⁎⁎⁎ Corresponding authors at: Department of Clinical Biochemistry, Afzalipour School of Medicine, Kerman University of Medical Sciences, Iran.
Received 2021 Mar 9; Revised 2021 Apr 10; Accepted 2021 Apr 11. Copyright © 2021 Elsevier GmbH. All rights reserved.Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the control of virus spread has remained challenging given the pitfalls of the current diagnostic tests. Nevertheless, RNA amplification techniques have been the gold standard among other diagnostic methods for monitoring clinical samples for the presence of the virus. In the current paper, we review the shortcomings and strengths of RT-PCR (real-time polymerase chain reaction) techniques for diagnosis of coronavirus disease (COVID)-19. We address the repercussions of false-negative and false-positive rates encountered in the test, summarize approaches to improve the overall sensitivity of this method. We discuss the barriers to the widespread use of the RT-PCR test, and some technical advances, such as RT-LAMP (reverse-transcriptase-loop mediated isothermal amplification). We also address how other molecular techniques, such as immunodiagnostic tests can be used to avoid incorrect interpretation of RT-PCR tests.
Abbreviations: cDNA, complementary DNA; CLIA, chemiluminescence immunoassay; COVID-19, Coronavirus Disease 2019; CRISPR, clustered regularly interspaced short palindromic repeats; CT, computed tomography scan; DNA, deoxyribonucleic acid; EDTA, ethylene-diamine-tetra-acetic acid; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; iLACO, isothermal LAMP-based method for COVID-19; NP, nasopharyngeal; NPV, negative predictive value; OP, oropharyngeal; RBD, receptor binding domain of the virus; RNA, ribonucleic acid; RT-LAMP, reverse transcription loop-mediated isothermal amplification; RT–PCR, reverse transcription of polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus; SRT, sampling-to-result time; TCEP, tris (2-carboxyethyl) phosphine
Keywords: SARS-CoV-2, COVID-19, Reverse transcription polymerase chain reaction, Diagnostic tests, Reverse transcription loop-mediated isothermal amplification, Serologic tests
In early December 2019, the first case of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in Wuhan, China, and ever since, there has been a growing spread of the coronavirus disease 19 (COVID-19) all over the world [1]. While the disease is widely known to be a deadly disease, some patients are asymptomatic but can still transmit the virus [2]. This has made tracing the disease difficult solely based on clinical symptoms, and undetected SARS-CoV-2 infection has posed serious challenges regarding control of the disease spread. Currently, the virus outbreak has reached pandemic proportions with over 3 million deaths across the world [2], underlining the rapid spread and the urgent need for control of disease transmission. Therefore, because widespread vaccination against SARS-CoV-2 will take some considerable time, keeping the disease transmission under control is a high priority, and there is a need to drastically improve the efficiency of the present diagnostic tests.
Currently, diagnostic techniques based on viral RNA amplification, specifically qRT-PCR (quantitative real-time polymerase chain reaction), are the gold standard diagnostic methods for COVID-19 [3,4]. Unlike other molecular tests that do not have perfect diagnostic specificity, qRT-PCR is highly specific with a specificity of almost 100 % [5]. This has led to RT-PCR becoming the gold standard molecular diagnostic test.
However, the RT-PCR test for SARS-CoV-2 virus does have some pitfalls that necessitate improvements in the way the method is used. As with immunodiagnostic tests, the RT-PCR test can have difficulties in distinguishing between true positive and true negative COVID-19 infected individuals [6]. The test fails in a considerable proportion of suspected and confirmed patients with clinical implications; as a result, it is a wise precaution not to rely on PCR test results alone, and to consider other clinical and molecular evidence [5]. This means that one always should take into account a combination of clinical and molecular evidence before sending a suspected patient home as disease-free. Furthermore, given the challenges with RT-PCR test results, repetition of the test over time and on multiple samples enhances the overall sensitivity of the test.
Moreover, it is necessary to improve the RT-PCR methodology to tackle the problem of less than perfect sensitivity. This could be achieved by designing more simple versions of the test. Simple tests provide opportunities for more wide-spread application among different components of the health-care system. A simple test requires less training and could enable other health-care staff to use the test correctly. It also minimizes the risk of disease transmission to the staff, and test failure due to improper manipulation of the clinical samples. Furthermore, simplification of the test can shorten the gap between sampling and results, allowing the repetition of the test over time or on multiple samples if needed. Finally, the simpler the test is, the more likely it can be offered at a lower cost per test [7].
