Episode 99 Recurrence persistence reinfection and mutations

Dear colleagues,  

There is a lot of questions about recurrence, persistence and re-infection by SARS-CoV-2.  These are diagnostic  problems, partly because “viral “shedding” in respiratory or other samples, as measured by (genomic) RNA PCR, can be prolonged or intermittent, without or with minimal symptoms. Are these patients still infectious?  When can we speak about “re-infection”?

In this episode, I try to summarize the existing literature.  It is complicated and the better you look, the more virus you see.  But is it clinically relevant? 

At the very end, I share also two papers that my attention was called upon:  on the significance of particular mutations for transmission, pathogenicity and T cell immunity….

1. Examples of recurrence
   1. A paper of Jing Lu on recurrence (EBioMed Aug Ep 99-1): This is about patients re-testing positive shortly after discharge from hospital (= after 2 consecutive PCR negative naso-pharyngeal or NP swabs within 24 H)

Findings: Among 619 discharged COVID-19 cases, 87 re-tested as SARS-CoV-2 positive in circumstances of social isolation within a few weeks (see slide 2).

• All re-positive cases had mild or moderate symptoms at initial diagnosis and were younger on average (median 28 yrs) than those who remained negative (Table 1 slide 2)
• Re-positive cases exhibited similar neutralization antibodies (NAbs) titre distributions to other COVID-19 cases. (Slide 3)
• No infectious strain could be obtained by culture and no full-length viral genomes could be sequenced from re-positive cases

Interpretation: It was recurrence (no reinfection), since they remained in isolation.  The virus was apparently defective and non-infectious(!?)

• 2. Another example Recurrence in HCW in nursing homes (Gallichotte medRxiv 5 Nov 2020 - Ep 99-2):
• Housekeeping staff more infected than nursing > administration > therapists (slide 4)
• A strong correlation between viral RNA and infectious virus (slide 5)
• A strikingly high degree of asymptomatic/mildly symptomatic infection, (slide 6).
• Variable duration of PCR positivity (1-4 wks) and recurrence in some individuals (slide 7)

2. Reviews on duration of shedding: viral load and infectiousness?
   1. Infectiousness in English patients: effect of viral load and  duration since onset (Singanayagam Eurosurveillance Aug 2020 Ep 99-3): Side 8

• Probability of culturing virus declines to 8% in samples with Ct > 35
• and to 6% 10 days after onset.
• It is similar in a-sympt-,  pre-sympt and symptomatic persons.

Asymptomatic persons represent a source of transmissible virus

1. 2. Review on duration viral shedding in resp.and stool sample (Fontana Oct 2020 Ep 99-4)

Respiratory samples

• The pooled median duration of viral RNA shedding is 18.4 d (95% CI, 15.5–21.3).
• Intermittent RNA shedding up to 92 d after symptom onset has been observed.
• Viable virus has been isolated via culture from −6 to 20 d relative to symptom onset.
• Duration of RNA shedding 13-45 days longer than duration of viable virus shedding

Stool samples:

• Viral RNA shedding in stool has not been consistently observed and may lag behind detection of respiratory RNA by PCR.
• The pooled median duration of viral RNA shedding is 22.1 d (95% CI, 14.4–29.8).
• Viral RNA shedding up to 55 d after diagnosis has been observed.
• Viable virus has been isolated via culture of stool on day 19 of illness.

1. 3. Review in The Lancet Nov 2020: SARS-CoV-2 RNA shedding duration (Ep 99-5)

• • upper-resp tract mean 17·0 days (maximum shedding 83 days);
  • lower resp tract  14·6 days (maximum 59 days)
  • stool 17·2 days (maximum 126 days). 
• Factors: RNA shedding increases with age and clearance delayed in symptomatic
• Live virus (by culture) was only recovered during first 9 days (even if RNA load persisted)

• 4. Review by Jefferson (CID Dec 2020) on relation PCR and infectious virus (Ep 99-6)
• In 12 studies Ct  (cycle threshold) values were lower (hence log copies higher) in specimens producing live virus culture. Two studies reported the odds of live virus culture reduced by approximately 33% for every one unit increase in Ct.
• Six of eight studies reported detectable RNA for longer than 14 days but infectious potential declined after day 8 even among cases with ongoing high viral loads.
• Four studies reported viral culture from stool specimens.

