Abstract
Objectives
In December 2019, a novel coronavirus (SARS-CoV-2)-infected pneumonia (COVID-19) occurred in Wuhan, China. Laboratory-based diagnostic tests utilized real-time reverse transcriptase polymerase chain reaction (RT-PCR) on throat samples. This study evaluated the diagnostic value to analyzing throat and sputum samples in order to improve accuracy and detection efficiency.
Methods
Paired specimens of throat swabs and sputum were obtained from 54 cases, and RNA was extracted and tested for 2019-nCoV (equated with SARS-CoV-2) by the RT-PCR assay.
Results
The positive rates of 2019-nCoV from sputum specimens and throat swabs were 76.9% and 44.2%, respectively. Sputum specimens showed a significantly higher positive rate than throat swabs in detecting viral nucleic acid using the RT-PCR assay (p = 0.001).
Conclusions
The detection rates of 2019-nCoV from sputum specimens were significantly higher than those from throat swabs. We suggest that sputum would benefit for the detection of 2019-nCoV in patients who produce sputum. The results can facilitate the selection of specimens and increase the accuracy of diagnosis.
Introduction
In December 2019, a novel coronavirus, since then named severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), emerged in Wuhan, Hubei Province, China [1], causing an acute febrile illness with acute respiratory distress syndrome (ARDS), which the World Health Organization (WHO) has named 2019 novel coronavirus disease (COVID-19) [2], [3], [4]. The type of pneumonia caused by the 2019 novel coronavirus (2019-nCoV, equated with SARS-CoV-2) is a highly infectious disease, and the WHO declared the outbreak a public health emergency of international concern on January 30, 2020 [5]. At the time of this writing, the WHO estimated that COVID-2 has already been diagnosed in as many as 509,000 people from 201 countries worldwide, causing nearly 23,400 deaths [6]. The rapid global expansion and rising fatalities raised concerns about global spread.
Full-genome sequencing and phylogenic analysis indicated that 2019-nCoV was a distinct clade from the beta-coronaviruses associated with human severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) [2]. The 2019-nCoV is closely similar to bat coronaviruses, with a homology of 85–96% to a bat SARS-like coronavirus (bat-SL-CoVZC45) at the whole genome level [7], and it has been postulated that bats are the primary source. However, the origin of 2019-nCoV is still under investigation.
A study in The Lancet by Huang et al. [3] firstly reported 41 cases of COVID-19 in which most patients had a history of exposure to Huanan Seafood Wholesale Market. The clinical manifestations of patients included fever, cough, dyspnea, myalgia, fatigue, normal or decreased leukocyte counts, and radiographic evidence of pneumonia. Organ dysfunction (such as shock, ARDS, acute cardiac injury and acute kidney injury) and death can occur in severe cases [3]. Signs of infection are highly nonspecific; thus, diagnostic test based on detection of the viral sequence by real-time reverse transcription polymerase chain reaction assay (RT-PCR) is the main means of confirmation.
Appropriate specimen selection is important for the diagnosis of respiratory viral infections [8]. We collected paired specimens of throat swabs and sputum for the first time. The objective of this study was to compare the positive rates of 2019-nCoV between throat swabs and sputum specimens in the detection of viral nucleic acid by RT-PCR. This facilitates the selection of specimens for the detection of 2019-nCoV, and improves the efficiency of diagnosis for patients suspected of having COVID-19.
Materials and methods
Study design and participants
For this retrospective, single-center study, we recruited 52 patients suspected of having COVID-19 from February 7 to February 16, 2020, at Jinyintan Hospital, which is located in Wuhan, Hubei Province, the endemic areas of COVID-19. Presumptive patients were diagnosed according to WHO interim guidance [9]. All patients enrolled in this study received detection of viral nucleic acid assays (RT-PCR) in both throat swabs and sputum specimens at the same time to make a definite diagnosis. Jinyintan Hospital is a hospital for adults (i.e. aged ≥14 years) specializing in infectious diseases, and as well the first designated hospital for COVID-19 patients in Wuhan. The study was approved by Jinyintan Hospital Ethics Committee, and oral consent was obtained from patients involved before enrollment when data were collected retrospectively.
