False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®). rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Cq between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.80% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.


Introduction
The first confirmed case of Coronavirus disease 2019  in Latin America occurred in Brazil, on February 25, 2020 (1) . Since then, until August 2020, Brazil has recorded about 4.1 million cases and about 126 thousand deaths due to COVID-19 (2) .
Early detection of infected individuals with large-scale testing, immediate isolation of cases with tracking and preventive self-isolation of close contacts, and prompt treatment of severe cases are essential measures to reduce the spread of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (3) . Therefore, to quickly diagnose infections and mitigate transmission of SARS-CoV-2, the Real-Time Reverse Transcription Polymerase Chain Reaction (rRT-PCR) is being used as the primary method in research and hospital laboratories to identify the virus in respiratory samples such as sputum or nasal, throat, nasopharyngeal swabs (4) .
rRT-PCR tests typically take 4 to 6 hours to complete, with RNA extraction, amplification, and detection (5) . Considering the limited supply of extraction reagents and test kits worldwide, extraction kits without RNA purification aim to solve this limitation and short the extraction time, thereby shortening the response time (4,5) .
However, amplification inhibitors, organic and inorganic substances, can be present in original samples or be introduced in samples transportation, processing, or RNA extraction, causing partial amplification inhibition, leading to a decrease of PCR sensitivity or total inhibition and consequently false-negative results (6) .
Extraction kits without RNA purification may need to optimized the rRT-PCR by sample dilution when there are problems with rRT-PCR amplification, thus minimizing the presence of amplification inhibitors (7) , allowing amplification even if there are inhibitors or some sample degradation, avoiding the need to request a new sample from the patient, however, it is necessary to know the diluted amplification patterns, avoiding false-negative diagnosis.
Due to the severity of the pandemic, test kits were and are being developed and approved quickly, to meet the worldwide demand for large-scale tests, creating the need for information on real data on the use of these kits in diagnostic laboratories (8) . Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence.
The results were evaluated by the rRT-PCR amplification standards, amplification values, and submitted to descriptive analysis, normality test and variance analysis (ANOVA), to detect differences between the results before and after dilution.

Efficiency of the rRT-PCR
The analytical efficiencies for detecting SARS-CoV-2 from the normal methodology and after dilution were performed by serial dilution in the following proportions: 1, 1:2, 1:4, 1:8, and 1:10. The results were evaluated by scatter plots and the efficiency values calculated from the R² of the linear regression.

Results
The results are described in Table 1

Discussion
The positive control showed amplification for the 3 targets evaluated in all tests (Cq ≤ 35) and the negative control did not show any amplification for the three evaluated targets, according to the mix manufacturer's protocol, validating the results.
The tests performed to demonstrate 1:2 dilutions were interesting to obtain a reliable amplification in samples with inhibitors, as shown in Figure 2. However, it is important to pay attention to the result curve patterns after dilution. In the example shown in figure 2.a, the sample without dilution did not obtain amplification of the internal control (Cq = undetermined value) and OF patterns (Cq = undetermined value), and NG (Cq The importance of performing rRT-PCR in kits that provide internal control marking has already been reported by Kim et al. (10) , generating conclusive results about the extraction process, avoiding the release of false-negative results in samples that were not amplified with precision since the interpretation of results is not always straightforward. The sensitivity of the rRT-PCR is negatively impacted by compounds present in the clinical sample that may partially or completely inhibit rRT-PCR chemistries (11)(12)(13)(14)(15) .
Protocols with purification steps can avoid the presence of amplification inhibitors, removing potential endogenous rRT-PCR inhibitors such as detergents, chelating compounds, and guanidinium HCl (11,13,(16)(17)(18)(19) . The efficiency of removing inhibitors in patient samples may be related to the intrinsic properties of the method used to extract the RNA (20) , which does not happen in the kit used in this study.
The Easy Extract™ kit does not completely purify the inhibitor compounds, which significantly reduces the extraction time, however, non-amplification by inhibitors may occur.
A diagnostic error can lead infected patients to non-COVID-19 areas with the subsequent risk of infection for others; or patients which are negative SARS-CoV-2 sent to COVID-19 areas (21) , generating possible contamination to uninfected patients and also the spread of viruses in the disinfected areas, which can lead to viral spread within hospitals and treatment centers, and contaminate the health workers. Knowing the rRT-PCR standards of diluted samples can help in the identification of falsenegative cases, and consequently avoid a wrong diagnosis.

Conclusion
The 1:2 dilution of the sample with inhibitors in UltraPure® H2O RNAse free generated amplification in 100% of the tested cases, being an alternative to avoid new sample collection in the patient. However, we emphasize that in this study we obtained 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.8% for detecting SARS-CoV-2. It is possible to infer that the dilution helps in cases where a new sample collection is not feasible, but caution is needed in the evaluation of the result of the rRT-PCR.
It is important to assess the pattern of the amplification curves after dilution to avoid inaccurate diagnosis. If the sample with inhibitors is positive with a high viral load, the result will be reliable if Internal Control and Gene N amplification occur up to Cq 30 and ORF-1ab up to Cq 35. In the case of non-amplification of the N Gene and ORF-1ab curve after dilution, we recommend assessing the need for a new sample and new analysis.
The authors declare no conflict of interest.