Conclusions:

• Complete live viruses are necessary for transmission, not fragments identified by PCR.
• Samples with high cycle threshold (low viral load) unlikely to have infectious potential.   

• 5. Persistent Detection and Infectious Potential ofSARS-CoV-2 in adults and children (Zapor Viruses Dec 2020 Ep 99-7):
• Despite the fact that children are considered much less frequently as a source of transmission, they can also shed viral RNA for prolonged time via  the respiratory and especially the stools (Side 9).
• Because the persistently shed RNA in non-immunocompromised subjects was repeatedly shown NOT to be cultivable in vitro, it is now generally considered non-infectious .  Therefore, the CDC adapted its guidelines for precautions to be taken from a (RNA) test based approach towards a symptom-based-approach, as summarized on slide 10.  More details can be found on https://www.cdc.gov/coronavirus/2019-ncov/hcp/disposition-hospitalized-patients.html

3. Is the presence of subgenomic RNA (measured by PCR) a proxy for viral culture and infectivity?

All the papers, quoted until now, suggest that prolonged shedding of RNA in immunocompetent subjects is NOT infectious virus, because it cannot be cultivated in vitro. There are however several caveats:

• One has to realize that the RNA targeted in the routine RT-PCR assays is genomic RNA, which could be associated with a non-replicating virus. 
• I could not find a real proof that there is no chance of transmission if genomic RNA is positive and culture is negative.  
• Microbiologists and virologists know that it is not easy to cultivate virus from nasopharyngeal swabs and there can be many technical reasons for failure.

Nevertheless, non-cultivability usually is associated with low viral loads indeed and therefore one can accept that chances on transmission are low, if the culture remains. 

Clearly, some researchers doubt about that presumption and try to develop an easier way to investigate whether in some individuals, the virus still replicates in vivo.  To that end, they measure various species of subgenomic RNA. 

1. 1. Kim explains the viral cycle (Cell May 2020 Ep 99-8), shown in slides 11, 12, 13.  Clearly, if you can show that subgenomic RNA is present, you have an indication that the virus replicates, even below the threshold level for culture.  Measuring this subgenomic RNA species (in addition to genomic RNA) is also easier, faster and probably more reproducible than culture. 

Let’s investigate the evidence until now

1. 2. Perera (EID Nov 2020 - Ep 98-9) shows that virus isola­tion and sgRNA detection were positive within the first 8 days after onset of illness and mainly for specimens with >6 log10 virus N gene copies/mL of clinical specimen.

• • Slide 14  shows that positive cultures were limited to first 8 days and samples with high viral load (genomic Nucleocapsid RNA).  After > 8 days, samples with high VL (107 log) remained negative in culture, confirming earlier data
  • Slide 15 shows the incomplete correlation between viral culture and the presence of subgenomic viral RNA.  The brown dots = culture (-) sgRNA (+) persist a bit longer than red dots culture(+) sg RNA (+), but after > 20 days only green dots N-RNA (+) culture and sgRNA (-) persist.

In this study, it is not clear from the methods (in Appendix) which species of sgRNA was measured

1. 3. Rodriguez-Grande (JCM Nov 2020 – Ep 98-10) investigated the genomic and subgenomic (sg) RNA of the E protein in parallel  in 60 patients with prolonged shedding. 

• • SG viral RNA was detected in 12/60 (20%) of the persistent cases, 28-79 days after the onset of symptoms.  Some examples are shown in slide 16.
  • Seven out of 12 of sgRNA(+) subjects were immune suppressed (see slide 17), however no clinical information on sgRNA(-) cases. 
  • Culture was not performed, but in the sgRNA(+) samples the genomic RNA was detected at < 30 Ct, consistent with a medium-to-high VL.   