Specimen collection
Throat swabs and sputum specimens were collected for extracting 2019-nCoV RNA from patients suspected of having 2019-nCoV infection. The procedure for collecting throat (oropharyngeal, OP) swabs entails swabbing the posterior pharynx and each tonsil area at least 3 times separately using a nylon-flocked swab, avoiding the tongue, and immediate placement of the swab into a sterile tube, containing 2~3 mL of sterile saline. The procedure for collecting sputum specimens requires the patients to cough up the deep sputum from the lower airways, and the sputum is collected in the sterile 50-mL plastic tube. Sputum specimens were microscopically screened for squamous epithelial cells (SECs) and polymorphonuclear cells (PMNs), with <10 SECs and >25 PMNs per low-power field being regarded as high quality. Before the assay, sputum specimens were added to an equal volume of acetylcysteine (10 g/L) and shaken at room temperature for 30 min to be fully liquefied.
RT-PCR assay for SARS-CoV-2
The diagnosis of SARS-CoV-2 relies on the detection of the virus by RT-PCR for in vitro qualitative detection. Viral RNA purification kit (QIAamp Viral RNA Mini Kit, Qiagen) was used for RNA, as instructed by the manufacturer. For all RNA extractions, 40 μL of elution was prepared for the RT-PCR assay of 2019-nCoV RNA. Then, n×19 μL mixed reagent of fluorescence PCR detection and n×1 μL RT-PCR enzyme (n is the number of reaction tubes) were mixed and vortexed for a few seconds. The aforementioned mixture of 20 μL was put into the PCR reaction tube, respectively, and after that 5 μL of the prepared elution was added. A panel of positive (pseudovirus-containing target fragment and internal control separately) and negative (pseudovirus-containing internal control) controls were used to evaluate the assays. The extracted positive (5 μL) and negative (5 μL) controls were included in each RT-PCR reaction. The pseudovirus used for positive control contained ORF1ab, N and E gene, and the pseudovirus used for internal control contained human RNase gene, and both of them were finished product included in the 2019-nCoV nucleic acid detection kit (Shanghai ZJ Bio-Tech Co Ltd). The cycle threshold value (Ct-value) of the positive control was required to be ≤30. To ensure the integrity of 2019-nCoV RT-PCR results by indicating potential RT-PCR inhibition, an internal control was analyzed in parallel for each patient sample and positive and negative controls.
The RT-PCR analysis was conducted using the ABI 7500 Real-Time PCR System. The PCR parameters were 45°C for 10 min, 95°C for 3 min, followed by 45 cycles of 95°C for 15 s, 58°C for 30 s, and a single fluorescence detection point at 58°C. Two target genes, including an open reading frame 1ab (ORF1ab) and nucleocapsid protein (N), were simultaneously amplified and tested during the RT-PCR assay. The RT-PCR assay was performed using a 2019-nCoV nucleic acid detection kit according to the manufacturer’s protocol (Shanghai ZJ Bio-Tech Co Ltd). A Ct-value less than 37 was defined as a positive test result, and a Ct-value of 40 or more was defined as a negative test result. A medium load, defined as a Ct-value of 37 to less than 40, required confirmation by retesting. These diagnostic criteria were based on the recommendation by the National Institute for Viral Disease Control and Prevention (China) [10].
Statistical analysis
All analyses were performed using SPSS 22.0. Continuous variables were described using mean (SD) if they are normally distributed or median (IQR) if they are not. Categorical variables were expressed as frequencies (percentages), and performed using McNemar’s test. p<0.05 was considered statistically significant.
Results
Presenting characteristics
The study population included 52 hospitalized patients suspected of having COVID-19. The average age was 57.3 years (SD, 12.5; range, 34–84 years), and 27 (51.9%) were men. All patients received RT-PCR assays in both throat swabs and sputum specimens (Table 1).
