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. 2022 May 13;17(5):e0268187. doi: 10.1371/journal.pone.0268187

Dealing with inconclusive SARS-CoV-2 PCR samples—Our experience

Zhivka Stoykova 1,2, Tsvetelina Kostadinova 2,3, Tatina Todorova 1,2,*, Denis Niyazi 1,4, Milena Bozhkova 1,4, Svetomira Bizheva 4, Temenuga Stoeva 1,4
Editor: Nei-yuan Hsiao5
PMCID: PMC9106147  PMID: 35560147

Abstract

Purpose

Early confirmation of SARS-CoV-2 is a key point in the timely management of infected patients and contact persons. Routine diagnostics of COVID-19 cases relies on RT-PCR detection of two or three unique sequences of the virus. A serious problem for the laboratories is how to interpret inconclusive samples which are positive for only one of the SARS-CoV-2 specific genes.

Materials and methods

A total of 16364 naso-oropharyngeal swabs were collected and tested with SARS-CoV-2 Real-TM kit (Sacace Biotechnologies, Italy) between May and September 2020. We retrospectively analyzed their amplification plots to determine the number of inconclusive samples. We also reviewed the medical records to summarize the patient’s COVID-19 testing history and basic demographic characteristics.

Results

We obtained 136 (0.8%) inconclusive samples with amplification signal only for the N-gene. Thirty-nine of the samples were excluded from further analysis as no additional data were available for them. Of the rest of the samples, the majority– 48% (95% CI 38–59%) had a previous history of SARS-CoV-2 positivity, 14% (95% CI 8–23%)–a subsequent history of positivity and 37% (95% CI 28–48%) were considered as false positive.

Conclusion

A substantial proportion of the inconclusive results should be considered as positive samples at the beginning or the end of the infection. However, the number of false-positive results is also significant and each patient’s result should be analyzed separately following the clinical symptoms and epidemiological data.

Introduction

Since the end of 2019, we have witnessed an unprecedented public health crisis [1, 2]. In less than six months, the new coronavirus disease (named COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has turned from an exotic and barely known infection to one of the most discussed and analyzed. The disease is still hardly identified because of the diverse clinical picture–symptoms (if present) can mimic other respiratory viral diseases and vary widely from totally asymptomatic cases to life-threatening pneumonia and respiratory failure [3, 4]. This, together with the aggressive transmission of the infection, makes the virological testing of suspicious individuals the most robust tool for precise COVID-19 management.

In the field of laboratory diagnosis, the real-time polymerase chain reaction (RT-PCR) of nasopharyngeal and/or oropharyngeal swabs has been rapidly accepted as the gold standard for accurate and timely control of the infection [5]. Thе technique has been considered decisive for both confirmation of symptomatic cases and screening of contact individuals, especially during the first months of the pandemic before the extensive introduction of SARS-CoV-2 antigen and antibody detection tools [6]. Despite the general agreement for the preferred testing method, there are still some controversies about result interpretation of the RT-PCR assays. The majority of commercially available kits rely on the detection of two, three (or even four) SARS-CoV-2 genes to reduce the risk of false-negative results. Preferred for their unique sequences are regions of the E, RdRp, N and ORF1A genes (for a review on available RT-PCR techniques see [7]). The question of how to interpret reactions with an amplification signal of one of the target genes is currently unclear. According to most of the guidelines and operation manuals, when only one specific SARS-CoV-2 gene is detected, especially at the end of the reaction (high cycle threshold (Ct) values) the result is inconclusive and the patient needs retesting [5, 6]. Thus, the test duration increases and the diagnostic decision is delayed. Moreover, the uncertain results pose questions about the sensitivity and specificity of the RT-PCR assay, as any conclusion for the status of the patient could be wrong and introduce additional risk.

The presence of samples positive for only one SARS-CoV-2 specific gene attracted our attention and we decided to analyze how frequent were inconclusive SARS-CoV-2 samples in the practice. We also tried to find the most feasible interpretation of such inconclusive results.

Materials and methods

This was a retrospective study of the SARS-CoV-2 RT-PCR results obtained between 01.05.2020 and 30.09.2020 in the Virology laboratory of St. Marina University Hospital, Varna, Bulgaria. As one of the first accredited SARS-CoV-2 laboratories in the country, it has been testing samples from most of the hospitals and regional Health Inspectorates in North-East Bulgaria. Between March and December 2020, it was the principal COVID-19 laboratory for the whole territory of North-East Bulgaria serving a total population of around 1351621 (according to the National Statistical Institute of Bulgaria, 2020 estimation). We were routinely testing samples from hospitalized patients and health care workers that showed respiratory infection symptoms, as well as from ambulatory individuals who were contact persons of verified COVID-19 cases. Asymptomatic no-contact persons who needed RT-PCR result for travelling or working purposes were not investigated.

