SARS-CoV-2 infection or COVID-19 pandemic was started as an outbreak in Wuhan, Hubei province, China. On March 11, 2020, the WHO declared COVID-19 as a global pandemic1. Real time reverse-transcriptase-polymerase chain reaction (rRT-PCR) was the most common test for SARS-CoV-2 detection by targeting the ORF 1ab, N or E genes. Oropharyngeal (OP) and nasopharyngeal (NP) swabs were the most frequently used samples for diagnosis2. However, a negative result from OP or NP swabs could not rule out COVID-19, as some patients were tested positive SARS-CoV-2 using other types of specimens, including bronchoalveolar lavage fluid, anal swab, stool and urine3.
SARS-CoV-2 viral load declines gradually since the onset of symptoms4. Low SARS-CoV-2 viral load in patients at late infection stage may often result in false-negative nucleic acid test results, creating challenges to current detection methods. Appropriate sample collection and reliable testing are the key factors in enhancing the detection rate of SARS-CoV-2.
Upper respiratory swabs (NP or OP) have remained the mainstay of obtaining samples for diagnosing virus infection. The initial studies of COVID-19-specific data comparing OP with NP swabs came from two, non-peer-reviewed research articles5,6. To date, limited published data exist on the testing performance of OP swabs compared with NP swabs for SARS-CoV-2 RNA6. The primary objective of this study was to compare the performance of NP and OP swabs for the diagnosis of COVID-19 infection among 2250 concomitant samples (1125 OP and 1125 NP). The secondary objective was to compare the viral load between NP and OP swabs and to find out a relationship between viral load and disease severity.
Material & Methods
This study was conducted at the Government Medical College Manjeri, Kerala, India, from November 1 to December 31, 2020 after getting approval from the institutional research and ethics committees. All individuals who presented to the COVID-19 clinic either being symptomatic or having primary contact history with a COVID-19 patient, were included in the study. A total of 1125 individuals were enrolled in the study after obtaining written informed consent, and a total of 2250 concomitant samples were collected (1125 OP and 1125 NP) for the purpose of diagnosis.
Sample collection: A team of trained 14 otolaryngologists performed the sample collections at the swab collection centre of the institution to avoid sampling variability and errors. After explaining the procedure to the study participants and obtaining written informed consent, the NP and OP swabs were collected sequentially on the same day. Standardized protocols were used for sample collection. The NP and OP swabs were collected in separate vials containing VTM, Hi Viral™ Transportation media (Hi Media, Mumbai) and transported to the laboratory within 2-3 h in triple layer packing maintaining the cold chain7,8.
rRT-PCR test for SARS-CoV-2: All tests were performed in a single laboratory as per the standard protocols and following international guidelines9,10. Both specimens were processed separately by rRT-PCR in CFX 96-C1000-Touch™ using Allplex™ real time PCR detection system (Bio-Rad, Hercules, CA, USA) 2019-nCoV Assay Kit (Cat no RV10248X ; Seegene, Seoul, Republic of Korea). Nucleic acid extraction was done using MagMAX viral/Pathogen Nucleic Acid isolation kit, (Cat NoA42352, Applied biosystems™, CA, USA) and Kingfisher™ Flex Purification System, Thermo Fisher Scientific™ Inc., Worcester, Massachusetts, USA), an automated nucleic acid extraction system. Extraction control from RT-PCR kit was spiked into the sample in the nucleic acid extraction step to ensure proper efficiency of the nucleic acid extraction process. The rRT-PCR assay detected both nucleocapsid N gene and the RNA-dependent RNA polymerase RdRp genes simultaneously as per the kit protocol for a total of 45 cycles. Commercially provided negative and positive kit controls were used along with laboratory confirmed positive and negative in-house controls to validate results. RNase P was used as the internal control for the extraction process efficiency determination and subsequent RT-PCR. RNase P in clinical samples assesses specimen quality. Samples with a threshold cycle of less than 30 for RNase P were included in the study.
