key: cord-1026212-v92lm8lg
authors: Parada D, David; Peña, Karla B.; Gumá, Josep; Guilarte, Carmen; Riu, Francesc
title: Liquid‐based cytological and immunohistochemical study of nasopharyngeal swab from persons under investigation for SARS‐CoV‐2 infection
date: 2020-09-24
journal: Histopathology
DOI: 10.1111/his.14257
sha: 2bf548db88f4c4c4e69bb57f9cd7339492f651c5
doc_id: 1026212
cord_uid: v92lm8lg

INTRODUCTION: We describe cytologic and immunohistologic findings in virus transport medium on cases under investigation of SARS‐CoV‐2 infection. METHODS: Cytologic findings in cases under investigation of SARS‐CoV‐2 infection from one hundred consecutive nasopharyngeal swab were reviewed. Immunohistochemistry and SARSCoV‐2 RT‐PCR determination were performed to detect virus. RESULTS: No viral inclusions were noted in squamous cells obtained from virus transport medium. Immunohistochemestry with monoclonal antibodody against SARS‐CoV‐2 viral nucleoprotein was present in squamous cells. No positivity was present in others cellular components. CONCLUSIONS: SARS‐CoV‐2 predominantly localizes squamous cells in cytology samples of patients with RT‐PCR positive determination of SARSCoV‐2. The results of the current study support the notion that the nasopharyngeal region is the anatomical station that SARS‐CoV‐2 infects first, and the infection can lead to the migration of the virus into the lower airways.

In December 2020, a new type of coronavirus was identified as the causative factor in a series of cases of severe pneumonia in the city of Wuhan, province of Hubei, in the People's Republic of China (1) . The World Health Organization gave the official name "COVID-19" for this coronavirus disease, as well as the term "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2) for the virus (2). This virus is currently the cause of a global pandemic, producing hundreds of thousands of hospital admissions and deaths, with enormous effects on the health and life of the population and serious economic consequences for society.

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The tests currently used for the direct identification of SARS-CoV-2 are mainly aimed at detecting viral RNA through nucleic acid amplification, generally using the polymerase chain reaction. The nasopharynx and/or oropharynx are the most tested sampling sites using swabs, with a slightly greater sensitivity shown by the nasopharyngeal swab (3) . This test uses virus transport medium to carry it out, however to the best of our knowledge no cytological studies of the virus transport medium have been carried out.

In this article, we report the cytological findings of the universal transport medium used to investigate cases of SARS-CoV-2 infection, as to cell types present and whether there is a viral cytopathic effect in the infected cells. In addition, we included the immunohistochemical study for the SARS-CoV-2 viral nucleoprotein and viral RNA.

This is a prospective and descriptive cohort study conducted on consecutive cases under investigation (CUI) of SARS-CoV-2 infection in Hospital Universitari de Sant Joan, in Reus, Spain, between 1st April 2020 and 30th July 2020. The study protocol followed the principles of the Declaration of Helsinki 1964 and its subsequent modifications. We included 100 samples of nasopharyngeal swab in universal transport medium (UTM) for SARS-CoV-2 infection submitted to molecular pathology unit of our pathology department. SARS-CoV-2 infection detection was performed by reverse transcriptionpolymerase chain reaction (RT-PCR) using swab samples from the upper respiratory tract (nasopharyngeal exudate). Tests were carried out with the VIASURE SARS-CoV-2 Real Time PCR Detection it that detects ORF1ab and N genes (CerTest Biotec, Zaragoza, Spain). RNA was extracted in a QIAcube apparatus with RNeasy reagents (Qiagen N.V., Hilden, Germany) according to the manufacturer's instructions, and analyses were carried out in a 7500 Fast RT-PCR System (Applied Biosystems, Foster City, CA,USA). Patient clinical, and laboratory data were extracted from medical records.

The cycle threshold (Ct) values were reported by the cobas SARSCoV-2 test as either "positive" (N and ORF1a genes detected, Ct values <38), "presumptive positive" (N gene or ORF1a gene detected, Ct values <38), or "negative" (N and ORF1a genes not detected, Ct value > 38).

This article is protected by copyright. All rights reserved After SARSCoV-2 RT-PCR determination, the UTM was processed to liquid-based cytology with the Thin Prep 5000TM LBC method (Hologic Co., Marlborough, MA, USA).

All the UTM material was fixed with the hemolytic and preservative solution CytolytTM.

The UTM material was spun at 3000 rpm per 5 min.; the sediment was then transferred to 20 ml PreservcytTM solution, keeping for 15 minutes at room temperature, to be processed with a T5000 automated processor according to the manufacturer's recommendations. Two slides for each sample were obtained and were fixed in 95% ethanol. The second slide was stained with Papanicolaou, whilst the first slide was processed to immunocytochemistry. This was done to try to preserve as much material as possible for the immunohistochemical study. Cell types were assessed and quantitative analysis was performed, taking 10 consecutive fields at 10x. 

This article is protected by copyright. All rights reserved SARS-CoV-2 test results were unknown at the time of evaluating both the cytological and the immunohistochemical studies.

The results of the cellular analysis are shown as means+SD and percentage. The differences in the results of cell count and SARS-CoV-2 test were examined by the nonparametric Mann-Whitney test for two independent groups. p<0.05 was considered statistically significant. All of the analyses were performed using IBM.SPSS version 23.

