COVID-19 re-infection: Diagnostic challenges and proposed diagnostic criteria

Infection with SARS-CoV-2 causing COVID-19 has affected people worldwide. SARS-CoV-2 Infection can be symptomatic or asymptomatic. Re-infection is common with other corona viruses whereas the exact potential for re-infection with SARS-CoV-2 is still not clear [1]. Animal studies show that there is no re-infection with same strains of SARS-CoV-2 [2]. We still have only limited knowledge about the level of protective immunity to subsequent infection with SARS-CoV-2. Poor humoral immune response or decreasing immunity over time may be the reason for re-infection. Antibody response to mild infections is lower than that of severe disease and 20% does not seroconvert [3]. Re-infection can occur even in the presence of protective antibodies [4]. Reports from various parts of the world show that people, who had infection with SARS-CoV-2, can subsequently develop re-infection if exposed to COVID-19 cases [[5], [6], [7]].

Acute infection with SARS-CoV-2 is mainly diagnosed based on throat swab RT-PCR positivity. Swab report depends upon time of illness, site of sample collection, technical factors, methods of transport and testing techniques. Prolonged shedding from upper respiratory tract is reported up to 104 days [8]. SARS-COV-2 RNA persist in stool specimen 5 weeks after respiratory specimen become negative [9,10]. Virus persists in sputum and feaces. Hence these samples can be positive even after throat swab RT-PCR negativity. Shedding of non-viable virus remnants (RNA from non-viable virus) has to be differentiated from persistent infection with a viable virus (RNA from live virus). RT-PCR can be positive in both situations (Fig. 1 ). Sensitivity of RT-PCR is about 66–80% [11]. Early and late RT-PCR can be false negative [11,12]. RT-PCR detects genetic material. So RT-PCR positivity can be due to infective live virus or dead material. Or in other words, RT-PCR positivity doesn’t always mean viable virus in the individual and hence need not be infectious. Persistent RT-PCR positivity can be due to prolonged viral shedding, reactivation, relapse, co-infection or true re-infection. Increased viral replication after stopping the drugs following clinical recovery, presence of dead virus and viral genomics fragments can also cause persistent RT-PCR positivity [9]. Dynamic RT-PCR or Intermittent negative PCR results (oscillating positive and negative tests) due to low viral concentration in the sample or inaccurate testing can result in positive RT-PCR after a negative report [13].

Fig. 1.

Approach to persistent RT-PCR positivity.

In a typical case with two separate episodes of symptoms of acute SARS-CoV-2 infection with positive RT-PCR and genetic studies showing infection with two different variants after two separate episodes of exposure to positive cases of COVID-19, with clinical recovery and RT-PCR negativity in between indicate re-infection with SARS-CoV-2. But in clinical practice, there are lots of challenges in the diagnosis of first infection with SARS-CoV-2 and COVID-19 re-infection (Table 1 ).

Table 1.

Challenges in the diagnosis of SARS-CoV-2 infection and re-infection.

Challenges in the diagnosis of SARS-CoV-2 infection
•Variation in testing policies in different areas and also in the same area at different stages of the pandemic
•Significant proportion of asymptotic infection
•Test positivity depends upon various factors- the stage of the illness; (may get false negatives RT PCR at the early stages and late stages of illness), site of sample collection, technical factors, transport and testing techniques
•False positive RT PCR results
Challenges in the diagnosis of SARS-CoV-2 re-infection
•One or both episodes of SARS-CoV-2 infection may be asymptotic, resulting in difficulties in identification of cases
•One or both episodes may be associated with false negative RT-PCR
•Difficult to differentiate re-infection from persist viral shedding. Viral culture my help to differentiate
•Viral genome sequencing facilities and other genetic tests to document, two episodes of SARS-CoV-2 infection due to different strains are not freely available
•When the viral load decline may get a false negative RT PCR in between two positive RT PCR results
•False positive result is rare, but possible due to contamination or human error during sample collection, transport or analysis

Based on our clinical experience and after reviewing relevant literature we are proposing criteria to diagnose COVID-19 re-infection (Table 2 ).

Table 2.

Proposed diagnostic criteria for COVID-19 re-infection.

Proposed diagnostic criteria for COVID-19 re-infection
Confirmatory (genetic/essential) criteria
Two episodes of infection with SARS-CoV-2 caused by different genetic strains/variants, documented by any one of the following
•SNVa
•Lineage/clades
•Phylogenetic
Clinical criteria
Two definite episodes of symptoms consistent with infection with SARS-CoV-2
Lab criteria
Two definite episodes of infection (symptomatic or asymptomatic) with SARS-CoV-2, confirmed by positive RT-PCR
Recovery criteria
It has two component clinical and lab
Clinical recovery criteria
Two definite episode of symptoms consistent with acute SARS-CoV-2, separated by asymptomatic time interval indicating clinical recovery
Lab/microbiological recovery criteria
Two definite episodes of infection (symptomatic or asymptomatic) with SARS-CoV-2, confirmed by two positive RT-PCR with negative RT-PCR in between
Epidemiological criteria
History of re-exposure to COVID-19 patients after recovery from first episode
Time duration between episodes
Longer the time interval between episodes, more in favor of re-infection