The cut-off cycle threshold (Ct) value was set at ≤40 for both genes. Based on Ct value, the viral load was graded into low (Ct more than 30 or <1.13 × 103 viral RNA copies/ml), moderate (Ct 25-30, or 1.13 × 103 to 3.01 × 104 viral RNA copies/ml), high (Ct 15-25 or 3.01 × 104 to 2.13 × 107 viral RNA copies/ml) and very high if Ct value is less than 15 or >2.13 × 107 viral RNA copies/ml.
The detection of E gene with either/both N and RdRp genes was defined as positive. When the target gene of SARS-CoV-2 RNA was not detected and internal control was valid, it was interpreted as negative.
Clinical data collection: Clinical data were collected from the individuals before sample collection, which included demographic variables, contact history, onset and duration of symptoms, if any, using a pre-validated questionnaire prepared by the study team. The data were systematically recorded. Only those individuals whose both NP and OP swabs were simultaneously taken and sent separately were included in the study for analysis.
Statistical analysis: The data including the test result of both NP or OP swab(s) and the respective viral loads were analysed. The continuous data were presented as median and range and categorical data as frequency and percentage. Comparison between unmatched proportions tested positive with respect to age group, gender, symptoms present or absent and duration of symptoms was done by the Chi-square test. Agreement between the two methods was assessed using the Kappa statistics. Absolute sensitivity, specificity, positive and negative predictive values (PPV and NPV) for OP and NP swabs relative to a positive result on either OP swab or NP swab were also calculated. All analyses were done using the SPSS software version 16.0 (SPSS Inc., Chicago, IL., USA).
Results
The median age of the participants (n=1125) was 31 yr (range 4 months-100 yr). There were 743 (66%) males and 382 (34%) females. Two hundred and seventy seven (24.6%) patients had some symptoms at the time of sample collection. The test positivity, viral load and demographic distribution and the results are summarized in Tables I-IV and Figures 1-3.
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Table I: Concordance and discordance between nasopharyngeal (NP) and oropharyngeal (OP) swabs among different age groups
Table II: Concordance and discordance between nasopharyngeal (NP) and oropharyngeal (OP) swabs with respect to symptoms
Table III: Concordance/discordance between nasopharyngeal and oropharyngeal swabs with respect to duration of symptoms
Table IV: Comparison of viral load with relation to clinical symptoms
Fig. 1: Viral load from 523 nasopharyngeal (NP) and 493 oropharyngeal (OP) samples of COVID-19 positive patients based on RT-PCR Tm value.
Fig. 2: Proportion of positives among different age groups in NP and OP swabs. NP, Nasopharyngeal; OP, Oropharyngeal.
Fig. 3: Proportion of positives among different duration of symptoms in NP and OP swabs. NP, Nasopharyngeal; OP, Oropharyngeal.
Five hundred and twenty three (46.5%) NP and 493 (43.8%) OP samples were tested positive. There was a fair degree of agreement between the methods (kappa = 0.275, P<0.001). Three hundred and six (27.2%) pairs were concordantly positive and 415 (36.9%) were concordantly negative giving an overall concordance of 64.1 per cent; 217 (19.3%) pairs were positive on NP but negative on OP swabs and 187 (16.6%) pairs were negative on NP but positive on OP swabs. When OP alone were used, the detection rate was 43.8 per cent (493/1125), NP alone 46.5 per cent (523/1125), if both OP and NP tests were used and either or both positive, the detection rate increased to 63.6 per cent (716/1125). The majority of patients tested positive presented with low-to-moderate viral load both in NP and OP swabs (Fig. 1).
The proportion of positives was significantly less in 21 - 40 yr both in NP and OP swabs, P=0.014 and 0.007, respectively (Fig. 2). There had been a fair degree of agreement between NP and OP swabs among all age groups (Table I). Three hundred and three (44.8%) males and 190 (49.7%) females were tested positive in NP swabs. The difference was not significant; 319 (42.9%) males and 174 (45.5%) females were tested positive in OP swabs. This difference also was not significant. A total of 475 (63.9%) pairs were concordant among males, kappa = 0.268, P<0.001 and 246 (64.3%) pairs were concordant among females, kappa = 0.288, P<0.001. There was a fair degree of agreement between NP and OP swabs among males and females.