Clinical data and nucleic acid detection results (RT-PCR) of the 52 patients.
| No. | Gender | Age, years | Throat swabs collection date | Throat swabs results | Sputum specimens collection date | Sputum specimens results |
|---|---|---|---|---|---|---|
| 1 | Female | 49 | 2020/2/14 | Positive | 2020/2/13 | Positive |
| 2 | Female | 47 | 2020/2/12 | Negative | 2020/2/13 | Positive |
| 3 | Male | 80 | 2020/2/13 | Positive | 2020/2/13 | Positive |
| 4 | Female | 73 | 2020/2/12 | Negative | 2020/2/13 | Positive |
| 5 | Female | 54 | 2020/2/13 | Negative | 2020/2/13 | Positive |
| 6 | Female | 63 | 2020/2/12 | Negative | 2020/2/13 | Positive |
| 7 | Male | 67 | 2020/2/14 | Positive | 2020/2/13 | Positive |
| 8 | Male | 47 | 2020/2/13 | Positive | 2020/2/13 | Positive |
| 9 | Female | 72 | 2020/2/7 | Negative | 2020/2/13 | Positive |
| 10 | Male | 49 | 2020/2/12 | Negative | 2020/2/13 | Positive |
| 11 | Male | 69 | 2020/2/11 | Negative | 2020/2/13 | Positive |
| 12 | Female | 57 | 2020/2/12 | Negative | 2020/2/13 | Positive |
| 13 | Male | 79 | 2020/2/13 | Negative | 2020/2/13 | Positive |
| 14 | Female | 34 | 2020/2/13 | Negative | 2020/2/13 | Positive |
| 15 | Female | 67 | 2020/2/13 | Positive | 2020/2/13 | Positive |
| 16 | Male | 54 | 2020/2/9 | Negative | 2020/2/14 | Positive |
| 17 | Female | 64 | 2020/2/14 | Positive | 2020/2/14 | Positive |
| 18 | Female | 35 | 2020/2/14 | Negative | 2020/2/14 | Positive |
| 19 | Female | 47 | 2020/2/14 | Positive | 2020/2/14 | Positive |
| 20 | Female | 66 | 2020/2/14 | Positive | 2020/2/14 | Positive |
| 21 | Male | 57 | 2020/2/9 | Positive | 2020/2/14 | Positive |
| 22 | Female | 66 | 2020/2/13 | Positive | 2020/2/14 | Positive |
| 23 | Female | 39 | 2020/2/15 | Negative | 2020/2/14 | Negative |
| 24 | Male | 59 | 2020/2/14 | Negative | 2020/2/14 | Positive |
| 25 | Male | 52 | 2020/2/12 | Negative | 2020/2/14 | Positive |
| 26 | Male | 64 | 2020/2/13 | Negative | 2020/2/14 | Positive |
| 27 | Male | 62 | 2020/2/14 | Negative | 2020/2/14 | Positive |
| 28 | Male | 55 | 2020/2/14 | Positive | 2020/2/14 | Positive |
| 29 | Female | 63 | 2020/2/14 | Positive | 2020/2/14 | Positive |
| 30 | Female | 71 | 2020/2/13 | Positive | 2020/2/14 | Negative |
| 31 | Male | 58 | 2020/2/14 | Negative | 2020/2/14 | Positive |
| 32 | Male | 51 | 2020/2/16 | Positive | 2020/2/14 | Negative |
| 33 | Female | 44 | 2020/2/14 | Positive | 2020/2/14 | Positive |
| 34 | Male | 55 | 2020/2/14 | Negative | 2020/2/14 | Negative |
| 35 | Male | 40 | 2020/2/13 | Negative | 2020/2/14 | Negative |
| 36 | Male | 49 | 2020/2/13 | Positive | 2020/2/14 | Negative |
| 37 | Male | 36 | 2020/2/12 | Negative | 2020/2/14 | Negative |
| 38 | Female | 59 | 2020/2/12 | Negative | 2020/2/14 | Negative |
| 39 | Female | 46 | 2020/2/13 | Negative | 2020/2/14 | Positive |
| 40 | Female | 59 | 2020/2/13 | Negative | 2020/2/14 | Negative |
| 41 | Female | 72 | 2020/2/13 | Positive | 2020/2/14 | Positive |
| 42 | Female | 56 | 2020/2/15 | Negative | 2020/2/14 | Positive |
| 43 | Female | 51 | 2020/2/13 | Negative | 2020/2/14 | Negative |
| 44 | Male | 75 | 2020/2/13 | Positive | 2020/2/14 | Positive |
