DIFFICULTIES IN THE DETECTION OF GENOMES OF THE NEW CORONAVIRUS 2 (SARS COV-2)

DIFFICULTIES IN THE DETECTION OF GENOMES OF THE NEW CORONAVIRUS 2 (SARS COV-2)

Pablo Goldschmidt. Pharmacist, Biochemist, Medical Biologist specialized in Virology. Ret. Laboratoire du Center Hospitalier National des Quinze-Vingts, Paris, France.

SUMMARY 

The management of respiratory viral infections, both nationally and globally, requires quality scientific results. The reverse transcriptase polymerase chain reaction (rRT-PCR) is considered the “gold standard” to detect the genome of the new coronavirus 2 (SARS-CoV-2), the causal agent of the disease by the new coronavirus (COVID-19) especially in the acute phase of the infection. Its use is controversial outside of a viral exposure context. 

The objective of this work is to analyze pitfalls found during the detection of the SARS-CoV-2 genome that can produce false results. False negatives from rRT-PCR may be due to the timing and efficiency of sample collection, freezing, storage and thawing, and thermal inactivation of virulence. Furthermore, delayed signals from internal controls override negativity. On the other hand, samples with little biological material lead to false negative conclusions, so determining a threshold (minimum number of epithelial cells) will help reduce them. However, most kits detect human DNA, but were not calibrated to quantify cellular load. Viral nuclear ribonucleic acids (RNA) attached to gloves, tubes and caps, - among other elements - are a source of false positives. 

Pharmacopoeias suggest that external contamination be controlled in series of 100 samples with at least 10% representativeness. If this approach is extrapolated to the clinical analysis laboratory, instead of one, at least 10 negative controls adjacent to 10 positive controls should be processed every 100 tests. Improving detection by rRT-PCR implies an increase of at least 20% in the cost of reagents, so additional resources are needed

INTRODUCTION

In the context of a pandemic such as COVID-19, caused by the new coronavirus 2 (SARS-CoV-2), it is necessary to have solid data to support policies and measures to protect the population. 

Nucleic acid amplification techniques (NAAT), particularly reverse transcription reactions followed by polymerase chain reactions (rRT-PCR), are valuable tools for detecting infectious agents2. However, researchers and health policy decision makers lack arguments to justify, for example, the high number of positive rRT-PCR tests in paucisymptomatic (those with mild symptoms) or asymptomatic people who do not trigger specific immune responses3 ,4. On the other hand, the technical difficulties faced with clinical presentations with negative rRT-PCR results require a specific analysis. 

This work aims to analyze certain difficulties detected during the study of viral genomes and propose solutions to minimize the error when drawing conclusions. 

TESTS FOR THE DETECTION OF SARS-CoV-2 

The NAATs used for confirmation and surveillance of the spread of SARS-CoV-2 were developed mainly by researchers in Germany, China, the United States of America and Japan5-8. 

In clinical practice, especially in the acute phase of infection, rRT-PCR (RNA reverse transcription followed by real-time PCR) is considered the “gold standard” for the detection of the SARS-CoV-2 genome, but its use in cases in which there is no confirmed viral exposure is still controversial. In fact, current rRT-PCR tests were validated with panels of material in an idealized situation and with patient samples that contained viral loads at levels different from those found in population screening9, 10.

LAMP MEDIATED ISOTHERMAL AMPLIFICATION

Nucleic acid isothermal amplification (LAMP) is a reaction that allows nucleic acid sequences to be amplified with four to six primers under isothermal conditions (63°C-65°C) and produces results that can be determined visual form. For several respiratory viruses, this technique has demonstrated detection limits similar to those of rRT-PCR (1,000 copies/ml)11, 12. 

RISKS OF FALSE RESULTS DURING PREANALYTICAL PROCEDURES

Despite continuous improvements in automation, it is common to see false results in the pre-analytical phase13. 

Virus detection is affected by the timing of sample collection (before or after the onset of symptoms), the quality of sampling procedures (nasopharyngeal and oropharyngeal swabs), and solutions for transport and viral conservation14. 

After sampling, tubes containing RNA viruses require special care, due to RNA degradation. The inactivation of infectiousness that is carried out in several laboratories at 56ºC alters the integrity of the viral RNA and affects the quality of the results, especially in samples with low viral load15. 