For this study, we analyzed the frequency of inconclusive SARS-CoV-2 samples during the first wave of COVID-19 in Bulgaria (from May 2020 to September 2020) [8]. During this period, the initial criteria of WHO for releasing COVID-19 patients from isolation [9] were followed in the country (see also Order № РД-01-371/30.06.2020 of Bulgarian Ministry of Health). To discharge people from isolation, the protocol required two negative RT-PCR results on samples taken at least 24 hours apart. As a result, positive individuals accumulated several PCR results and we were able to analyze the “history of testing” and duration of positivity for most of them. With the pandemic expansion, the initial recommendation has become extremely difficult to follow and it was changed (Order № РД-01-604/13.10.2020 of Ministry of Health)–since October 2020 most of the suspicious patients were tested only once.

During the above-mentioned period, a total of 16364 naso-oropharyngeal swabs were collected from hospitalized, out-of-hospital patients or contact individuals and transported to the laboratory in saline water or viral transportation medium.

RNA extraction was performed with SaMag Viral Nucleic Acid Extraction Kit using SaMag-12 instrument (Sacace Biotechnologies, Italy). The initial extraction volume was 400 μL and the elution volume– 50 μL. Isolated RNAs were amplified with SARS-CoV-2 Real-TM kit (Sacace Biotechnologies, Italy). This test allows the reverse transcription and amplification to be performed in a single, one-step reaction and detects three genes: a region of the E gene common for all SARS-like coronaviruses (Fam channel), E and N genes specific for SARS-CoV-2 (Rox and Cy5 channels). To exclude inhibition of RNA extraction and amplification, an internal control RNA is also amplified and detected in the Hex channel. The sample is considered as SARS-CoV-2 positive if there is an amplification signal with defined Ct value for SARS-like coronaviruses and E- and (or) N-genes of SARS-CoV-2; positive for other coronaviruses if amplification is detected only in the Fam channel and inconclusive if there is an amplification signal for only one of the specific SARS-CoV-2 genes. When this was the case, a new sample was requested from the corresponding clinical unit or health inspectorate and the test was repeated in 48 hours. Samples without identified fluorescence signals in all channels, including that for the internal control, were considered invalid and the test was repeated starting from the extraction step. The quality of each RT-PCR assay was confirmed by valid positive and negative controls. Sacace Biotechnologies sets the analytical sensitivity of the assay to 500 copies viral RNA/ml and the diagnostic sensitivity and specificity to 100%.

All data were retrospectively analyzed using the assigned laboratory number for each patient and no additional sampling or patient intervention was performed. Only basic demographic data (age, gender and hospitalization status) were retrieved from the laboratory information system and examined for each patient because of the heterogeneity of the samples–they were obtained from different hospitals and regional health inspectorates in North-East Bulgaria, and clinical data were often incomplete. No sensitive personal data (names, ethnicity, disease outcome, etc.) were used.

Descriptive statistics–Kruskal-Wallis analysis of variance and Mann-Whitney U test for independent measures, and Chi-square test for categorical variables–were the preferred statistical methods with a significance level of 0.05. To test the possible association between the overall positivity rates and the number of inconclusive samples, the Pearson correlation coefficients were calculated. The statistical program used was the R project for statistical computing (version 4.0.4/2021-02-15).

Ethical approval was obtained from the Ethics Committee of the Medical University Varna (Protocol Approval Number 114).

Results

For the period between May 2020 and September 2020, we tested 16364 samples collected from COVID-19 suspicious individuals or contact persons. The proportion of positive results with N and E SARS CoV-2 specific genes amplified was 12.6% (2054 out of 16364). A total of 136 naso-oropharyngeal swabs (0.8%) had shown a positive amplification signal only for the N gene with an average Ct of 32.8 (range 27.7–38.2). We did not detect samples with isolated amplification of the E gene.

Fig 1 shows the dynamics of the PCR testing and the change in the positive rate during the studied period. The number of inconclusive samples correlated well with both the total number of tested samples (Pearson correlation coefficient of 0.64, p = 0.001) and the rate of SARS-CoV-2 positivity (Pearson correlation coefficient of 0.80, p < 0.001).

