coughing (4).The oral cavity plays a vital role in virus entry and transmission.Oral signs and symptoms include dry mouth, burning mouth, taste loss, Candida Albicans infection, dysgeusia and ulceration (5,6).The buccal mucosa is lined by nonkeratinized stratified squamous epithelium and exhibits angiotensin converting enzyme2 (ACE2) and transmembrane serine protease2 (TMPRSS) expression (7,8).The virus consists of the envelope protein (E), spike protein (S), transmembrane protein (M), and nucleoprotein (N).The (E, M, and S) proteins have a role in virus entry, assembly, and pathogenesis.The(N) protein plays a major role in RNA replication, suppressing the host cell's innate immune response, and aiding in virions assembly (9,10).The principal mode entry of (SARS-CoV-2) is via binding of the viral spike glycoprotein (S) to ACE2 host cell receptor and activated by host cell-derived transmembrane serine protease2 (TMPRSS2) to gain entry for replication (11).The oral cavity is one of the main routes of virus entry, and the mucosa is the first site of infection and exposure to cellular changes (5).Several studies demonstrated the colonization of oral mucosa by (SARS-CoV-2) (7), and recently there are ongoing studies on oral mucosa and saliva as a rapid screening test for the disease (10).The study of cytomorphometric changes due to the virus will help in a better understanding of virus pathogenesis and diagnosis, however, no quantitative studies were conducted for exfoliated cells from the oral mucosa.
Therefore, the present study aimed to determine the cytomorphometric change of oral mucosa exfoliated Pap-stained smears for COVID-19 recovered patients at Khartoum state 2022.

MATERIAL AND METHOD Study design/Setting:
This was a cross-sectional comparative, community-based study conducted at Khartoum State, Sudan from December 2021 -January 2022.

Participants:
Eighty (80) participants who recovered from COVID-19 enrolled in the study as cases and twenty (20) healthy participants who were never exposed to COVID-19 as the control group.

Inclusion Criteria:
Regardless of gender and age, all COVID-19-recovered patients were their infection confirmed by RT-PCR enrolled in the study.

Exclusion Criteria:
Diabetes Mellitus, women with menstruation, alcoholism, tobacco user, acute diseases such as acute respiratory infection, and the patient exposed to computed tomography were excluded from the study.

Sample collection:
Under sterile and antiseptic procedures each participant was requested to rinse his mouth with water and dried it with sterile gauze to remove excess saliva and debris.Using a moist wooden spatula with distilled water to obtain a smear from clinically normal appearing buccal mucosa for each group with gentle pressure for 2-3 minutes.Scrapping material was smeared on clean labelled slides.Slides were then fixed in 95% alcohol for 30 minutes.Air drying of the smears was avoided, and smears were stained using conventional pap stain.All quality control measures were adopted during the specimen's collection and processing.The smears were then stained with Papanicolaou stain as the following procedure.Demographical information was collected from the cases as well as the control group.

Papanicolaou staining procedure (Pap stain):
The smears were hydrated through descending concentrations of ethanol, 90%, 70%, and distilled water for 3 minutes for each.Then the nucleus was stained with Harris's hematoxylin for 4 minutes, bluing for 4 minutes, counterstained with orange G6 for 4 minutes, differentiated with 95%alcohol, followed by cytoplasmic stain with EA50 (eosin azure 50) for 4 minutes, differentiated with 95%alcohol.Finally, the smears were dehydrated through ascending concentration ethanol for 3 minutes for each, cleared and mounted.

Cytomorphometric Analysis:
The smears were examined under x40, the cells were selected by moving the microscope stage in a "Z" shape to avoid recounting the same cell.The cells were identified based on morphology and staining characteristics.Nuclear, cytoplasmic diameter, and nuclear/cytoplasmic ratio were obtained by the Optika optical microscope camera using a digitizer cursor in both axes and about ten (10) well-defined, unfolded, good stained cells were counted.The means for the nuclear diameter, cytoplasmic diameter, and nuclear/cytoplasmic ratio were calculated for each sample and expressed in micrometres.

RESULTS
In the present study, the ages of the participants ranged between 19-75 years.There were 52 males and 36 females (Table 1).Mean was significantly different in cytoplasm diameter (CD), nucleus diameter (ND), and nuclear/cytoplasmic ratio (N/C R) between the patients who recovered from COVID-19 versus the healthy group (69.71 μm versus 37.45μm, P. value= 0.01; 10.02μm versus 6.05μm, P. value= 001; and 28.9 μm versus 18.0μm, P. value= 0.016) respectively (Table 2 and Figure 1).There were no significant differences in cytomorphometric analysis according to gender (Table 3).Although, no significant correlation between the duration of infection and cytomorphometric changes of COVID-19 recovered patients, all cytomorphometric parameters significantly direct correlated with female age (Table 4).
The data were analyzed using Statistical Package for the Social Sciences 20 (SPSS Inc., Chicago).The differences in cytomorphometric between the two groups were tested by independent sample T-test.A P. value of <0.05 was considered significant.

Figure 1 .Figure 2 .
Figure 1.Means of cytomorphometric in the study population

Table 1 .
Descriptive statistics for the study population

Table 4 .
Correlation between duration and female age of recovered patients and cytomorphometric changes