The World Health Organization and local health authorities recommend that all operating procedures ensure staff are trained and highlight the potential risk of infection. To this end, biosafety standards establish physical distancing, with appropriate protective equipment for each of the health workers. Masks, eye protectors, aprons, caps, gloves and other necessary items should be available in clinical and laboratory settings. However, mechanical barriers do not protect samples from cross-contamination (for example, from viral RNA that can adhere to gloves). Swabs with mucous secretions, potentially infected cells, and free viral particles should be placed in tubes and closed tightly (generally, this occurs with gloves contaminated with viral droplets). Many sampling centers disinfect gloves, but the substances used for this purpose do not eliminate adhered nucleic acids. 

Positive PCR tests were obtained by rinsing gloves used during eye infection detection campaigns16, so it is necessary to highlight the risk of false positive results if viral genomes are shed at a later stage. 

Changing gloves after taking samples from each person is not feasible with restricted healthcare budgets, and the cost of products that destroy nucleic acids is very high. One option is to have a standard solution of hydrochloric acid (normal 1 hydrochloric acid, useful for destroying nucleic acids) prepared daily. Pieces of sterile gauze are soaked with the solution and wiped over potentially contaminated surfaces after disinfecting them with biocidal agents. This procedure is viable from a cost point of view, although it may complicate the sector's routine.

SAMPLE PREPARATION FOR RRT-PCR

The genomes of coronaviruses (single-stranded RNA) must be prevented from being exposed to RNases, enzymes abundant in the environment and on the hands that degrade RNA molecules. For this reason, RNA isolation and purification procedures require aseptic techniques with RNase-free solutions and materials. 

Sample preparation aims to extract nucleic acids to make them accessible to retrotranscription and amplification, with the elimination of possible inhibitors of these reactions.

At the beginning of preparation in the laboratory, the tube caps are opened to remove the swabs from the transport medium, and RNA sequences are introduced in specified volumes, which will serve as internal control (IC). These sequences must not be related to coronaviruses. For all PCRs associated with clinical contexts, ICs will be extracted, retrotranscribed and amplified simultaneously and in the same tube with the sample to validate that all procedures and reactions have been carried out correctly17.

For the extraction of viral nucleic acids, for example, silicon-coated magnetic microspheres (automatic or manual systems) can be used, which allow the element to be amplified to be extracted in the presence of concentrations of proteins and lipids, among other compounds. Activated silicon particles have the ability to capture nucleic acids that, once washed to remove unfixed material, dissociate the nucleic acids with special solutions that are transferred to a plate to continue with the amplification reactions. Manual RNA extraction is less efficient than that performed with automated robotic systems18.

REVERSE TRANSCRIPTION

PCR amplifies deoxyribonucleic acid (DNA), so the extracted and purified RNA must be transformed into complementary DNA (cDNA) thanks to the action of reverse transcriptase endowed with the activity of the rTth enzyme, derived from the bacterium Thermus thermophilus. The rTth enzyme is a thermostable DNA polymerase that can in turn exert reverse transcriptase activity.

For this process, the purified nucleic acids are mixed with reverse primers from SARS-CoV-2 and the internal control (IC), which fix on their respective molecular targets and extend. The newly formed double strand is opened by the action of heat and more primers fixate on the cDNAs (of the IC and the molecular target) and extend. Extraction and reverse transcription procedures require serial pipetting of solutions that are introduced into reaction tubes. These procedures carry risks of cross-contamination due to splashing of imperceptible microdroplets.

THE NEED FOR ENDOGENOUS CONTROLS

Epithelial cells lining the nasal passages and oropharyngeal respiratory epithelium express SARS-CoV-2 receptors, and must be present in the samples to be analyzed.

Using conjunctival scraping samples from children with clinical signs of active trachoma, samples containing less than 50 cells/μl of swab transport fluid were determined to give false negative results. This situation was reproduced for other infectious agents of mucosal or epithelial tissues19.

rRT-PCR for SARS-CoV-2 uses signals produced by the R-actin gene or the cellular ribonuclease p30 subunit (RPP30) gene. These markers were not calibrated with serially diluted epithelial cells to determine the cell load directly in each sample. However, mere qualitative assessment showed a strong association between positive rRT-PCR results for SARS-CoV-2 and elevated RPP3020 levels.

On the other hand, quantifying the number of cells in a sample allows us to standardize a denominator for the viral load (copies of viral RNA/number of cells present in a sample). False negative results were reliably identified in samples with low expression of the RPP30 gene, as well as in the majority of contradictory rRT-PCR results21. Therefore, negativity for SARS-CoV-2 cannot be validated in samples that contain cellular loads (determined with endogenous controls) lower than the threshold to be determined.