They conclude: Our results suggest that a percentage of persistent positive SARS-CoV-2 PCR positive cases might still be contagious.

1. 4. Prolonged Infectious SARS-CoV-2 Shedding from an Asymptomatic Immunocompromised Individual with Cancer (Ep 99-11)

Patient with chronic lymphocytic leukemia and acquired hypogammaglobulinemia

• Shedding of infectious SARS-CoV-2 was observed up to 70 days, and of genomic and subgenomic RNA up to 105 days, after initial diagnosis.
• First convalescent plasma treatment: no clearance of infection.
• Several weeks after a second convalescent plasma transfusion, SARS-CoV-2 RNA was no longer detected.
• Marked within-host genomic evolution of SARS-CoV-2 with continuous turnover of dominant viral variants, which were equally “fit” in vitro. 

Certain immunocompromised individuals may shed infectious virus very long.

Detection of subgenomic RNA is s a proxy for shedding of infectious virus.

4. Other evidence of persistence
   1. Scutari Life Dec 2020 (Ep 99-12): Evidence from droplet-digital PCR: Long-Term SARS-CoV-2 Infection Associated with Viral Dissemination in different body fluids including bile in two Patients with acute cholecystitis (slide 21)

For both patients, ddPCR revealed persistent viral RNA in the nasopharyngeal swab, despite triple-negative or single-positive results by qRT-PCR.

• In Patient 1, SARS-CoV-2 RNA dropped more rapidly in bile and rectal-swab and declined slowly in NP swab and plasma, becoming undetectable in all compartments 97 days after symptoms onset.
• Conversely, in patient 2, SARS-CoV-2 RNA was detected, even if at low copies, in all body samples (with the exception of urine) up to 75 days after the onset of symptoms.

Thus SARS-CoV-2 RNA can persist and be detected by ddPCR for a prolonged time in respiratory samples and in several biological samples despite negativity to qRT-PCR, supporting SARS-CoV-2’s ability to provoke persistent and disseminated infection and therefore to contribute to extra-pulmonary clinical manifestations.

1. 2.  Remember also the paper by Gaebler in bioRxiv Nov 2020 (Ep 99-13), where evolution of antibody responses is seen months after recovery.  This evolution of antibodies in vivo was explained by the persistent presence of virus in GI biopsies in 7/14 asymptomatic individuals for at least 3 months. Unfortunately, there are no precise data on these subjects: antibody responses were analyzed in great detail, but no data on viral PCR or evolution….

5. Review on reinfections: Babiker JCM 23 Dec 2020 (Ep 99-14): Few have been well documented, because you need sequence proof.  Many more cases suspected but not proven.

Criteria are shown in slide 22:

1. “Investigative” evidence = clinical and PCR indication

• Either: positive PCR (<33Ct) > 90 days after last positive sample
• Or: positive PCR 45-90 days AND typical COVID symptoms or close contact with case

2. “Laboratory” evidence = proof by sequence:

• 1st and 2nd belong to different clades
• AND/OR > 2 nucleotides difference per month since last positive sample (because up to 2 nucleotides may change during persistent infection).

Characteristics of 16 published cases of well-documented reïnfection:

• Epidemiological: mostly young (20-30 yrs) and HCW (but this may be a bias, bc better follow-up than in general population). 
• Clinical: second episode: mostly milder or similar than first; viral load also similar. 
• Immunity status: unclear, since insufficient data on neut Ab.