Fig 1. Weekly distribution of SARS-CoV-2 positive and inconclusive samples between May and September 2020.

Fig 1

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The blue line represents the total number of tested samples (left vertical axis); the green line–the positive cases (left vertical axis), and the purple bars–the number of inconclusive SARS-CoV-2 samples (right vertical axis).

A short description of the demographic characteristics of the groups of patients with the positive, negative and inconclusive results is shown in Table 1. The sex ratio, the proportion of hospitalized patients and the age distribution were similar between the groups of patients with positive and inconclusive samples: 56% females (95% CI 54 to 58%) vs. 57% females (95% CI 49 to 66%); 65% hospitalized patients (95% CI 63 to 67%) vs. 61% (95% CI 52 to 69%) and mean age of 53 years (95% CI 52 to 54) vs. 54 years (95% CI 51 to 57), respectively.

Table 1. Basic demographic characteristics of individuals with positive, negative and inconclusive SARS-CoV-2 RT-PCR results (01.05–30.09.2020).

Positive samples (N = 2054) Negative samples (N = 14174) Inconclusive samples (N = 136)
Sex
Female 1146 (56%) 9127 (64%) 78 (57%)
Male 908 (44%) 5047 (36%) 58 (43%)
Age
Mean±SD 53±20 51±19 54±20
Median 56 53 55
Hospitalization
Yes 1332 (65%) 7750 (55%) 83 (61%)
No 722 (35%) 6424 (45%) 53 (39%)
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The analysis of the inconclusive samples (Fig 2) showed that for 39 patients, the RT-PCR test with the inconclusive result was the only one recorded in the database and we excluded them from further analysis. For the rest 97 patients, at least one additional sample was obtained and analyzed after the inconclusive result. For 36 of these patients, no history of positive tests or hospital admission existed and the testing of the new sample did not show any amplification–we considered them as samples with false-positive signal for the N gene. Among the other 61 patients, 47 had one or several previous positive results, while 14 –one or several subsequent positive results. The inconclusive result was the very last or first in their SARS-CoV-2 positive testing history. Only two samples were associated with patients with both previous and subsequent positive tests. As the previous positivity was significantly longer than the positivity after the inconclusive result (13 versus 7 and 8 versus four days, respectively, see also S2 Table), we considered them into the group of patients with previous positive results.

Fig 2. Patients’ flowchart.

Fig 2

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The interval between the first positive sample and the inconclusive result varied widely–from 6 to 38 days with an average of 14 days. A large timeframe was also detected for the individuals who had an inconclusive result and then one or more positive results–subsequent positivity lagged from 1 to 29 days with an average of 11 days. The Mann-Whitney U test revealed that the difference between a randomly selected value of the group with the previous positivity history and the group with subsequent positivity was not big enough to be statistically significant (p = 0.17).

The average Ct detected among the samples with previous SARS-CoV-2 positivity was 32.7±1.8; among the samples with subsequent positivity– 32.9±1.5 and among the samples with presumably false positive signal– 32.9±1.7 (p = 0.63).

Discussion

From May to September 2020 (which roughly covers the first wave of COVID-19 in Bulgaria) [8] we detected around 1% of inconclusive RT-PCR tests with amplification of only one of the two SARS CoV-2 specific genes. The proportion of single gene-positive samples differs widely across the scarce literature data with a prevalence rate between 0.3% [10] to 21% [11]. In a study from the Republic of Korea, conducted also in the first months of the pandemic, Lim et al. obtained similar to our results number of inconclusive samples of around 1% [12]. However, comparing the results among the studies is challenging because a wide variety of test systems is currently in use and laboratories are working with different patients groups.

We also tried to clarify the clinical importance of samples positive for one SARS CoV-2 specific gene–if such amplification indicated very high cycle thresholds or was a false-positive result. Our experience tips the balance towards the former interpretation– 48% of the inconclusive samples for the period of the study were obtained from patients who previously had shown positivity for SARS-CoV-2 and 14%—from patients with subsequent positivity. Viral loads are lower before the onset of the symptoms and at the end of the infection [13, 14]. Therefore, the detection of only one of the SARS-CoV-2 specific genes could result from the natural decrease in the amount of SARS-CoV-2 RNA in the course of infection kinetics.