In NP swabs, the proportion of positives was significantly more among patients with symptoms of COVID-19 compared to those who did not have any symptoms. However, in OP swabs, no such association was noted (P=0.024 and 0.139, respectively). The proportion of positives in NP swabs was 52.3 and 44.6 per cent in symptomatic and asymptomatic individuals, respectively, whereas the proportion of positives in OP swabs was 47.7 and 42.6 per cent in symptomatic and asymptomatics, respectively.
There was a fair degree of agreement between NP and OP swabs among individuals with and without symptoms (Table II). There was no significant difference in the proportion of positives with respect to the duration of symptoms among NP and OP groups, P=0.621 and 0.411, respectively (Fig. 3). There was fair degree of agreement between NP and OP swabs with respect to duration of symptoms, though in more than 2 wk of symptom duration the agreement could not attain significance due to small number of patients in this group (Table III).
Absolute sensitivity, specificity, PPV and NPV for OP and NP swabs relative to a positive result on either OP swab or NP swab were calculated. NP swabs had better sensitivity (73.7 vs. 69.4%) and NPV (68.9 vs. 65.9%) when compared to OP swabs. Specificity and PPV of both NP and OP swabs were 100 per cent. No significant relationship could be observed between the viral load and the presence of clinical symptoms (Table IV).
Discussion
The primary and preferred method for diagnosis of COVID-19 is the collection of upper respiratory samples using NP or OP swabs. US Centres for Disease Control and Prevention (CDC) reported earlier that NP swab seemed to be more sensitive than OP swab for COVID-19 diagnosis11. LeBlanc et al12 have reported that a combined OP and NP swab is better than only NP swab for the diagnosis of COVID-19 infection with sensitivity of 94.4 per cent (combined swab) compared to 91.7 per cent sensitivity of NP swab alone. The study by Zhang et al13 in August 2020, with a sample size of 43 COVID-19 confirmed patients, concluded that NP and OP swabs had similar yield, and the consistency of results between OP and NP swabs was low when the disease duration was <14 days or more than 21 days. There were two studies published in 202114,15, having the sample size of 240 and 309, respectively. One was looking at comparative evaluation of NP and OP swabs based on rapid SARS-CoV-2 antigen detection and rRT-PCR (sample size 240). The other was comparative analysis of naso/oropharyngeal swab and saliva for RT-qPCR.
In a study conducted by Wang et al16 among 353 patients, higher positivity was found for NP swab as compared to OP swab. Another study with 120 paired NP and OP swabs suggested that NP swab had significantly higher SARS-CoV-2 detection rate, sensitivity and viral load than OP swab17. Patel et al18 found that among 146 NP and OP swab pairs collected ≤7 days after illness onset, RT-PCR diagnostic results were 95.2 per cent concordant. In our study, the positivity rate of NP swabs from symptomatic patients was found to be more than the positivity rate of NP swabs from asymptomatic patients. However, in OP swabs, no such association was noted. There was a fair degree of agreement between concordant OP and NP swabs among patients with and without symptoms.
Various studies suggest that the viral shedding from upper respiratory tract specimen is maximum during the early days after illness onset so that early testing might be ideal to increase the yield for SARS-CoV-2 detection in NP and OP swabs19,20. However, in our study, there was no significant difference in the proportion of positives with respect to the duration of symptoms between NP and OP swabs.
In a study21, comparable clinical sensitivity was seen between OP and NP swab at quantitative level. In our study also, a reasonable degree of agreement was seen between the methods (kappa=0.275, P<0.001). There was no significant difference in the proportion of positivity with respect to gender or age. Furthermore, the number of patient samples and outcome variables can limit the prediction based on the expected test-positivity rate. A similar study used logistic regression mixed-effect model analysis on 19,110 samples with 1.5 per cent test-positive rate and detected only 25.6 per cent variation among OP, NP and saliva samples22. Although rRT-PCR may not be the ideal tool to diagnose infectivity or for mass screening, our study provided an insight into the equal utility of OP and NP swabs if done appropriately in the real-world settings.
In conclusion, our study showed that both NP and OP swabs samples had similar sensitivity and specificity for detecting the presence of SARS-CoV-2 in an infected individual. The detection rate was more if both OP and NP swabs were combined.
Financial support & sponsorship: None.
Conflicts of Interest: None.
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