To the Editor:
Despite many efforts in understanding the pathogenesis of coronavirus disease 2019 (COVID-19), numerous unsolved questions contribute to the increasing sense of uncertainty about this disease. A compelling problem is the management of previously infected patients with a persistent SARS-CoV-2 nasopharyngeal swab polymerase chain reaction (PCR) positivity after an appropriate period of quarantine and proved “immunization” as confirmed by seroconversion.1
Although PCR positivity may not drive public health concerns in terms of contagiousness, it may be detrimental for health care workers and people on waiting lists for elective procedures or surgeries. The question that arises is: What is the biological mechanism underlying the prolonged persistence of test positivity in absence of symptoms? Considering the pathogenicity of SARS-CoV-2, three potential mechanisms have been hypothesized: (i) reinfection, (ii) residual fragments of viral RNA and (iii) genomic integration.
Reinfection is not a well-defined event in COVID-19. In patients with reinfection, analysis of viral genome sequences documented that distinct isolates of SARS-CoV-2 can cause a second infection. Most cases of reinfection had worse clinical outcomes despite the absence of presumed defects of the immune system. Although the scenario can seem worrisome in terms of disease severity and spread of the virus, these data are largely biased by patient selection.2 Testing has indeed been performed mainly in symptomatic patients; therefore, the rate of subjects with asymptomatic recurrence of the disease remains undiagnosed in the absence of a systematic surveillance program. Recently, the entity of immune response has been evaluated in a large study conducted on 30,082 patients. The findings of this study showed that more than 90% of the screened patients with mild or moderate COVID-19 were able to mount a robust and prolonged (at least for 5 months) neutralizing antibody response (anti-spike protein) against the virus. Although it is too early to draw firm conclusions about the protective role of these antibodies, the authors of this study claimed that neutralizing antibodies may reduce the risk of reinfection or mitigate the symptoms of recurrence.3
Concerning the issue of viral remnants, SARS-CoV-2 nasopharyngeal swab PCR positivity does not rule out the possibility to detect genetic material without infectious potential. As in other viral infections (SARS-CoV, Middle East respiratory syndrome coronavirus, influenza virus, Ebola virus, Zika virus and measles virus), viral RNA can be detected long after the resolution of the infection. Noninfectious small fragments of viral genetic material are slowly degraded by the immune system and remain detectable on the mucosal surface for many weeks. Similarly, SARS-CoV-2 shedding has been detected for more than 2 months in adults and pediatrics.4
Nevertheless, persistent viral shedding paves the way to a new hypothesis and raises questions about the possibility that viral RNA may integrate into the host genetic material. On this issue, Zhang et al5 have recently documented in vitro that SARS-CoV-2 RNA may be reverse transcribed and integrated into human cells, and the integrated sequences can be subsequently transcribed to produce new viruses. This condition, which has alerted public opinion, seems to be difficult—if not improbable—to realize outside of the optimized experimental conditions. Integration is a rare phenomenon in nature involving few viruses, including HIV, and other retroviruses. To the best of our knowledge, it is unlikely that SARS-CoV-2 may drive integration. Similar to the viruses responsible for MERS and SARS, SARS-CoV-2 replicates into the cytoplasm using an RNA-dependent RNA polymerase without entry into the cell nucleus, where integration takes place. More importantly, the virus lacks 2 key enzymes in the process of viral integration: reverse transcriptase that generates a double-stranded linear viral DNA by the viral RNA, and integrase that insert the viral DNA into the host chromosomal DNA. Even though host or retrovirus-encoded reverse transcriptase can theoretically be used by SARS-CoV-2 to retro-integrate in human cells, the likelihood remains low, and further studies are required to verify this process under standard conditions.
Based on this background, the best way to evaluate the infectivity of specimen test remains the cell culture-based system, but preanalytic (source of specimens and storage) and analytic biases (cell line culture environment, equipment and expertise) limit its application as a routine clinical practice.
In conclusion, the etiological mechanism underlying prolonged RNA shedding after recovery is still unclear and reflects the poor understanding of the interplay between the immune system and SARS-CoV-2 infection. Reinfection seems to be the unique condition that carries a high risk of severe infection and virus spreading and therefore warrants a high clinical suspicion, especially in patients who present symptoms after 90 days from SARS-CoV-2 infection (Centers for Disease Control and Prevention recommendations).
Annachiara Ferrari, MD
Department of Specialistic Medicine
Azienda Unità Sanitaria Locale di Reggio Emilia
Gaetano Alfano, MD
Medical and Dental Department of Morphological Sciences
Section of Nephrology
University of Modena and Reggio Emilia
Clinical and Experimental Medicine PhD Program
University of Modena and Reggio Emilia
Giovanni Guaraldi, MD
Clinic of Infectious Diseases
University Hospital of Modena
1. Lu J, Peng J, Xiong Q, et al. Clinical, immunological and virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR. EBioMedicine
. 2020;59:102960. doi:10.1016/j.ebiom.2020.102960.
2. Iwasaki A. What reinfections mean for COVID-19. Lancet Infect Dis
. 2021;21(1):3–5. doi:10.1016/S1473-3099(20)30783-0.
3. Wajnberg A, Amanat F, Firpo A, et al. Robust neutralizing antibodies to SARS-CoV-2 infection persist for months. Science
. 2020;370(6521):1227–1230. doi:10.1126/science.abd7728.
4. Atkinson B, Petersen E. SARS-CoV-2 shedding and infectivity. The Lancet
. 2020;395(10233):1339–1340. doi:10.1016/S0140-6736(20)30868-0.
5. Zhang L, Richards A, Khalil A, et al. SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome. bioRxiv
. Published online December 13, 2020. doi:10.1101/2020.12.12.422516.