A comparative study of SAR-CoV-2 detection using RT-PCR based testing from a nasopharyngeal swab versus indwelling tracheostomy in patients with a tracheostomy

Introduction

The novel coronavirus disease (COVID-19), the latest pandemic since the Spanish Flu, has impacted the entire globe. Until recently, the number of infected cases continued to increase exponentially. According to the latest information from August 2023, the World Health Organization (WHO) reported over 770 million infected patients worldwide, with a death toll of over 6.9 million people. In Thailand, up to 4,756,406 infected patients have been found, with a death toll of 34,459 people since January 2020, when the first infected patient in Thailand was found (incidentally the first outside of mainland China).¹ Despite improved control of infection rates, demotion to surveillance instead of concern, and acceptance of inexpensive ATK (Antigen Test Kit) as a priority in non-exposed patients without symptoms, it is still undeniable that precise and accurate COVID-19 RT-PCR testing will still be necessary in exposed asymptomatic and symptomatic patients.² It may also be required in patients who may need immediate treatment. It is still necessary to support the development of appropriate treatments and to identify variants that continue to mutate from the original. Additionally, efficient screening methods may reduce infection in medical personnel with inevitable close contact during the care of COVID-19 patients.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) is still the most accurate and standardized COVID-19 test utilized for collecting specimens from various sample sites.^3,4 A study found differences in the percentage of positive infection rates depending on the sampling site. For instance, the highest rate of viral detection was observed in bronchoalveolar lavage specimens (93%), followed by sputum (72%). In nasopharyngeal swabs, the current standard collection method, the virus was detected at a rate of 63%.^5–10 In patients with a tracheostomy, whose altered airway essentially bypasses the nasopharynx, yield of detection from this site in this patient population has not been clearly understood^5,9,11–13 According to previous studies, it was found that discordant results of infection rates by 30% in post-tracheostomy testing of COVID-19 patients. The test results from nasopharyngeal swabs did not detect the virus, whereas those from tracheal secretions were able to detect the virus.^14–17 The patients with a tracheostomy because of their stoma had a closer proximity to the alveolus containing high viral content, resulting in an increased risk of aerosolization.^18–21 Therefore, the current recommendation for utilizing nasopharyngeal swabs for tracheostomized patients may not be ideal.^18–21 Because there have been no comparative studies on concordance and discordance between nasopharyngeal swabs and tracheal secretions by specific RT-PCR for this group of patients, this research was implemented to clarify this inquiry. Although the WHO announced an end to COVID-19 as a public health emergency, an accurate technique for collecting adequate COVID-19 samples with RT-PCR testing remains essential for disease diagnosis, treatment, and spread control. Furthermore, this on-site information and how samples are necessary for this patient population can be applied for future respiratory infection outbreaks that may occur.

Methods

This was a cross-sectional study. Nasopharyngeal swabs and tracheal secretion samples were collected from 100 patients who underwent RT-PCR at Thammasat University Hospital from February 1, 2022, to October 19, 2022. The participants were aged between 1 and 96 years, had not been previously diagnosed with COVID-19 prior to the test, or were clinically unstable. The criteria for performing COVID-19 RT-PCR testing in our hospital during data collection in this study included patients who required screening for admission or intervention, persons under investigation (PUI), and patients with respiratory symptoms. The sample collection in this study was conducted by a single otolaryngologist using full PPE. Tracheal secretion samples were collected by closed-circuit suction. All the samples were sent to the hospital’s central laboratory unit for PCR testing.

The study protocol was reviewed and approved by The Human Research Ethics Committee of Thammasat University (Medicine) Review Board and was approved on October 20 2021, with the Number of COA 265/2021 and project number MTU-EC-OL-2-270/64. This study was conducted in accordance with the ethical standards set in the Declaration of Helsinki 1975. The patients provided written informed consent to participate in the study, for participants under 18 years old, written informed consent was obtained from their parents/guardians.

The demographic data were studied and presented as means, standard deviation (SD), median, or max-min, depending on the data distribution. Discordance and concordance were compared using an independent t-test or Mann-Whitney U test. The detection results and Cycle threshold (Ct values) values from each site were presented as numbers and percentages, and analyzed by McNemar’s test, Kappa coefficient and agreement with a 95% confidence interval at a significance level of 0.05. The data from the collection form were verified, entered into Excel, and analyzed using SPSS.

Results

According to data collected at Thammasat University Hospital from February 1, 2022, to October 19, 2022, there were 100 subjects recruited. According to the demographic data, there were 57 males (57%) and 43 females (43%) aged between 1-96 years. The average age was 66.50 years. Common underlying diseases include hypertension and diabetes mellitus. Nearly 75% of the participants did not have a history of smoking. Nine patients had respiratory symptoms on the day of specimen collection. The duration from tracheostomy to specimen collection was–1-156 months, with an average of approximately 12 months. It was also found that 51% of the patients underwent tracheostomy due to upper airway obstruction, followed by prolonged intubation (46%). Three patients underwent tracheostomy because of pulmonary toilet needs (Table 1).²⁶ Most patients were tested for RT-PCR COVID-19 due to screening admission requirements of 85%, while a smaller percentage had respiratory symptoms (9%) or were persons under investigation given close contact (PUI) (6%) (Table 2).