An additional interesting point that merits discussion was that all of the inconclusive results we had obtained were with amplification of the N-gene. This is not surprising as the N protein and the corresponding mRNA are the most abundant in the replication cycle of coronaviruses [15, 16]. Prolonged SARS-CoV-2 positivity has been widely reported in the literature [17–19], as well as positivity before the onset of the clinical symptoms [20]. We presume to speculate that the N-gene was the first and the last to be expressed in the infected cells. The remnant N gene subgenomic RNA, which are abundant in inconclusive samples [12, 21], could be partially amplified in RT-PCR assays causing weak positive signals. The different analytical sensitivity of the RT-PCR assay for the individual genes could also play a role–each primer-probe set has a specific target and even single-point mutations could lead to amplification failure of the corresponding gene [22, 23]. Most likely, a large proportion of the inconclusive results should be considered as positive samples with a high cycle threshold or E gene target failure.

Nevertheless, around 37% of the inconclusive samples were classified as false-positive. Causes for the false positivity could be found in the non-specific binding during the late phases of the PCR reaction [24]–an assumption that is supported by the relatively high Ct values obtained. An additional source for inconclusive results could be contamination of the samples from the other highly positive samples in the same assay. A strong correlation between the rates of inconclusive and positive results existed, and we should admit that during the first months of the pandemic, the burden for rapid and accurate testing was enormous. However, both inconclusive and positive detection rates depended on the total number of tested samples. If the contamination is a real reason for the majority of the uncertain results, someone should expect to have inconclusive samples with the E-gene signal too, which wasn’t the case. Inconclusive samples were also detected in weeks when positive samples were not found (Fig 1 and S1 Table).

Finally, pre-analytical factors could also lead to an inconclusive result in an infected person: poor quality of the collected sample (too little material or inappropriate sampling); poor handling and shipment of the specimen; or technical issues, such as inhibition during the assay are well-recognized reasons for an unclear testing conclusion. Pre-analytical causes could explain why inconclusive results were obtained in two cases with previous and subsequent positivity.

To the best of our knowledge, the current work is the sole analysis of this kind in Eastern Europe and one of the rare studies trying to resolve an actual practical issue that many COVID-19 diagnostic laboratories face. However, the study has some limitations, among which the major are its retrospective nature and the short-term period of analysis. We evaluated just the first COVID-19 wave when multiple results were available for most of the positive individuals. In this way, we could not validate our data for all of the SARS-CoV-2 waves and variants detected later. In addition, the number of inconclusive samples stood for less than 1% of all tested samples, and this made the statistical comparison between the groups uncertain. A significant number of the inconclusive samples were also excluded from further analysis because no additional information was available. This definitely could impact the final results and indicates possible gaps in the management of COVID-19 cases.

Conclusion

The present study enlightens one of the major problems in the current laboratory identification of SARS-CoV-2 –the inconclusive RT-PCR results received in a time of significant pressure towards the laboratories for rapid and correct diagnostic. In most of the clinical units, algorithms on how to interpret such samples are still missing. Based on our experience, we recommend when it is not possible to repeat the test, to consider inconclusive samples as positive, especially when clinical symptoms are present.

Supporting information

S1 Table. List of samples tested for SARS-CoV-2 RNA between 01.05.2020 and 30.09.2020 in the Virology laboratory of St. Marina University Hospital, Varna, Bulgaria.

To ensure fully anonymization the laboratory number of each sample was replaced with an arbitrary given number. Sheet 1 contains the raw data and Sheet2 the weekly summary of the same data.

(XLSX)

Click here for additional data file. (472.1KB, xlsx)
S2 Table. List of inconclusive samples obtained between 01.05.2020 and 30.09.2020 in the Virology laboratory of St. Marina University Hospital, Varna, Bulgaria.

To ensure fully anonymization, the laboratory number of each sample was replaced with an arbitrary given number.

(XLSX)

Click here for additional data file. (13.8KB, xlsx)

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Nei-yuan Hsiao

4 Feb 2022

PONE-D-21-31632Dealing with inconclusive SARS-CoV-2 PCR samples – our experiencePLOS ONE

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In a retrospective laboratory-based study Stoykova et al. describe inconclusive results from the St. Marina University Hospital, Varna, Bulgaria. 136 of 16364 samples tested inconclusive. These results were further investigated against other tests from the same patients, of these 39 had no additional tests, 47 had prior positive tests, 14 subsequent positive tests and 39 subsequent negative tests.

The authors conclude that most inconclusive results are true positive and should be considered as positive, especially when symptoms are present. The manuscript is of importance to the field and is generally well-written and easy to follow. Tables and figures are appropriate.

I have some comments for improvement:

1. Please provide more detail about the laboratory: Is this a public health or private health laboratory? What population does it serve?