Our analysis included 54 male and 46 female patients with a median age of 59 years (range, 27-93 years). Sixty-four (64%) cases were asymptomatic. Thirty-six cases (36%)

showed symptoms related to SARS-CoV-2 infection, such as fever, dry cough, shortness of breath, fatigue, rhinorrhea and respiratory distress. Four (4%) patients died from COVID-19. Forty (40%) cases were negative to RT-PCR SARS-CoV-2 test. Thirty-three (33%) cases showed N and ORF1a genes positivity (positive test). Gene N or ORF1a positivity was seen in twenty-seven (27%) cases (presumptive positive test) (Gene N positive: 24; Gene ORF1a positive: 3). Table 1 and 2, summarized the results. Table 3 .

We performed a cytological and immunohistochemical study of the universal transport medium for RT-PCR determination of SARSCoV-2 in one hundred patients admitted to our center, regardless of the diagnosis. Reverse transcription-polymerase chain reaction (RT-PCR) has emerged as the primary mode of diagnosis of acute infection with SARS-CoV-2 (4), but there is currently no gold standard for the diagnosis of COVID-19; therefore, sensitivity and specificity could not be calculated (5) . The diagnostic strategy recommended by the CDC to identify patients with COVID-19 is to test samples taken from the respiratory tract to assess for the presence of one or several nucleic acid targets specific to SARS-CoV-2 (6). A nasopharyngeal specimen is the preferred choice for SARS-CoV-2 testing (7) . In the present study of the viral transport medium on cases under investigation (CUI) of SARS-CoV-2 infection, variable cellular composition was evident, with predominance of squamous cells. Only in seven samples material was limited for cytological evaluation. However, in cases with limited cytological evaluation, adequate results were obtained for analysis RT-PCR determination of SARSCoV-2.

These findings confirm that the nasopharyngeal swab is an adequate source of material for the diagnosis of COVID-19.

This article is protected by copyright. All rights reserved A finding of our study was the presence of 3 cases with a negative SARS-CoV-2 RT-PCR test, which showed immunohistochemical positivity for viral nucleoprotein. Currently, RT-PCR is the valid diagnostic method for the diagnosis of COVID-19, however different studies have shown a high rate of false negatives of RT-PCR technique (8) (9) (10) . This may be caused by in-sufficient viral material in the specimen, laboratory error during sampling, or restrictions on sample transportation (8) (9) (10) . The difference with the three cases found in our series could be explained by a lack of sampling, low amount of RNA template of the sample near or below the detection limit of the reactions, mutations in the nucleic acid sequences targeted, slightly different amplification yield of the targets regions, or other factors related to the RT-PCR test.

Another point of the present study is the diagnostic category of presumptive positive RT-PCR test, when one of the studied genes was positive. The possibility that these are false positive results, arising either from spurious amplification or from detection of a closely related virus, cannot be excluded (5) . Another possibility is that these samples contain low levels of virus, near the limit of detection for the cobas SARS-CoV-2 tests but below the threshold of detection. However, in the 33 presumptive positive cases, cytoplasmic reactivity was demonstrated for the viral nucleoprotein SARS-CoV-2. This finding could indicate that cases classified as presumptive positives should be classified as positive.

In the present study, one of our initial approaches was to demonstrate whether there were characteristic viral cytopathic changes related to SARS-CoV-2 infection in cells analyzed from nasopharyngeal swab in universal transport medium, and whether these changes could serve as an indicator of infection. The cytological study showed that no changes were observed associated with the SARS-CoV-2 infection. This could imply that in order to demonstrate the presence of SASR-CoV-2 infection, more specific tests are required to demonstrate genetic targets using molecular biology, RT-PCR, or the demonstration of viral products by immunohistochemistry, such as nucleoproteins.

Histopathological studies on SARS-CoV-2 infection have shown involvement of different organs, being the most relevant findings at pulmonary level (11) (12) (13) (14) (15) . Non-specific findings are described in early stages, and included oedema, pneumocyte hyperplasia, focal inflammation and multinucleated giant cell formation (11) . In advanced stages, histopathological characteristics included diffuse alveolar damage with exudates (10-15; 7-11). However, there is no characteristic viral cytopathic effect of SARS-CoV-2 in the

This article is protected by copyright. All rights reserved different organs studied in patients with this infection (11) (12) (13) (14) (15) . In our study, no definitive viral inclusions were noted in squamous cells correlated with the immunohistochemical study.

The immunohistochemical analysis of the present study showed granular cytoplasmic positivity in squamous cells, without other cellular components showing this reactivity.

This finding seems to indicate that SARS-CoV-2 would have the ability to infect squamous cells and, therefore, produce viral replication in these cells, and ACE2 receptors in the nasopharyngeal and oral mucosa could support the infectious capacity of squamous cells (16) (17) (18) . The ACE2 receptor is necessary for the virus to enter the cell, through endocytosis, to release its RNA, and it uses cellular machinery to replicate and assemble more virions (17) 

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Accepted Article This article is protected by copyright. All rights reserved

Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus-2: A Narrative Review

Dynamic profile of RT-PCR findings from 301 COVID-19 patients in Wuhan, China: A descriptive study

False negative of RT-PCR and prolonged nucleic acid conversion in COVID-19: Rather than recurrence

Stability issues of RT-PCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19

Autopsy in suspected COVID-19 cases

Insights into pathogenesis of fatal COVID-19 pneumonia from histopathology with immunohistochemical and viral RNA studies

In situ detection of SARS-CoV-2 in lungs and airways of patients with COVID-19

COVID-19 Autopsies

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

Transmission, Diagnosis, and Treatment of Coronavirus Disease