| 45 | Male | 47 | 2020/2/16 | Positive | 2020/2/15 | Positive |
| 46 | Male | 76 | 2020/2/10 | Positive | 2020/2/15 | Positive |
| 47 | Male | 38 | 2020/2/13 | Positive | 2020/2/15 | Negative |
| 48 | Female | 84 | 2020/2/14 | Positive | 2020/2/15 | Positive |
| 49 | Male | 60 | 2020/2/15 | Negative | 2020/2/15 | Positive |
| 50 | Male | 47 | 2020/2/14 | Positive | 2020/2/15 | Positive |
| 51 | Male | 72 | 2020/2/15 | Negative | 2020/2/15 | Positive |
| 52 | Male | 41 | 2020/2/13 | Negative | 2020/2/15 | Negative |
Comparison of throat swabs and sputum specimens
The viral nucleic acid by RT-PCR showed that 23 cases (44.2%) of throat swabs were positive while 29 cases (55.8%) were negative, and 40 cases (76.9%) of sputum specimens were positive while 12 cases (23.1%) were negative (Figure 1). The positive rate of sputum specimens was almost two-fold that of throat swabs.
Distribution of RT-PCR results on throat swabs and sputum specimens in patients with suspected COVID-19.
Most of the patients (51.9%) had the same results of RT-PCR assay on throat swabs and sputum specimens, 36.5% with both positive and 15.4% with both negative. However, quite a few patients (40.4%) showed positive sputum specimens and negative throat swabs, while only a tiny minority of patients (7.7%) showed negative sputum specimens and positive throat swabs. The findings showed that positive rates displayed a significant statistical difference (p=0.001) between throat swabs and sputum specimens (Table 2).
Comparison of RT-PCR results between throat swabs and sputum specimens.
| No. (%) of sputum specimens result | ||
|---|---|---|
| Positive | Negative | |
| No. (%) of throat swabs result | ||
| Positive | 19 (36.5%) | 4 (7.7%) |
| Negative | 21 (40.4%) | 8 (15.4%) |
p-Value=0.001 by McNemar’s test.
Discussion
Adequate specimen collection is important for the diagnosis of respiratory viral infections [8]. At present, the sample collection for viral nucleic acid detection of suspected patients with COVID-19 is mostly upper respiratory tract samples (mainly throat swabs) [11]. The collection process is extremely risky for medical staff. Sputum is representative of the lower respiratory tract but is rarely used for viral testing. The aim of this study was to compare the detection rates of 2019-nCoV RNA from throat swabs and sputum specimens using the RT-PCR assay.
In our research, most of the patients were middle-aged and elderly men, which was similar to previous studies [3], [4]. Paired specimens of throat swabs and sputum were obtained from 52 subjects. The positive rates of 2019-nCoV from sputum specimens and throat swabs were 76.9% and 44.2%, respectively. The present study found that the overall positive rate from sputum specimens in adults was significantly higher than that from throat swabs using RT-PCR assay. This finding was consistent with the result of a previous study that sputum showed an obviously higher positive rate than throat swabs in detecting SARS-CoV-2 [12]. Han et al. [13] also suggested that sputum samples might be more helpful than throat swabs for the detection of SARS-CoV-2 RNA.