2. The difference in patient characteristics were described as” between the groups of positive and negative samples.” It should however be noted that far fewer inconclusive cases were included which would mean that the analysis might be underpowered when comparing inconclusive with other categories. Line 105 and 106 appear to be a table legend – please indicate this clearly.

3. Please elaborate on the causes of single gene positive inconclusive results including pre-analytical and analytical causes – such as true low viral loads, inhibition, sample contamination, primer mismatches etc. and in the discussion of how to interpret inconclusive results. Please also consider pre-analytic factors that might change over the course of the epidemic such whether patients are seen during an epidemic wave upswing or thereafter, when many more patients may have residual low viral loads during the recovery phase.

4. Please provide data about the time-difference between the initial inconclusive and subsequent tests (for cases that tested positive vs negative on subsequent samples) and in the limitations please consider to discuss that samples that initially tested inconclusive and subsequently negative might have become negative due to the natural kinetics of SARS-CoV-2 RNA in infected patients, with true low viral loads declining to becoming undetectable over time.

Language:

Line 19: Routine diagnostic – replace with “Routine diagnostics”

Line 131: “scare” meaning is unclear; please replace with the correct term, perhaps “scarce”

Reviewer #2: 1. This is an important topic that requires study. There are various different real time PCR assays for SARS-CoV-2 and it has been challenging interpreting and dealing with inconclusive results.

2. Abstract:

Line 20: “detection of at least two unique sequences of the virus” is not entirely accurate, rather use two SARS-CoV-2 specific gene targets or three gene targets.

In results, lines 31-33, the absolute values are stated, it would be preferable to use the percentage with 95% confidence intervals.

It is also noted that a large number of inconclusive results were excluded from the analysis due to lack of data, this could have impacted on results.

3. Keywords line 41-42: viral load is not suitable as a keyword as SARS-CoV-2 PCR is primarily a qualitative test and not reported as a viral load.

4. Introduction: Noted to be quite a general introduction with few references.

Lines 54-57 refers to guidelines and operation manuals but this is not referenced.

Line 58 – Provide clarification as to how detection of single gene targets poses questions on the sensitivity of the RT-PCR assay. Specificity can also be affected as some of these can be false positive.

5. Methods:

Line 65 – why was the timeframe May-September 2020 chosen? Was this during a peak period of COVID-19 or between the waves as this can also impact on testing and subsequent results eg. many samples with high viral loads during the peak can cause cross contamination and false positive single N gene target detection. It is only subsequently noted in the discussion that this period roughly covers the first wave of COVID-19 in Bulgaria (lines 128-129). The PCR positivity rate during this period was noted to be 12.6% (line 92). What was the case definition for testing during this period? – did it also include testing of asymptomatic exposed cases, screening of all hospitalized patients regardless of exposure or symptoms.

Line 86-87 – I am not sure if https://www.socscistatistics.com/ is suitable or acceptable to use for statistical analysis in biomedical studies, however the website indicates that it has been audited for accuracy against the output produced by a number of established statistics packages, including SPSS. Most published studies use established statistical softwares such as STATA, SPSS, etc.

Lines 92-95 – describes the result distribution of the PCR assay. It is also interesting that there were no invalid results with internal control failure. In my experience, beside the issue of inconclusive results, the problem with invalid results has been challenging.

Line 27-28 & 84-85 - Was the demographic data taken from electronic medical records or patient files or from a laboratory information system as there is minimal clinical data.

No ethics application or clearance is noted - Even though the study was a retrospective analysis of laboratory data, ethics approval is still required from the relevant institution as patient data is being used and for publication purposes.

6. Results:

Lines 112-113 – the interval/timeframe between the inconclusive and positive test results would also be useful to be indicated to give an understanding how long and when in the course of infection single N gene positivity can occur.

Line 148 – “Causes for these false-positivity could be found in the different analytical sensitivity of the RT-PCR assay for the individual genes” – please clarify how this can cause false positivity. The analytical sensitivity/limit of detection of the RT-PCR assay used is not mentioned.

Line 150 – what were the Ct values obtained for 37% of inconclusive results that were false positive in comparison to the true positive results (48% of the inconclusive samples from patients who previously had shown positivity for SARS-CoV-2 and 14% from patients with subsequent positivity) and was there a significant difference? A graphical comparison would be useful.

7. Discussion: Noted to be brief.

There is no mention of other factors that can cause inconclusive results such as pre-analytical factors eg. specimen type, quality, handling & storage conditions.