In the present paper, we will review publications discussing the diagnostic ability of the RT-PCR test as well as the implications of its failure, and some ways of maximizing the current molecular diagnosis for COVID-19 will be addressed. We cover the clinical evidence for RT-PCR results in COVID-19 patients, approaches adopted to enhance the test efficacy, and recent technological developments in the design of the test. The false-negative and false-positive results of the RT-PCR test are discussed, along with the advantages of the combined use of immunodiagnostic tests together with RT-PCR.
False-negative results in a screening test can have serious implications during a pandemic, such as COVID-19 because a proportion of true infected cases are categorized as disease-free and can unintentionally transmit the disease. Unfortunately, there is no single molecular test that can guarantee the infection free status for a suspected case; therefore, the clinical history and social contacts of the individual should be always taken into account in the assessment of the infection probability. Repetition of the molecular tests over time also helps to increase the selectivity.
Reports have described RT-PCR on various specimens obtained from the respiratory tract; however, there are accumulating reports indicating the lack of adequate sensitivity for the test. RT-PCR examination of nasal and oropharyngeal swabs, nasopharyngeal washing or aspirate is recommended as the gold standard for the diagnosis of COVID-19 [[8], [9], [10]], However, the overall sensitivity of the RT-PCR test is reportedly between 45–60% in nasopharyngeal aspirate and swab samples [11]. For instance, Yang et al. reported a false negative rate of 11 % for sputum, 27 % for nasal, and 40 % for throat swabs within the first seven days from onset of illness in 213 patients hospitalized with COVID-19 [12]. Similarly, Zhao et al. reported a false negative rate of RT-PCR tests within the same range. These researchers reported that 33 % of respiratory samples gave false-negative results in 173 hospitalized patients with COVID-19 as diagnosed with typical chest CT scans and acute respiratory symptoms [12]. The false-negative rate ranged from 2% to 29 % in a systematic review of five other studies containing a total of 957 patients suspected or confirmed with COVID-19 [13].
One of the main reasons for such a high false-negative rate in RT-PCR results, is the time of sampling after the onset of symptoms. The time of sampling is important because it was shown that the false-negative rate of the test varies over time [14]. The false-negative rate of RT-PCR testing on nasopharyngeal (NP) and oropharyngeal samples was described as "shockingly high" in a study of 1330 confirmed cases. In their investigation, the authors pooled the data on the confirmed COVID-19 cases from seven previously published studies. They analyzed these data using a Bayesian hierarchical model to estimate the false-negative rate from 5 days before the onset of symptoms up to 21-days post-emergence of symptoms. They found that the median false-negative rate reduced gradually from 100 % to 20 % during days 3 and 4 post-onset of symptoms. The false-negative rate was 67 % one day before the onset of symptoms and 38 % on the day of symptom manifestation, and returned gradually to 66 % on day 21 post-onset of symptoms. Consequently, the false-negative rate of the test changes over time depending on when the samples were obtained from the onset of symptoms, and even at best, the RT-PCR fails to detect a considerable fraction (one out of five) of the infected cases [14].
Another explanation for the high negative rate of RT-PCR tests for COVID-19 could be related to the viral load present in the sampling site from each patient. This could vary among different specimens and patients. The highest viral loads are found in the lower respiratory tracts of COVID-19 patients compared to the upper respiratory tract [15]. However, sampling from the lower respiratory tract is difficult in patients with severe respiratory symptoms who are receiving oxygenation intervention [16]. In the upper tract, nasopharyngeal and oropharyngeal swaps or aspirates are recommended for early diagnosis of the infection. NP samples exhibited much higher viral loads compared to OP samples, giving a better chance detecting SARS-CoV-2 infection and lowering the risk of missing the infection [17]. Moreover, false-negative results occurred in some patients with gastrointestinal symptoms. In these patients, sampling from fecal samples revealed the presence of SARS-CoV-2 [16]. Therefore, some false-negative results are inevitable depending on the specimen chosen and the patient clinical symptoms.