GENE AMPLIFICATION

The SARS-CoV-2 genome is structured with a strand of positive RNA. The 3' terminal end of the genome encodes four structural proteins: the spike glycoprotein (S), the envelope protein (E), the membrane glycoprotein (M), and the nucleocapsid phosphoprotein (N) with accessory proteins. The probes used for the detection of the amplified sequences are designed and synthesized with single-stranded DNA oligonucleotides labeled with fluorophores at the 5' end and fluorescence quenchers at the 3' end. Each probe is labeled with a different fluorophore to detect the different amplified products in the same microtube simultaneously. In the absence of the molecular target recognized by the probe, the fluorescence remains quenched, but if it is recognized, the complementary sequences hybridize, and the fluorophore is separated from the component that masks its signal. As new strands of DNA are copied, the computer program tracks the fluorescence produced. rRT-PCR (RNA reverse transcription followed by chain amplification reaction) requires 40 cycles of amplification of the desired molecular target (2 raised to the 40th power). Signals exceeding a threshold (exponential amplification region) define the threshold cycle or Ct22.

The rRT-PCR kits currently marketed did not show cross-reactions with the panel of other respiratory viruses, except for the E gene of SARS-CoV-116, 23. Therefore, the detection of a single signal is not sufficient to consider rRT-PCR positive. Samples that detect a minimum of two or three signals from the virus genome are considered positive: S, M, E and N. Discordant results in symptomatic patients require the processing of a new sample.

NEGATIVE RESULTS AND FALSE NEGATIVES

The signals produced in each sample by the IC must be the same as those obtained with the negative and blank controls. Delays in IC signals in a sample reveal deficiencies in extraction or retrotranscription, or the presence of Inhibitors of any of the rRT-PCR steps. In these circumstances, a negative result cannot be reported and a new sample is required.

For this type of laboratory techniques, extremely small volumes are pipetted (5 or 10 pl), so precautions are required to avoid the entry of air microbubbles into the tips of the pipettes (difficult to detect by the human eye). since little material available for amplification can lead to false conclusions.

Positive rRT-PCR for asymptomatic cases have been described at levels of 30.8% in Japan22, 50% on the Diamond Princess cruise ship and 56.5% in the United States23, 24.

Seven studies presented rRT-PCR results from the onset of symptoms or possible contacts from the day since infection with SARS-CoV-2 was estimated (exposed people and healthcare workers). These studies showed that during the first four days of infection and before the time of symptom onset (day 5), the probability of a false negative result in an infected person is high. On the day of symptom onset, the mean false negative rate was 38% and decreased to 20% (12%-30%) on day 8 (three days after symptom onset) and then began to decrease. increase again, from 21% (13%-31%) on day 9 to 66% (54%-77%) on day 21. From the above it is deduced that rRT-PCR added little diagnostic information in the days immediately after exposure to the virus. To reduce erroneous conclusions it was then suggested to take samples one to three days after the onset of symptoms. However, in cases of high clinical suspicion and in contexts of viral transmission, the presence of the virus should not be ruled out solely on the basis of negative rRT-PCR results25.

On the other hand, reports from China showed false negative results for a period of up to two weeks post-exposure. In analyzes performed on repeated samples, false negative results were reported in between 2% and 33% of the samples analyzed26.

Based on the above, in the first days of viral infection with SARS-CoV-2 or any other respiratory virus, negative rRT-PCR test results should be considered necessary, but not sufficient, markers to eliminate the precautions designed to prevent the transmission of infection. In case of doubt, thoracic computed tomography (CT) provides pathognomonic images that help, in certain circumstances, to clarify the diagnosis (however, normal thoracic CT scans have been reported in people with positive rRT-PCR)27.

POSITIVE RESULTS AND FALSE POSITIVES

Positive rRT-PCR results reveal the presence of viral RNA, but do not predict virulence or transmissibility, nor do they rule out bacterial infections or coinfections with other viruses. This confusion could have led to considering the existence of "very numerous infected cases" when in reality it was the number of tests with positive results.

In New York City, studies in which rRT-PCR to detect the presence of the viral genome was followed by the detection of anti-SARS-Co-V2 antibodies, demonstrated that of the 624 participants with COVID-19 symptoms confirmed, all but three produced specific antibodies against the SARS-CoV-2 spike protein, while seroconversion could be verified only in 37% of rRT-PCR positive people with suspected infection. These results suggest that in these cases, the conclusions regarding the presence of the virus were erroneous28.

rRT-PCR performed on exposed healthcare workers in the Veneto region (Italy) also detected specific antibodies in 100% of severe clinical cases of COVID-19 but only in 83% of those with moderate symptoms . Asymptomatic subjects with positive rRT-PCR showed a significantly lower seroconversion rate (58%)29.