It would be helpful with the interpretation of results to know the background history such as if patient symptomatic or not, onset of symptoms in relation to testing, contact of confirmed case, etc. Several published studies on this topic have reported both clinical and epidemiologic data. This is lacking in this study.

The limitations of the study are not mentioned which is required.

**********

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Reviewer #1: Yes: Gert U. van Zyl

Reviewer #2: Yes: Aabida Khan

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 May 13;17(5):e0268187. doi: 10.1371/journal.pone.0268187.r002

Author response to Decision Letter 0

21 Mar 2022

Dear Editor,

Dear Reviewers,

We are grateful for the positive feedback and the possibility to revise our work. The academic editor and reviewers raised many interesting points and we hope that any concerns have been suitably addressed in the revised version and this letter. The responses to the specific commentaries and criticisms are as follows:

Editor Comments:

Please pay careful attention to the reviewers's comment both regarding contextualising the study and accuracy of the terminologies used. In particular, in both the methods and discussion, highlight the population being tested and provide an account of the change in positivity rate of PCR during the study period. Indicate whether positivity rate affect the number and likely cause of inconclusive result and suggest potential solution/interpretation to this changing trends.

We agree with the editor that all these features are crucial for the improvement of the manuscript – accordingly, we have strengthened different parts of the work to reflect these concerns. The population of interest is now described in the Materials and methods, and the weekly change in the rates of testing and positivity are present in the Results (Fig 1).

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

The style requirements were carefully checked and observed in the new version of the manuscript.

2. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available.

The study’s minimal underlying data set is uploaded as Supporting Information files. The Supporting files consist of two tables: S1_Table, which contains the raw data of all samples tested in the studied period and S2_Table, which contains the data associated with the inconclusive RT-PCR samples. All results were retrospectively analyzed using the assigned laboratory number for each patient and no sensitive personal data were used. To ensure fully anonymization, the laboratory number of each sample was replaced with an arbitrary given number before the upload of the data.

3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

We included this phrase in the previous version of the manuscript because the information was not considered to be crucial for the main conclusions of the work. Now, the phrase was removed from the revised version of the paper and the average Ct of the different groups of samples are given in the last paragraph of the Result Section.

4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

The reference list is complete and follows the style of the Journal. Because of the necessary enlargement of Introduction and Discussion Sections (as suggested by the reviewers), 11 new references were included in this Section.

Reviewer 1:

We thank the reviewer for the generally positive reaction to the paper.

1. Please provide more detail about the laboratory: Is this a public health or private health laboratory? What population does it serve?

We have provided more information about the laboratory. The first paragraph of the Materials and methods Section is now improved with details about the region and the population covered by the laboratory.

2. The difference in patient characteristics were described as” between the groups of positive and negative samples.” It should however be noted that far fewer inconclusive cases were included which would mean that the analysis might be underpowered when comparing inconclusive with other categories.

We realize that the number of the inconclusive samples is significantly lower than the negative and positive results. However, this is the only way to compare their characteristics in a statistically sound manner. In the Discussion of the revised version, this was indicated as a limitation of the study.

Line 105 and 106 appear to be a table legend – please indicate this clearly.

The Table legend is now separated from the main body of the text.

3. Please elaborate on the causes of single gene positive inconclusive results including pre-analytical and analytical causes – such as true low viral loads, inhibition, sample contamination, primer mismatches etc. and in the discussion of how to interpret inconclusive results. Please also consider pre-analytic factors that might change over the course of the epidemic such whether patients are seen during an epidemic wave upswing or thereafter, when many more patients may have residual low viral loads during the recovery phase.

To address this concern, causes for inconclusive results are now discussed in the Discussion. This section of the manuscript was entirely rewritten and amended with emphasis on the possible reasons for single gene amplification.

4. Please provide data about the time-difference between the initial inconclusive and subsequent tests (for cases that tested positive vs negative on subsequent samples) and in the limitations please consider to discuss that samples that initially tested inconclusive and subsequently negative might have become negative due to the natural kinetics of SARS-CoV-2 RNA in infected patients, with true low viral loads declining to becoming undetectable over time.

This suggestion is of particular importance and we included in the new version the time from the first positive result to the inconclusive one and between the initial inconclusive and the last positive sample. The natural kinetics of SARS-CoV-2 infection is also discussed.

Line 19: Routine diagnostic – replace with “Routine diagnostics”

Line 131: “scare” meaning is unclear; please replace with the correct term, perhaps “scarce”

The language is now corrected.

