Variability between testing methods for SARS-CoV-2 nucleic acid detection 16 days post-discharge: a case report

To the Editor,

By March 17, 2020, there were 80,881 cumulative confirmed cases, 128 suspected cases and 3226 deaths reported by the National Health Commission of the People’s Republic of China [1]. Early diagnosis and isolated treatment have been effective strategies to control the spread of disease because of the lack of effective treatment. According to the diagnosis and treatment protocols from the National Health Commission of the People’s Republic of China, the diagnostic criteria for COVID-19 are as follows: (1) epidemiological history – travel/history of residence in Wuhan or history of exposure to fevered patients with respiratory symptoms from Wuhan within 14 days before the onset of illness; (2) clinical manifestations – fever, imaging characteristics of pneumonia and/or normal or decreased white blood cell count or decreased lymphocyte count; and (3) laboratory diagnosis – qualitative reverse transcription polymerase chain reaction (qRT-PCR) revealing positive detection of SARS-CoV-2 in throat swabs or the lower respiratory tract [2], [3]. From trial version 3 to trial version 6, nucleic acid detection by qRT-PCR has always played a definite role in the diagnosis of COVID-19. Based on this background knowledge, many enterprises and scientific research institutions have urgently developed different brands of nucleic acid testing kits, which provide additional choices during clinical investigations.

A 36-year-old woman presented with fever and cough for 7 days and was admitted to the fever clinic at the Hospital of Chengdu University of Traditional Chinese Medicine, complaining of chest congestion, fatigue and headache. A week before admission, the patient had a fever, with the highest body temperature recorded as 38.5°C after contact with staff returning from Wuhan. The routine blood examination showed only an increased monocyte count (13.20%) at that time. She then took oseltamivir by herself but had no significant improvement in symptoms. On the day of admission, she denied any history of smoking or drinking and had no major illness. She had no respiratory symptoms, a blood pressure of 130/80 mmHg, a pulse rate of 76 beats per minute, a respiratory rate of 18 breaths per minute and a body temperature of 36.9°C upon complete check-up. CT showed patchy infection and ground-glass opacities with indistinct borders in the upper lobe of the right lung, the middle lobe of the left lung and the lower lobes of both lungs. The laboratory examination results were as shown in Table 1 on the day of admission (23 Jan 2020). Finally, she was diagnosed with COVID-19 via qRT-PCR amplification of SARS-CoV-2 nucleic acid from a throat swab. On January 24, the patient was transferred to a designated hospital for treatment. After treatment, the patient’s temperature returned to normal, lasting for more than 3 days, and her respiratory symptoms improved as well. During this time, the results of the SARS-CoV-2 nucleic acid detection test from her throat swab were negative on the fourth day, the sixth day and the eighth day in the designated hospital. In addition, re-examination of chest CT showed that the lesions demonstrating bilateral lung infection were improved compared with the results of the CT scan on January 23. Her laboratory results on January 31 were as shown in Table 1.

All of the aforementioned results met the discharge criteria of the ‘Diagnosis and treatment protocols for pneumonia caused by SARS-CoV-2 (trial version 4)’ [4]. Thus, the patient was discharged after hospitalisation and isolated treatment for 8 days. Subsequently, the patient was isolated at home alone for 16 days. On February 16, alarmingly, her re-examination for SARS-CoV-2 nucleic acid showed detectable nucleocapsid protein (N) gene from a throat swab using a different testing kit Daan (DAANGENE, Guangzhou, China, Lot: 20200809, genes: N and Open reading frame 1 ab: ORF1ab) in the Department of Laboratory Medicine, the Hospital of Chengdu University of Traditional Chinese Medicine.

On February 17, throat and sputum samples were processed and tested using two different kits Daan (N and ORF1ab) and Genuo (GENEODX, Shanghai, China, Lot: GZTRM 21202002, genes: N, ORF1ab and Envelop E gene: E). The results were positive for both throat swab and sputum by Daan (N and ORF1ab), while they were positive for only sputum by Genuo (N, ORF1ab and E) (Table 2). Because the Daan (N and ORF1ab) detected a single viral nucleic acid in the throat swab (Sample number: 2020021707) as shown in Table 2, the interpretation was considered not indicative of COVID-19 according to the ‘Laboratory guidelines for novel coronavirus pneumonia (version 4)’ [5]. Moreover, amplification efficiency and system problems led to a low-detection fluorescence signal and atypical amplification curve, which were not conducive to assessing the results by Daan (N and ORF1ab) on February 17 (Figure 1). For example, as shown in Figure 1A, the amplification curve of Daan (N) was atypical, with a detection value of 38.00 (cut-off value: 40), which was determined as positivity. Similarly, the amplification curve of Daan (ORF1ab) was atypical, and the detection value of 41.23 (cut-off value: 40) was negative. In Figure 1B, the curves were all normal amplification curves. The detection value of Daan (N) and Daan (ORF1ab) were 31.38 and 33.32, respectively, which were both determined as positivity, according to the cut-off value of 40. The general characteristics of SARS-CoV-2 detection reagents and the partial test results are shown in Supplementary Table 1, Table 2 and Figure 1 for qRT-PCR. The same sample number meant that the sample was tested twice at different dates using the same reagent or parallelly tested at the same time using different reagents. As a supplement, we also measured the anal swab by Daan (N and ORF1ab) and Genuo (N, ORF1ab and E), and both results were negative on February 17. On February 21, we obtained positive IgM results from serum with the SARS-CoV-2 IgM antibody detection kit (colloidal gold immune chromatography) (Beijing Hot King Biotechnology Co., LTD, China, Lot: 20200205). The CT scan showed that some lesions had obvious absorption and tended to show local fibrosis. In addition, physical and other examinations of the patient basically appeared normal. On February 18, the patient was isolated again, and her complete clinical course is shown in Figure 2.