^aSNV- Single Nucleotide Variation

1. Essential criteria

Demonstrating the two episodes of infection by different strains of SARS-CoV-2 confirms the diagnosis of re-infection. Single nucleotide variation (SNV) more than 10 or demonstration of two different variant by lineage, clades or phylogenetic analysis confirms that two infections are caused different virus [[14], [15], [16]]. Considering the possibilities of intra-host mutation (mutation occurring in the virus while inside the host body) SNV more than 10 is taken significant because, virus can undergo 2 nucleotide variations per month. The two episodes of infection if proved different by genetic clades or lineage it is a stronger indicator of re-infection [15,16].

2. Clinical criteria

Two separate episodes of symptoms suggestive of acute COVID-19 also favor the diagnosis of re-infection. But there are lots of infections which can present with similar symptoms. Presence of “long COVID-19” symptoms has to be differentiated from the symptoms of acute infection. In addition to that a significant proportion of individuals infected with SARS-CoV-2 are asymptomatic. If both episodes of infections are asymptomatic, we may miss the case without laboratory screening.

3. Laboratory criteria

Two episodes of infection with SARS-CoV-2, confirmed by positive RT-PCR have the advantage of including both symptomatic and asymptomatic infections. But prolonged viral shedding is reported even up to 104 days, resulting in RT-PCR positivity months after the first positive result [8]. It has to be differentiated from re-infection. Presence of viral RNA fragments, which are non-infectious, can also cause false positive RT-PCR, which can be differentiated from re-infection by viral culture. Cycle threshold (CT) value also helps to differentiate these two. CT value is the number of cycles of PCR amplification required to detect the gene target. It is high in those with low viral load and vice versa. High CT value in persistent viral shedding helps to differentiate it from re-infection, where CT value is low. A person who is found to be positive for antibodies, without any history of vaccination if subsequently develop RT-PCR positivity or evidence of acute SARS-CoV-2 infection, it also favors the diagnosis of re-infection. But all the people who had infection will not develop antibodies and it will gradually disappear even in those who had antibodies. 33% of COVID-19 recovered patients are negative for antibodies during convalescent period (39 days average) 40% at 8 weeks [17,18]. Viral culture helps to assess infectivity and cell culture, its cytopathic effects which will help to differentiate prolonged PCR positivity due to infection from persistence of viral fragments.

4. Recovery criteria

Demonstration of definite period of recovery in between the episodes also favors re-infection. Longer the time gap between 2 episodes, higher the chance of re-infection. It can be clinical recovery (where patients will be asymptomatic before the onset of second episode) or laboratory recovery or microbiological recovery (where RT-PCR is negative). Those with “long COVID” symptoms after the first episode will not be asymptomatic during the recovery period. Differentiation of “long COVID” symptoms from symptoms of acute SARS-CoV-2 infection helps to solve this confusion. When the viral load is declining and reaching near the detection threshold level, we may get negative or positive RT-PCR report depending upon that particular sample characteristic. A negative RT-PCR between two positive RT-PCR is possible in such scenario causing confusion about re-infection. Viral culture and CT value helps to differentiate.

5. Epidemiological criteria

The one who recovered from SARS-CoV-2 infection develops symptoms of acute COVID-19 again after exposure to COVID-19 cases also favors re-infection

6. Time duration between episodes

Persistent viral shedding can lasts up to 104 days. Even after the respiratory samples become negative, gastrointestinal shedding can continue for weeks. A long gap between first and second episode favors re-infection.

With the proposed criteria, clinicians will be able to categories re-infection as confirmed, probable or possible cases, which will give uniformity in reporting and managing cases of re-infection (Table 3 ).

Table 3.

Interpretation of proposed diagnostic criteria for COVID-19 re-infection.

Interpretation of proposed diagnostic criteria for COVID-19 re-infection
1st episode of SARS-CoV-2 infection			Recovery period			2nd episode of SARS-CoV-2 infection		Demonstration by two different strains	Inference
Symptoms	RT-PCR report	Antibody testing	Time gap between episodesa	Symptoms	RT-PCR report during recovery period	Symptoms	RT-PCR
±	+	±	+	–	–	±	+	+	Confirmed
±	–	+	+	–	–	±	+	–	Probable
+	–	–	+	–	–	±	+	–	Possible
±	+	–	+	–	–	+	–	–	Possible
+	–	±	+	–	–	+	–	–	Doubtful

^aThe longer the duration, more in favor of re-infection.

The major limitation of this criterion is that in laboratory test negative individuals, if one or both episodes are asymptomatic, we will miss the diagnosis of re-infection and it cannot be categorized based on the proposed criteria.

To conclude, re-infection with SARS-CoV-2 is reported from various parts of the world. Genetic studies and viral cultures are helpful to confirm re-infection, which is not commonly available. Practicing doctors worldwide face lot of challenges in diagnosing COVID-19 re-infection. We hope that the criteria proposed will be useful for the medical fraternity all over the world.