Reviewer #2:

1. This is an important topic that requires study. There are various different real time PCR assays for SARS-CoV-2 and it has been challenging interpreting and dealing with inconclusive results.

We acknowledge the positive feedback and the detailed comments of the reviewer.

2. Abstract:

Line 20: “detection of at least two unique sequences of the virus” is not entirely accurate, rather use two SARS-CoV-2 specific gene targets or three gene targets.

In results, lines 31-33, the absolute values are stated, it would be preferable to use the percentage with 95% confidence intervals.

We agree with the reviewer and the Abstract was changed accordingly.

It is also noted that a large number of inconclusive results were excluded from the analysis due to lack of data, this could have impacted on results.

The concern about the significant number of excluded results is discussed as one of the limitations of the study.

3. Keywords line 41-42: viral load is not suitable as a keyword as SARS-CoV-2 PCR is primarily a qualitative test and not reported as a viral load.

We admit that viral load is not the best choice for a keyword and we had dropped it from the list of the keywords.

4. Introduction: Noted to be quite a general introduction with few references.

We realize that the previous version suffered from a short Introduction and Discussion Section. In the new version, new paragraphs with additional background information were added to make the text more ‘reader-friendly’ and complete.

Lines 54-57 refers to guidelines and operation manuals but this is not referenced.

References are now added.

Line 58 – Provide clarification as to how detection of single gene targets poses questions on the sensitivity of the RT-PCR assay. Specificity can also be affected as some of these can be false positive.

The sentence ‘Moreover, this poses questions about the sensitivity of the RT-PCR assay.’ is now changed to “Moreover, the uncertain results pose questions about the sensitivity and specificity of the RT-PCR assay, as any conclusion for the status of the patient could be wrong and introduce additional risk.”

5. Methods:

Line 65 – why was the timeframe May-September 2020 chosen? Was this during a peak period of COVID-19 or between the waves as this can also impact on testing and subsequent results eg. many samples with high viral loads during the peak can cause cross contamination and false positive single N gene target detection. It is only subsequently noted in the discussion that this period roughly covers the first wave of COVID-19 in Bulgaria (lines 128-129). The PCR positivity rate during this period was noted to be 12.6% (line 92). What was the case definition for testing during this period? – did it also include testing of asymptomatic exposed cases, screening of all hospitalized patients regardless of exposure or symptoms.

The second paragraph of the Materials and methods section now contains more information on why this period was chosen for analysis. We also clarified the groups of tested patients and the region served by the laboratory.

Line 86-87 – I am not sure if https://www.socscistatistics.com/ is suitable or acceptable to use for statistical analysis in biomedical studies, however the website indicates that it has been audited for accuracy against the output produced by a number of established statistics packages, including SPSS. Most published studies use established statistical softwares such as STATA, SPSS, etc.

Although the listed online calculator is a really good (and free) statistical tool, and the performed statistical tests are quite simple to generate serious inconsistency, we agree that a more rigorous program should be used in science. The same analyses were redone with the R project for statistical computing (version 4.0.4/2021-02-15) software. This led to negligible differences in the obtained results.

Lines 92-95 – describes the result distribution of the PCR assay. It is also interesting that there were no invalid results with internal control failure. In my experience, beside the issue of inconclusive results, the problem with invalid results has been challenging.

We admit that more clarification is needed about our choice to discuss only valid RT-PCR results. The issue of invalid samples is significant for the laboratory practice, but in our experience, it is more related to technical problems during the assay – we have noticed that invalid results are not equally distributed in the time and correlate with the kit lot used for either extraction or amplification. Additionally, the Bulgarian National COVID-19 information system does not allow invalid results to be uploaded (only three options are available – positive, negative or inconclusive result). This leads to completely different management of invalid samples and if such a result is obtained the sample is repeatedly processed starting from the extraction step. Only in case, when the second or the third extraction and amplification are unsuccessful, a new sample is requested from the corresponding clinical or out-of-hospital unit. The new sample has been processed and registered under the laboratory number of the original sample, which means that it is almost impossible to perform correct retrospective analysis for the number of invalid results.

Line 27-28 & 84-85 - Was the demographic data taken from electronic medical records or patient files or from a laboratory information system as there is minimal clinical data.

The demographic data were retrieved from the laboratory information system. In the new Materials and methods section the reasons for the minimal clinical data are now explained – during these first months of the pandemic we have been receiving samples from approximately 20 different hospitals and health inspectorates often with incomplete patient data.

No ethics application or clearance is noted - Even though the study was a retrospective analysis of laboratory data, ethics approval is still required from the relevant institution as patient data is being used and for publication purposes.