Example:  Goldman medRxiv Ep 99-15:   

• Slide 23 illustrates normal distribution of shedding: up to 90 days.  The far-right is a patient with documented reinfection original infection with D614 and reinfection > 120 days later with 614G,
• Slide 24 shows that in this patient neut was weak  shortly after reinfection and increased thereafter. Suggestive, but no formal proof of failure (induction or persistence?) of antibody response after first infection 

6. MUTATIONS OF SPECIAL INTEREST
   1. Farkas medRxiv Oct 2020: Viral evolution and spike mutations associated with elevated mortality rates. (Ep 99-16)

• Nsp2, 3C-like proteinase, ORF3a and ORF8 are under active evolution, as evidenced by their increased ratios of non-synonymous mutations (giving rise to changes in amino acids)
• Two important mutations in Spike: V1176F in co-occurrence with D614G and S477N, located in the Receptor Binding Domain (RBD) are associated with high fatality rates and are increasingly spreading throughout the world.
• Mutation in ORF3a (Q57H) and Nucleocapsid (I292T, RG203KR) also associated with increased mortality.
• Evidence of hypermutation (by activity of host APOBEC mechanism) in 2 %: is a “natural immunity” defense mechanism.  

1. 2.   Agerer bioRxiv Dec 2020: Mutations in MHC-I epitopes as a consequence of escape from CD8 T cell immunity (Ep 99-17)

Starting from deep sequencing exercise 197 non-synonymous mutations in predicted epitopes for HLA-A 02:01 and B 40:01 were investigated.  Mutants with predicted lower affinity to HLA were further analyzed in vitro with PBMC from SARS-CoV-2 pt

• • with HLA-tetramers: lower binding to CD8 T cells (slide 25 3C-E)
  • restimulation: lower production of Interferon-gamma (slide 25 3G-I).

The fact that these mutations tend to get fixed in the circulation SARS-CoV2 population pleads for pressure by cytolytic T cells.  

Conclusions:

1. Recurrence of (genomic) RNA in respiratory samples on the short term is relatively frequent, even in a “low-risk” group (healthy relatively young people).  If RNA load is low (Ct > 33) considered as non-infectious.
2. Persistence of viral shedding (in respiratory and stools) usually in older subjects and/or people with underlying risk factors (immune deficiency, cancer). During persistence, the virus will evolve with up to 2 nucleotides per month.  Viral evolution implies replication, even if not proven by culture.
3. Reinfection only to be considered if (1) > 45 days since last positive sample and symptoms  or close contact with case OR (2) > 90 days without symptoms. Proof is by sequencing (at least evolution of > 2 nucleotides per month).  

The traditional tool  to evaluate infectiousness while viral shedding recurs or persists is viral culture, but this is not a routine test and it can be false negative for technical reasons. Clearly also, a genuine negative culture does not mean that there is no replicating virus.  It is unlikely that viral RNA could be shed for weeks without any viral replication, but it is quite possible that it is a low infectious load that would normally be below the threshold for transmission.

The measurement of “subgenomic RNA” species is being suggested as a more easy and reproducible alternative for viral culture, but this clearly needs to be further validated.  

The distinctions between persistence recurrence and reinfection are somewhat arbitrary.  These are clinical definitions, meant to facilitate decisions about isolation, treatment etc. The underlying presumption is that persistence = pathological.  That is, however, not sure in light of the data by Gaebler and Nussenzweig (Ep 99-13): they  show antibody evolution in asymptomatic recovered patients, which suggested to them that it was driven by continuous presence of replication-competent virus.  They demonstrated virus  in gut biopsies from 7/14 asymptomatic subjects.

Therefore, it is possible that low-grade  persistence of the virus in extra-respiratory site, i.e. gastro-intestinal lymph nodes,  may be “normal”, even in healthy, immune competent subjects and rather beneficial, because it stimulates continued antibody production, which according to Gaebler also evolve and “mature”, without a high risk for transmission.    No persistence would result in more rapid loss of (neut) Ab, hence susceptibility to reinfection.  High-level persistence would be the result of immune deficiency (or immaturity in infants or immune senescence in elderly?) and then could be associated with recurrent pathology and infectiousness.  

Clearly, at present, this is an hypothesis.  I’m sure we will know more in a few months.

Best wishes,

Guido