Host Genetic Analysis Should Be Mandatory for Proper Classification of COVID-19 Reinfections

Dear Editor,

A number of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfections [1–5] have been communicated since the first reported case in August 2020 [6]. Confirmation of reinfection has always been based on differences between sequential SARS-CoV-2 strains in the first and second episodes, either major, different lineages/a large number of single nucleotide polymorphisms (SNPs) or moderate, lower diversity, but still relevant, between the strains.

A recurrence is not classified as reinfection without the support of viral genomic data; however, there is a lack of genomic rigor when documenting whether the samples used to characterize the strains involved in the sequential episodes correspond to the same patient. This is essential to support viral genomics findings and demonstrate that different SARS-CoV-2 strains have sequentially infected a single patient. To the best of our knowledge, only 2 SARS-CoV-2 reinfection studies have taken this point into consideration; these have shown, supported by short tandem repeat (STR) analysis, identical host markers for the nasopharyngeal samples collected for the analyses [4, 7].

With the striking increase in laboratory workload once the first coronavirus disease 2019 (COVID-19) wave hit, collecting sample remnants for future studies was a real challenge. Mistakes in sample labeling or in aliquoting were more likely to occur under the experienced stress. Thus, when documenting a reinfection in COVID-19, genomic data ensuring that the stored sample belongs to the same patient should be a requirement before a SARS-CoV-2 reinfection report is admitted as such.

Here, we present a COVID-19 recurrence case that met all the clinical and microbiological requirements of a SARS-CoV-2 reinfection. The patient is a 61-year-old man with a history of chronic obstructive pulmonary disease, obesity, and ankylosing spondylitis, who had a first COVID-19 episode in March 2020 (positive SARS-CoV-2 reverse transcription polymerase chain reaction [RT-PCR] on March 17; cycle threshold [Ct] 19) and a second episode 6 months later, in September (positive RT-PCRs on September 13, 17, 28, and October 4; Ct values 17, 18, 23, and 35, respectively). Both episodes were characterized by mild symptoms; the second occurred in the context of a SARS-CoV-2 nosocomial infection. Three negative SARS-CoV-2 RT-PCRs were obtained between the first and second episodes (March 30, August 4, and September 3). Serology was not available for the first episode, but it was negative in August, and 10 days after the second episode, the patient showed a positive antibody titer.

Genomic viral analyses determined 6 SNPs for the SARS-CoV-2 strain, lineage 19A, obtained from the first episode, with respect to the Wuhan-1 reference sequence (Figure 1A). The strain from the second episode did not show any of the SNPs for the first episode strain and carried 16 other SNPs (Figure 1A, Table 1) and belonged to a different lineage, namely 20A.EU1, the primary lineage in Spain and many European countries since the end of June [8]. Based on major genomic differences between the 2 strains, we labeled the recurrence a reinfection.

Figure 1.

A, Schematic single nucleotide polymorphism distribution (vertical black lines) along the SARS-CoV-2 chromosome for the 2 COVID-19 episodes. B, Human identity testing analysis was done by short tandem repeat PCR (Mentype Chimera Biotype, Germany) on the same samples used to perform the SARS-CoV-2 RT-PCR, and they were subsequently sequenced. Representative loci of the 12 noncoding short tandem repeat loci and the gender-specific locus amelogenin were analyzed. They were labeled with 3 different dyes (6-FAM, BTG, or BTY) using the Mentype Chimera PCR amplification kit (Biotype, Germany). Abbreviations: COVID-19, coronavirus disease 2019; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Table 1.

Differential SNPs Between Episodes

	03/17/2020	09/13/2020
Position	First Episode	Second Episode
C|9360|T	1	0	ORF1ab	missense_variant	Thr3032Ile
G|11083|T	1	0	ORF1ab	missense_variant	Leu3606Phe
C|14805|T	1	0	ORF1ab	synonymous_variant	Tyr4847Tyr
T|17247|C	1	0	ORF1ab	synonymous_variant	Arg5661Arg
A|18077|G	1	0	ORF1ab	missense_variant	Lys5938Arg
G|26144|T	1	0	ORF3a	missense_variant	Gly251Val
C|241|T	0	1	ORF1ab	upstream_gene_variant	-25C > T
T|445|C	0	1	ORF1ab	synonymous_variant	Val60Val
C|3037|T	0	1	ORF1ab	synonymous_variant	Phe924Phe
C|6286|T	0	1	ORF1ab	synonymous_variant	Thr2007Thr
C|12119|T	0	1	ORF1ab	missense_variant	Pro3952Ser
C|13115|T	0	1	ORF1ab	synonymous_variant	Leu4284Leu
C|14408|T	0	1	ORF1ab	missense_variant	Pro4715Leu
A|21222|T	0	1	ORF1ab	synonymous_variant	Ala6986Ala
G|21255|C	0	1	ORF1ab	synonymous_variant	Ala6997Ala
C|22227|T	0	1	S	missense_variant	Ala222Val
A|23403|G	0	1	S	missense_variant	Asp614Gly
C|25889|T	0	1	ORF3a	missense_variant	Ser166Leu
C|26801|G	0	1	M	synonymous_variant	Leu93Leu
C|27944|T	0	1	ORF8	synonymous_variant	His17His
C|28932|T	0	1	N	missense_variant	Ala220Val
G|29645|T	0	1	ORF10	missense_variant	Val30Leu

Abbreviation: SNP, single nucleotide polymorphism.

We then performed STR analysis from the same total nucleic acid preparation that had been used to perform the viral genomic analysis (Supplementary Data). It revealed a different origin of the specimens due to completely different patterns for the first- and second-episode specimens (Figure 1B). In fact, the material from the first episode belonged to a woman and the second to a man, indicating mislabeling or mishandling of the specimen assigned to our (male) patient’s first episode. The certainty of our patient’s second episode was proved by (i) 4 sequential positive RT-PCRs, (ii) 0 SNPs between the sequences from 2 of the SARS-CoV-2-positive specimens from his second episode, and (iii) STR analysis indicating that these last 2 specimens belonged to the same person (both males). The STR analysis revealed that the reinfection was wrongly classified as reinfection, something that would have gone unnoticed if the host material had not been characterized.

Our data highlight the potential incorrect classification of SARS-CoV-2 infections when samples from COVID-19 sequential episodes, used for viral genomic sequencing, are not confirmed to belong to the same individual by host genetic analysis. STR-based analysis should be mandatory in all reports assessing SARS-CoV-2 reinfections.

Supplementary Data

Supplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.