The detection rate of 2019-nCoV from sputum samples was higher than that from throat swabs, which may be related to the novel coronavirus’s main invasion and infection of lower respiratory tract cells, resulting in clinical manifestations such as cough and pneumonia. In addition, several studies have shown that respiratory viruses were increasingly recognized as the cause of lower respiratory tract infections [8]. In these cases, specimens of the lower respiratory tract should be collected for the detection of 2019-nCoV, and caution should be taken when using the negative result of viral nucleic acid from throat swabs as the criterion for the exclusion of infection and conformation of cure.
It should be noted that the high viscosity of sputum can make it difficult to extract nucleic acid. Therefore, pre-treatment of sputum samples is required, but currently no standardized pre-treatment procedure is available for virus detection. In our study, acetylcysteine was used for the digestion of sputum, which may cause substantial loss of RNA due to the washing and pipetting steps in the procedures. Therefore, thorough mixing and homogenization of sputum samples might be important factors to obtain reliable test results [8].
In order to illustrate the diagnostic efficiency of sputum more persuasively, we made a detailed comparison and discussion of nasopharyngeal (NP) swabs, OP swabs and sputum samples based on an overall review of related papers published. According to recent studies on SARS-CoV-2 viral loads in respiratory specimens, sputum samples showed higher viral loads than throat swabs [14], and viral loads in the nose were also higher than those in the throat [15]. Pan et al. [14] reported that the viral loads of throat swab, nasal swab and sputum sample were 7.99×104, 1.69×105 and 7.52×105 copies per mL, respectively. Unfortunately, there was only one sample of the nasal swab. In a study of SARS-CoV-2 detection in different types of clinical specimens [12], bronchoalveolar lavage (BAL) fluid specimens showed the highest positive rates (93%), followed by sputum (72%), nasal swabs (63%) and pharyngeal swabs (32%). Moreover, Winichakoon et al. [16] reported that patients with negative NP/OP swabs could be positive in BAL fluid. Therefore, the aforementioned analysis and evidence have proven that specimens from the lower respiratory tract (e.g. sputum and BAL) would have a higher diagnostic yield of SARS-CoV-2 than an upper respiratory specimen (e.g. NP/OP swabs). This finding was previously observed in previous SARS and MERS outbreaks. Basic science evidence has supported that the target functional receptor of these viruses is angiotensin-converting enzyme 2 (ACE2) [17], [18]. Surface expression of ACE2 was found abundantly on both type I and type II alveolar epithelial cells, but minimally on bronchial epithelial cells and negative on oral, nasal and nasopharynx [16]. For all these reasons, we considered that the diagnostic efficiency of sputum samples could be higher than NP/OP swabs, and NP swabs might be a better choice than OP swabs for COVID-19 patients without sputum production.
This study has several limitations. First, only 52 patients with suspected COVID-19 were included, and further studies are needed to investigate the observation in a larger group of patients. Second, the date only involved throat swabs and sputum samples, and expanding collections of NP swabs and bronchoalveolar lavage fluid are necessary in the future. Although sputum is more sensitive than throat swabs for 2019-nCoV detection, the use of sputum may be limited because not all patients with COVID-19 produce sputum, especially elderly patients. During the submission of this paper, Han et al. [13] published a work which suggested that induced sputum (i.e. 10 mL of 3% hypertonic saline was inhaled through a mask with oxygen at a flow rate of 6 L/min for 20 min or until the sputum was produced) could be used for those patients who do not have sputum. Therefore, if a sputum sample is available, it might be a rather reliable specimen for nucleic acid detection of 2019-nCoV.
In conclusion, the detection rates of 2019-nCoV from sputum specimens are significantly higher than those from throat swabs using the RT-PCR assay. We suggest that sputum would benefit for the detection of the novel coronavirus in patients who produce sputum. The results can facilitate the selection of specimens and increase the accuracy of diagnosis of COVID-19.
Acknowledgments
This work was supported by the Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund under Grant znpy2017022.
Research funding: Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund, znpy2017022.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: Authors state no conflict of interest.
Informed consent: Informed consent was obtained from all individuals included in this study.
Ethical approval: The study was approved by Jinyintan Hospital Ethics Committee.
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