Figure 1:

The amplification curve of hallmarker genes from different samples.

A comparison of the atypical amplification curve (A) and normal amplification curve (B) for SARS-CoV-2 nucleic acid detection by qRT-PCR.

Figure 2:

Diagram showing the clinical course of the patient.

According to the disease course, the patient history included exposure to an infected person followed by symptoms consistent with COVID-19, specifically weakness and cough. Consequently, her CT scan and initial positive SARS-CoV-2 nucleic acid test both confirmed this disease.

Undeniably, early diagnosis becomes the key to controlling the epidemic, and qRT-PCR technology is the preferred screening method. Nucleic acid detection has the advantages of early diagnosis, high sensitivity and specificity, and easy operation, but the accuracy of nucleic acid detection results needs to be comprehensively analysed according to the sample types, quality, experimental factors, kit performance and patient infection cycles [6]. Whether assessing this case or other cases, most nucleic acid test results have been unsatisfactory (low detection rate). Faced with the problems of nucleic acid testing and substantial social pressure, the National Health Commission of the People’s Republic of China promptly updated the ‘Laboratory guidelines for novel coronavirus pneumonia (version 4)’ to final version 5 [7]. The new guidelines (version 5) make it clear that positive results can be determined by the simultaneously positive results of a single target (N or OFR1ab) from two samples with qRT-PCR or by the positive results of a single target from two samples of the same type [7].

The quality of nucleic acid detection kits is the key to determining the detection rate of the same sample in the same laboratory except for the error of the clinical laboratory practitioner’s inter-batch operation. Although both SARS-CoV-2 nucleic acid detection kits Daan (N and ORF1ab) and Genuo (N, ORF1ab and E) detect genes at least including N and OFR1ab, the primers were designed at different sites, which directly determined the amplification efficiency [8]. In addition, the selection of reverse transcriptase enzymes and reverse transcriptase primers can also affect reverse transcriptase efficiency because RT enzymes do not have a proofreading function to check for errors in newly synthesised DNA [9]. Both SARS-CoV-2 nucleic acid detection kits Daan (N and ORF1ab) and Genuo (N, ORF1ab and E) use one-step RT-PCR, and in this system, researchers should take the activity of reverse transcriptase and DNA amplification enzymes into account, which would affect the overall detection rate [10].

Except for the influence of nucleic acid detection kit performance, the dynamic balance between SARS-CoV-2 and the host is also an important factor affecting the outcomes of the disease. According to the disease course of the patient, when she was released from quarantine, her nucleic acid detection result was negative. Given that low viral loads below the minimum detection limit cannot be measured using the current kits, a small amount of virus might continue to replicate, especially after treatment is ceased; then, the number of viruses may increase to the detection level. For this case, we could not conclude whether she had relapsed or had not been cured. However, she remained in isolation until her nucleic acid detection was positive again. Therefore, there was very little chance for reinfection.

In summary, based on the current situation of novel coronavirus nucleic acid detection, the outcomes of patients with isolation treatment need to be supported by more clinical evidence. In terms of nucleic acid testing, in addition to examining respiratory specimens multiple times, the use of more types of samples is also recommended (such as serum, faeces and even urine). When selecting the types of samples, the samples from the lower respiratory tract (such as deep sputum, lavage fluid, etc.) were preferred, followed by samples from the upper respiratory tract (such as nasopharyngeal swabs, etc.), at last faeces, urine and blood samples were selected [7]. We can even choose other supplementary methods, such as the use of serum-specific antibodies IgM or IgG. Because there are no specific drugs for the treatment of COVID-19, it seems that the infusion of specific antibodies from cured patients may provide an exciting therapeutic strategy. However, we still know little about SARS-CoV-2. The definitions of incubation and recovery are also being revised. We believe that the use of serum-specific antibodies from cured patients for infused treatment requires more careful consideration. Finally, the management and timely review of discharged patients should be considered.