Given the imperfect selectivity of the RT-PCR test, other diagnostic information should be taken into account to achieve the desirable sensitivity for true-positives or true-negatives for COVID-19. These factors include the clinical symptoms, immunodiagnostic test results, and prevalence of the disease within the community. These factors can help clinicians to better estimate how likely any particular case is to have disease. For instance, whether or not a case demonstrates the typical clinical symptoms of COVID-19 can give a primary estimate of the probability of the case being infected, and successive addition of the molecular test results (e.g. RT-PCR and serologic tests) will increase the confidence to distinguish between disease-free or infected. Furthermore, RT-PCR in combination with an immunodiagnostic test will improve the overall selectivity [18]. For example, in a retrospective study of 375 patients, the combined selectivity of RT-PCR and antibody testing was significantly higher compared to each test alone. The diagnostic sensitivity of the RT-PCR and antibody test were 95.7 % and 92.2 % respectively, which rose significantly to 98.6 % when these tests were combined [17]. Lastly, the prevalence of the disease should be taken into account in deciding whether or not a particular result is enough to send a person home as disease-free. The low prevalence of disease implies a low probability of having the disease, and a post-test negative result with a given sensitivity (say 95 %) could suggest a person is highly likely to be disease-free. However, when the prevalence of disease increases throughout a community, that level of sensitivity is less valuable to ensure a suspected patient is disease-free. In technical terms, the negative predictive value (NPV) of the test decreases with an increase in the prevalence of the disease [19].
To sum up, the false-negative rate of RT-PCR is significant and varies across different specimens and time periods. However, the false-negative rate can be minimized when immunodiagnostic tests and clinical symptoms are considered along with the RT-PCR test result. Moreover, it is importannt to stick to social distancing and recommended hygiene protocols to keep the prevalence of the disease as low as possible, in order to maintain the NPV of the tests at a high level. Otherwise, the negative results of PCR tests will no longer give us enough confidence that the suspected case is disease-free.
Due to the high rate of false-negative results in RT-PCR of COVID-19 patients, and the epidemiological prevalence, some improvements can be suggested.
The false-negative rate of the RT-PCR test can occur due to several reasons. Firstly, the viral load can be low or absent within the samples [19]. The viral load governs the amount of RNA in the samples. The higher the viral load in the sample, the more RNA with a better chance for a test to get a truly positive result. Secondly, the viral RNA might be subjected to denaturation or degradation in the samples due to improper manipulation or storage, which lowers the final amount of intact RNA for the test [20]. Thirdly, a sufficient viral load is limited to specific time periods when the virus rapidly replicates itself and is shed from the cells. Fourthly, the viral load has also shown to vary in terms of the anatomical site from which the specimen is obtained Lastly, the virus is present at low numbers or is absent in some specimens from some patients, while other specimens might have a higher viral load in the same patients [21]. Therefore, the variability of the false-negative rate depends on the viral load, which in turn, fluctuates over the course of the disease, and between specimens from patients with different clinical characteristics.
The above-mentioned problems can be solved with optimized RT-PCR diagnostic protocols. Given the mentioned viral load variability over time, specimen, and patients, an improved RT-PCR test should be more simple, rapid, and cost-effective to allow frequent repettition [22]. This will increase the chance of detecting the infection if the test is repeated over time and on different samples. If the test can be made rapid and less labor-intensive, the sampling-to-PCR gap time will be shortened, which will reduce the loss of viral RNA due to denaturation during this period. Moreover, a simplified test will require less sophisticated laboratory equipment. These simplified tests could enable rapid point-of-care sample manipulation and analysis, with a higher throughput.
The current RT-PCR test could be used more effectively by conducting the test in pooled samples. Pooling different samples from either the same patient or the patient's family members can reduce the number of tests and lower the costs positive rate of the test. Because in some patients the viral infection is limited to the lower respiratory tract, combining sputum, nasal and pharyngeal swabs coulsd be useful. In other patients with gastrointestinal involvement, the virus was only found in fecal material, while RT-PCR of the NP swabs and sputum were negative. Therefore conducting the test on pooled samples from different specimens can improve the probability of getting a sample with sufficient viral load to increase the accuracy of RT-PCR. The other benefit of pooling samples is to allow better at-home quarantine decisions amongst communities. For instance, pooled samples from the whole family of a suspected case can provide guidance on strict quarantine for the entire family, to reduce disease transmission in the community [23].