In this study, four participants showed positive results for COVID-19. Three of them were female, and one was male. On the day of specimen collection, all four subjects all showed respiratory symptoms. The average duration of tracheostomy in this group was 1-48 months. With regards to indications, all received a tracheostomy due to prolonged intubation.

According to data collected from the subjects with COVID-19, two were detected via nasopharyngeal swabs and tracheal secretion aspiration via the tracheostomy tube. One tested positive via nasopharyngeal swab but did not detect COVID-19 via tracheal secretion aspiration via the tracheostomy tube. One individual tested positive via tracheal secretion but not via a nasopharyngeal swab. When comparing the groups with concordant results, no significant difference was found (p = 1.000) (McNemar’s test) (Table 3). The percentage of agreement in our study was 98% (95%CI 93-100), and the kappa coefficient value was 65.64% (95% CI 21.1-100.0) with a p-value <0.001.

For the Ct ratio obtained by the COVID-19 test in the subjects who revealed COVID-19 using the ORF1Ab/N gene by RT-PCR, the average Ct ratio (ORF1Ab/N gene) by nasopharyngeal swab was 31.67/30.76, and 22.24/24/22 by tracheal secretion aspiration via the tracheostomy tube (Table 4).

Discussion

Coronaviruses mainly infect the respiratory system via droplet and aerosol transmission,^17,22 particularly during respiratory medical procedures. In Thailand, the first case of infection was reported on January 13, 2020.²³ Subsequently, the World Health Organization formally announced it as a pandemic on March 11, 2020.²⁴ This has resulted in a serious need for a robust COVID-19 screening protocol to control viral transmission into communities and medical personnel with limited resources in a hectic environment.

Patients with a tracheostomy tube have a different respiratory physiology from their non-tracheotomized counterparts. Most or all air entering the lungs flow directly to the trachea and lungs, without the ability to dehumidify, which typically occurs in the upper airway. As a result, patients may experience irritation, respiratory mucosal injuries, and a higher risk of infection. Therefore, it is important to understand whether these physiological changes affect sampling for respiratory diseases in the tracheotomized patient population.^5,11–13

According to a previous study by Joshua et al. in 2021^5,14 reported that 13 (28.9%) COVID-19 patients with tracheostomies had at least one discordant severe-acute-respiratory-syndrome-related coronavirus-2 (SARS-CoV-2) detection result in the nasopharynx versus trachea, leading to concerns about the potential for missed diagnosis using nasopharyngeal swabs alone for screening and diagnosis. Furthermore, Burrel et al. in 2021² found that the detection rate of SARS-CoV-2 was significantly higher in LRT than in URT (53.6% vs. 13.4%, p<0.0001) and concluded that LRT samples increased the accuracy of diagnosis of COVID-19. Alternatively, Kitt et al. in 2021²⁵ compared COVID-19 RT-PCR testing between URT and LRT samples in an artificial airway including endotracheal and tracheostomy tubes, revealing that there was a high concordance rate, but no isolated positive result from an LRT sample. Our study supports a high concordance rate in negative cases. In addition, we present an example, contrary to Kitt et al. al.’s study with a positive tracheal site specimen, but no concordant nasopharyngeal swabs.

Therefore, precise screening is essential. This study found that COVID-19 screening in patients with a tracheostomy revealed discordant results between the collected specimens at the two positions, particularly those who were not identified via the routine nasopharyngeal swab site but were subsequently detected via tracheal secretion aspiration from the tracheostomy tube. This study supports the idea that there is a possibility of incorrectly labeling a negative case from only a negative nasal swab in a highly suspicious case. According to our results, the agreement and Cohen kappa value indicate that one sample from the nasopharynx or tracheal tube should be sufficient to determine the infection status in negatively tested and low-risk patients. For highly suspicious cases, multisite sampling should be performed.

The limitations of this study include the small number of positive cases. Since most patients were tested for admission and screening purposes without risk of infection and symptoms, 96% had negative results. Further studies should be conducted in a setting with a higher prevalence of respiratory infections, including a larger sample size, to increase the number of positive cases.

Searching for other precise screening methods, particularly in patients with tracheostomy and an altered airway, is imperative.

Conclusions

This study showed discordance in COVID-19 RT-PCR screening results for samples from nasopharyngeal and tracheal sites in patients with an indwelling tracheostomy. Multiple site sampling from both the upper and lower respiratory tracts is suggested in highly suspected patients with tracheostomy.

Ethics and consent

The study protocol was reviewed and approved by The Human Research Ethics Committee of Thammasat University (Medicine) Review Board and was approved on October 20 2021, with the Number of COA 265/2021 and project number MTU-EC-OL-2-270/64. This study was conducted in accordance with the ethical standards set in the Declaration of Helsinki 1975. The patients provided written informed consent to participate in the study, for participants under 18 years old, written informed consent was obtained from their parents/guardians.