Positive rRT-PCR performed on samples from people in close contact with COVID-19 patients in Jiangsu, China, correlated with pathognomonic chest imaging in 50% of asymptomatic subjects, and none of the asymptomatic patients developed severe pneumonia or died. . On the other hand, in asymptomatic subjects with positive rRT-PCR, laboratory results were normal in 55% of cases and the decrease in circulating lymphocytes (one of the markers of viral infection) was observed only in 16%. Levels of C-reactive protein (a biological marker of inflammation) were slightly above the normal range and only in 14% of cases. The results of these studies suggest that a significant number of positive rRT-PCR in asymptomatic people were not indicative of SARS-CoV-2 infection. On this point, it is worth noting that, since the beginning of 2020, the Center for Disease Control and Prevention of the United States and other international health entities removed several kits from commerce due to the high rate of false results30 without even having added the risks of the sampling or processing of samples.

From the above it can be inferred that, for asymptomatic individuals in whom antibodies directed against SARS-CoV-2 are not detected, the interpretation of positive results from rRT-PCR tests requires extreme caution.

CONCLUSIONS

As a general rule, the preparation of nucleic acids (mainly by manual techniques) and the mixing of reagents for rRT-PCR is carried out under a vertical laminar flow hood or in the open air. In these procedures there is a potential risk of contamination between samples due to imperceptible splashes.

The equipment available on the market indicates the use of controls in each series of determinations without mentioning the number of negative tests that must be taken into account (most explicitly specify a positive control and a negative control)31. A negative and a positive control would have scientific support only if the systems process a single sample at a time. However, and in another context, the pharmacopoeia establishes that at least 10% of representative samples of each batch of 100 elements must be controlled to evaluate external contamination32. If this approach is extrapolated to the procedures for the detection of viral genomes, a minimum of 10% of samples should be processed (pairs of 10 negative controls adjacent to 10 positive controls) distributed in each series of 100 tests.

On the other hand, the location of the samples in the experimental detection plan can help determine possible risks of cross-contamination. If, for example, positive results are obtained for two adjacent samples, and if for one of them the Ct is greater than 30 or less than 25, the clinical signs and the context of viral transmission must be verified for both. If three positive signals with Ct greater than 32 are detected for a sample neighboring a strongly positive one, the test must be repeated.

Furthermore, subjects infected with respiratory viruses develop specific immune responses, so the detection of antibodies (a month or more after clinical signs) confirms the veracity of positive rRT-PCR results. Therefore, the absence of antibodies in immunocompetent people would invalidate the positive rRT-PCR results in many cases33, 34 (see Table 1).

Table 1 Conclusions from the comparison of positive rRT-PCR results for SARS-CoV-2 with laboratory and clinical parameters. 

To take these interventions into account, additional resources are required, since the cost of each determination increases by at least 20% for the reagents alone.

In conclusion, the detection of airborne infections requires accurate testing and adequate protective measures. However, results obtained by spurious methods create confusion.

Knowing that the techniques used can amplify the signal of a searched element more than 10 billion times, each step of diagnosis requires knowing and minimizing the risks of false results.

rRT-PCR are reference instruments and are not in themselves the exclusive proof of infection or risk to public health (especially positive results in asymptomatic unexposed people or repeated negative results in exposed people)35, 36.

False positives can alter the effectiveness of public health strategies and preventive measures. Additionally, fear of a falsely detected illness can lead to unnecessary treatments and increased social anxiety.

The results obtained by rRT-PCR for SARS-CoV-2 have put health personnel in front of dilemmas in decision-making. For this reason, it is a priority to identify and resolve technical difficulties since it is inferred that serious complications and deaths may have been wrongly attributed to SARS-COV-2, when in reality they were other conditions.

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EDITOR'S NOTE: The authors are solely responsible for the opinions expressed in their articles, which do not necessarily reflect the opinion of the RASP publishing institution. 

DECLARATION OF CONFLICT OF INTEREST: There was no conflict of Interest during the conduct of the study.

Received: November 24, 2020; Approved: December 10, 2020

*CORRESPONDING AUTHOR: pablogol@aol.com

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