Therefore, the repetition of the RT-PCR test in pooled samples might offset the high false-negative rate of the test. Also, the conduction of the test in pooled samples appears to increase the utility of the test for screening purposes. To this end, recent cutting-edge technology has attempted to provide simple point-of-care or at home RT-PCR kits.
The preparation of a simple, rapid, and inexpensive RT-PCR test requires understanding the technical difficulties that have restricted the use of the RT-PCR method. By overcoming these obstacles, the laboratory RT-PCR test can be turned into a convenient, rapid, and budget-friendly kit that can be used more widely in clinics.
Technically, the RT-PCR procedure for SARS-CoV-2-infected samples consists of several steps, and needs laboratory equipment that makes the process tedious and difficult to be conducted outside the laboratory setting. First of all, the RNA material must be extracted from the cells and the virions (viral particles) and preserved from destruction by RNase enzymes. This step needs laboratory equipment such as a centrifuge and a laminar flow cabinet, and might lose some of the RNA materials due to denaturation. Secondly, the process of PCR requires thermal-cycling equipment for creating a cyclic temperature change during the process of RNA amplification. The third difficulty is the readout method used, which in most cases required expensive sophisticated spectrofluorometric equipment [24].
Before the RT-PCR step, the viral RNA has to be extracted from the samples. During this process, certain laboratory chemicals and equipment are used for specific purposes. Firstly, the infected cells and the virions are disintegrated by the addition of lysis buffer typically containing detergents (Tween 20 or Triton X100). The lysis of the cells and virions causes all the biomolecules, including viral RNAs to be released into the medium and be readily available for the test. The lysis buffer also contains salts such as sodium iodide (NaI) or guanidinium thiocyanate (GuSCN) that facilitate the separation of the viral RNA from other biomolecules (e.g. cell and viral proteins). Centrifugation of samples containing these salts assists in the separation of these proteins from the viral RNA fraction. Besides, cellular RNase enzymes are inactivated by the addition of detergents and thermal treatment. Some detergents such as TCEP or EDTA are added to the samples, which are incubated at 95 °C for 5 min. The RNase inactivation inhibits the process of RNA denaturation and causes the RNA to be preserved for the RT-PCR test [25].
Subsequently, the RNA samples are amplified during the RT-PCR process using a thermal cycling program. In each cycle, the temperature of the samples is alternatively decreased to 70 °C and increased to 95 °C for a certain time, and at each cycle the number of RNA replicates is doubled. At 70 °C, the single stranded C-DNA replicates are used as the template for the synthesis of new stands of C-DNA. During this step, Taq DNA polymerase synthesizes a new strand of C-DNA from the template which gives rise to a double-strand DNA composed of the old and new strands of C-DNA. At 95 °C, these strands must be detached from each other to give twice the number of single strand templates for the next round of C-DNA replication. This cycle is repeated several times (e.g. for 30 cycles) until the number of C-DNA replicates reaches an amount capable of being detected by spectrofluorimetric techniques. The thermocycler apparatus that provides this accurate cycle of temperature changes is expensive equipment that is often confined to a laboratory [25].
Finally, the increasing number of C-DNA replicates is monitored using a real-time spectrofluorimetric technique that is also expensive and not always available. This technique offers a readout of the C-DNA amplification on a computer screen based on the fluorescent signal that changes increases in line with C-DNA numbers. Different fluorescent probes are used for the quantification of C-DNA replicates in the PCR. For instance, in the SYBR green assay, the fluorescent probe intercalates into the C-DNA double strands that quenches the probe fluorescence. This fluorescent probe de-quenches upon the separation of the C-DNA strands from each other. In the TaqMan assay, the fluorescent probe is de-quenched by the binding of the TaqMan primer to the C-DNA templates. In both techniques, a spectrofluorimetric apparatus coupled to a computer is required for the final readout of the RNA amount in the samples. These pieces of equipment are expensive and may not be available everywhere in large numbers [25].
Given the aforementioned difficulties of the RT-PCR test, enormous efforts have been made to produce an easier, faster, and more convenient test capable of being used outside the laboratory environment. A simple and rapid test can reduce the sampling-to-result time (SRT) and encourage its wider application. The test procedure should require fewer steps and laboratory tools. A shorter SRT and easier manipulation of the sample will have some other benefits, including an increase in the test sensitivity.
