Journal of The Academy of Clinical Microbiologists

SHORT COMMUNICATION
Year
: 2020  |  Volume : 22  |  Issue : 1  |  Page : 59--61

Inconclusive SARS-COV-2 reverse transcription-polymerase chain reaction test reports: Interpretation, clinical and infection control implications


Sanjay Bhattacharya, Anju Vidyadharan, Vinitha Mary Joy 
 Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India

Correspondence Address:
Dr. Sanjay Bhattacharya
Consultant in Microbiology, Department of Microbiology, Tata Medical Center, Kolkata, West Bengal
India

Abstract

Inconclusive SARS CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) reports for the detection of infection in symptomatic patients or during screening of asymptomatic contacts can cause clinical, diagnostic and infection control uncertainty. It has been noted that up to 5% of COVID RT-PCR reports may be inconclusive. The reasons for these inconclusive reports are varied and may be classified into virological causes, sample collection and sample quality-related issues and finally technical issues related primarily due to problems in RNA extraction. The objective of this article is to discuss these causes and suggest corrective measures.



How to cite this article:
Bhattacharya S, Vidyadharan A, Joy VM. Inconclusive SARS-COV-2 reverse transcription-polymerase chain reaction test reports: Interpretation, clinical and infection control implications.J Acad Clin Microbiol 2020;22:59-61


How to cite this URL:
Bhattacharya S, Vidyadharan A, Joy VM. Inconclusive SARS-COV-2 reverse transcription-polymerase chain reaction test reports: Interpretation, clinical and infection control implications. J Acad Clin Microbiol [serial online] 2020 [cited 2023 Feb 9 ];22:59-61
Available from: https://www.jacmjournal.org/text.asp?2020/22/1/59/291889


Full Text



 Introduction



Inconclusive SARS CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) reports for the detection of infection in symptomatic patients or during screening of asymptomatic contacts can cause clinical, diagnostic and infection control uncertainty. It has been noted that up to 5% of COVID RT-PCR reports may be inconclusive. The reasons for these inconclusive reports are varied and may be classified into virological causes, sample collection and sample quality-related issues and finally technical issues related primarily due to problems in RNA extraction [Table 1]. The objective of this article is to discuss these causes and suggest corrective measures.{Table 1}

Inconclusive results due to sample collection or sample quality-related issues

The reasons that may adversely affect sample quality are improper collection techniques (inadequate swabbing), poor quality of viral transport medium, improper swabs (cotton/calcium alginate swabs with wooden stick instead of Dacron or Rayon swab with plastic shaft) and inadequate or absent cold chain during sample transport. These factors may result in inadequate genetic material in the sample causing the absence of amplification of the internal housekeeping gene (RNAse P). A study has reported sputum as the most accurate sample for laboratory diagnosis of COVID-19, followed by nasal swabs, while throat swabs were not recommended for the diagnosis.[1],[2],[3]

Inconclusive results due to problems associated with RNA extraction

RNA extraction problems are perhaps the most common reasons for an inconclusive result in SARS CoV-2 RT-PCR. The frequency of occurrence may vary with the extraction kit used (spin column vs. magnetic bead extraction) and systems used for nucleic acid extraction (automated or manual). Generally, magnetic bead extraction (Thermo Scientific or Qiasymphony automated extraction) has better extraction quality and efficiency than spin column extraction (Qiagen or Zymo Research). In addition, automated robotic extractions (Qiasymphony from Qiagen or Kingfisher from Thermo Scientific) not only have a higher throughput but also have a greater precision in terms of purity of the RNA extracts and extraction yield. These properties may be estimated using a NanoDrop spectrophotometer checking 260/280 nm and 260/230 nm wavelength absorbance ratios. The quality of extraction can also be checked by the internal control amplification curve. The internal control could be either endogenous (RNAsePin Black Bio TRUPCR assay) or exogenous (Altona Real-Star assay which uses MS2 bacteriophage). In case of exogenous internal control, the housekeeping gene is not checked, but the efficiency of extraction can be checked along with PCR failure. The exogenous internal controls (e.g., ALTONA assay) are spiked to samples and are designed in such a way that they do not interact with the primary assay (in this case, the detection of SARS CoV-2 target genes). Assays which detect endogenous internal controls such as RNAsePare able to detect sample quality and the presence of PCR inhibitors in the extract and indicate the possibility of PCR procedural failure if any (TRUEPCR assay).[4]

Virological causes of inconclusive results

This usually occurs if in a MULTIPLEX assay or in an assay where multiple (usually two) viral genes are detected – only one gene is positive by RT-PCR and the other is negative. This may happen due to various reasons such as – (a) betacoronavirus infections which are not due to SARS CoV-2, (b) different analytical sensitivity of individual viral gene PCR (E/RDRP/N2/S gene), especially at low viral load levels, and (c) non-specific binding of PCR primer or probe during the late phases of PCR cycles (e.g. after 35 cycles).

 Management of Inconclusive Results



This should involve a repeat extraction and a repeat PCR from the fresh RNA extract if there is failure of internal control. If the reasons are virological, a repeat sample should be sent after two days. In case of non-detection of housekeeping gene on E-extraction, a fresh sample should be collected for RT-PCR as soon as possible. It is also important that the clinician and various stakeholders (e.g., infection control experts) are educated about these possibilities. The counselling of the patient and relative is also important.

In conclusion, quality control and quality assurance of all processes from sample collection and RNA extraction to PCR are essential to prevent inconclusive result-related delays and uncertainties. Inconclusive results are expensive to deal with and are resource intensive for healthcare systems.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Collecting, Preserving and Shipping Specimens for the Diagnosis of Avian Influenza A (H5N1) Virus Infection. Available from: https://www.who.int/ihr/publications/CDS_EPR_ARO_2006_1.pdf?ua=1. [Last accessed on 2020 May 12].
2Dheda K, Jaumdally S, Davids M, Chang JW, Gina P, Pooran A, et al. Diagnosis of COVID-19: Considerations, Controversies and Challenges in South Africa. Wits Journal of Clinical Medicine. 2020;2:3–10. doi: 10.18772/26180197.2020.v2nSIa1. PMCID: PMC7187744.
3Tang YW, Schmitz JE, Persing DH, Stratton CW. The Laboratory Diagnosis of COVID-19 Infection: Current Issues and Challenges; J. Clin. Microbiol 2020. doi:10.1128/JCM.00512-20.
4Chan JF, Yip CC, To KK, Tang TH, Wong SC, Leung KH, et al. Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-PCR assay validated In vitro and with Clinical specimens. J Clin Microbiol 2020;58:e00310-20.