As the study is fully retrospective and all data were retrieved from standard-of-care tests without additional sampling, it was not considered as ethically controversial. However, we admit that ethic approval is necessary for publication of the results and accordingly the manuscript has been submitted to the institutional ethic committee and approved for publication (Protocol Approval Number 114 by the Ethics Committee of the Medical University Varna). The last paragraph of the Materials and methods presents the ethical approval.

6. Results:

Lines 112-113 – the interval/timeframe between the inconclusive and positive test results would also be useful to be indicated to give an understanding how long and when in the course of infection single N gene positivity can occur.

As suggested, a new paragraph discussing the time between the first positive or the last positive result and the inconclusive result is added to the Result section.

Line 148 – “Causes for these false-positivity could be found in the different analytical sensitivity of the RT-PCR assay for the individual genes” – please clarify how this can cause false positivity. The analytical sensitivity/limit of detection of the RT-PCR assay used is not mentioned.

Causes for the false positivity are now more elaborated in the Discussion section. The analytical sensitivity of the RT-PCR kit used is included at the end of the Materials and methods section.

Line 150 – what were the Ct values obtained for 37% of inconclusive results that were false positive in comparison to the true positive results (48% of the inconclusive samples from patients who previously had shown positivity for SARS-CoV-2 and 14% from patients with subsequent positivity) and was there a significant difference? A graphical comparison would be useful.

The Ct values were also added to the main text for the three groups of interest. However, because there was no any significant difference we consider graphical presentation unnecessary.

7. Discussion: Noted to be brief.

There is no mention of other factors that can cause inconclusive results such as pre-analytical factors eg. specimen type, quality, handling & storage conditions.

The Discussion has been completely rewritten and all these causes suggested by the Reviewer are now part of it.

It would be helpful with the interpretation of results to know the background history such as if patient symptomatic or not, onset of symptoms in relation to testing, contact of confirmed case, etc. Several published studies on this topic have reported both clinical and epidemiologic data. This is lacking in this study.

We agree that adding the background history of the patients would improve the quality of our work. Unfortunately, clinical units and regional health inspectorates in Bulgaria have not been obliged to submit the patient’s clinical background to the laboratory which has been tested the corresponding sample. We were unable to collect the complete clinical information and we decided to focus only on the basic demographic characteristics. Please, refer also to the response to the comment about the minimal demographic data of the patients.

The limitations of the study are not mentioned which is required.

We admit that in the previous version of the manuscript we have not focused in detail on the limitations of the study. To address this concern, they are now included at the end of the manuscript.

Once again thank you for your comments and suggestions, which we consider as a significant improvement of our work.

Sincerely yours,

Tatina Todorova

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (26.5KB, docx)

Decision Letter 1

Nei-yuan Hsiao

25 Apr 2022

Dealing with inconclusive SARS-CoV-2 PCR samples – our experience

PONE-D-21-31632R1

Dear Dr. Тодорова,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Nei-yuan Hsiao

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for amending the manuscript which is now accepted for publication. I suggest minor amendments of:

1) remove the p value column from table 1. If you wish to discuss the difference in population (in text) it is better to use confidence intervals and compare mainly positive vs inconclusive.

2) removing (low) "viral load" and replace them with (high) cycle threshold as assay used is not quantitative. Although Ct is a proxy to viral load there are situations where the two may differ and thus it is better to use a more precise language.

Reviewers' comments:

Acceptance letter

Nei-yuan Hsiao

5 May 2022

PONE-D-21-31632R1

Dealing with inconclusive SARS-CoV-2 PCR samples – our experience

Dear Dr. Todorova:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

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on behalf of

Dr. Nei-yuan Hsiao

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. List of samples tested for SARS-CoV-2 RNA between 01.05.2020 and 30.09.2020 in the Virology laboratory of St. Marina University Hospital, Varna, Bulgaria.

    To ensure fully anonymization the laboratory number of each sample was replaced with an arbitrary given number. Sheet 1 contains the raw data and Sheet2 the weekly summary of the same data.

    (XLSX)

    Click here for additional data file. (472.1KB, xlsx)
    S2 Table. List of inconclusive samples obtained between 01.05.2020 and 30.09.2020 in the Virology laboratory of St. Marina University Hospital, Varna, Bulgaria.

    To ensure fully anonymization, the laboratory number of each sample was replaced with an arbitrary given number.

    (XLSX)

    Click here for additional data file. (13.8KB, xlsx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (26.5KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

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