One important simplification in the nucleic acid amplification procedure was the invention of an isothermal PCR method that eliminated the need for a thermal cycling apparatus. This allowed the amplification of RNA or DNA using a widely available kitchen oven maintained at a specific constant temperature. In this method there is no need to increase the temperature up to 90 °C to unwind the double-stranded DNA. Instead, the DNA polymerase itself displaces one of the strands of the DNA as it acts on the other strand and synthesizes a new copy. During this process, primers fold back on themselves and create loops of DNA that provide 3′ starting points for the new round of DNA replication by DNA polymerases. Therefore, the technique is called the loop-mediated isothermal amplification (LAMP) technique, described in reference [26]. LAMP only needs an optimum temperature of 65 °C for DNA polymerase activity. The provision of a constant temperature is technically much easier than a temperature cycling program that is required for conventional PCR [19].
Another invention which has made the test more convenient and quicker is where the pre-PCR RNA extraction step is combined with the PCR step itself. This reduction in the number of steps of the test offers some advantages. Firstly, a single step preparation of RNA reduces the SRT and increases the potential of the test for wider application. A shorter SRT decreases the probability of disease transfer by individuals whose test results have yet to be determined. Secondly, a one-step preparation of the RNA samples is much easier for potential users to learn how to use the test correctly. Thirdly, during the extraction of RNA from the sample, there is a risk of viral transmission from the samples to the laboratory staff, and cross-sample contamination due to unintentionally errors in sample manipulation. A shorter and easier process of RNA preparation can minimize the mentioned risks. Fourthly, the use of an RNA purification protocol increased the sensitivity of the RT-LAMP test shown by a reduction in the detection limit of the test from 100 copies/μL to 1 copy/μL. Lastly, the combination of the steps has been shown to eliminate the need for apparatus that limits the test to a lab environment [19].
Furthermore, further simplification of RT-PCR can be achieved by providing RT-LAMP kits with simpler result readouts. In the case of COVID-19 infection, it is only necessary to know whether or not viral RNA is present in the samples; therefore, there is no need for expensive quantification methods like spectrofluorimetry. Instead of quantitation, qualitative readouts such as a color change are much easier to achieve, and are more appropriate for diagnosis of SARS-CoV-2 infection. By using these kinds of readout, one can simply observe the results with the naked eye [19]. For instance, Yu et al. [27]. recently developed an iLACO assay (isothermal LAMP-based method for COVID-19) with a fluorescent readout. Using this kit, they could achieve the results within 15−40 min and observe the results without any need for expensive spectrofluorimetric equipment. In this test, the positive samples with Genefinder dye turned bright white, while the negative samples remained blue under blue light. Another example was the Penn-LAMP technique that produced a color change upon the amplification of the viral RNA. In this technique, the sample color changes from white to blue if the samples contained the amplified RNA material. Also, the entire RT-LAMP process can be done in a single vial without any need for specific equipment.
Previous studies have suggested using CRISPR/Cas systems in the diagnosis and treatment of COVID-19 [28,29]. Perhaps the best solution to produce a "fast and simple test" may be the "integrated RT-LAMP and CRISPR-Cas-12 method' ( Fig. 1 ), that literally can be used anywhere by anyone. The method contains a kit with a lateral flow visual readout using a strip of paper. In this test, one just needs to dip the correct end of the designed strip in the vial of the final RT-LAMP product and wait to observe either a positive or negative result. These results appear in the form of a band at specific distances from the starting point. In the designed strip, the detection method relies on the CRISPR Cas-12 endonuclease enzyme and a trans-reporter molecule. The CRISPR Cas-12 detects a specific E gene and N gene sequence in the amplified viral DNA and acts as a “shredder” on other irrelevant RNA sequences. Therefore, all other RNA sequences are eliminated except for the target sequence that remains intact in the RT-LAMP product. The FAM-biotin trans-reporter is already placed and affixed to the strip. As the sample flows laterally across the strip, the remaining target sequence interacts with the FAM-biotin trans-reporter molecules on the strip producing the band. Using this DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTOR) technique, a fluorescently-labeled band can be observed in less than one minute [8].