key: cord-0715659-7evym4gs authors: da Silva, Mariana Soares; Demoliner, Meriane; Hansen, Alana Witt; Gularte, Juliana Schons; Silveira, Flávio; Heldt, Fágner Henrique; Filippi, Micheli; Pereira, Vyctoria Malayhka de Abreu Góes; da Silva, Francini Pereira; Mallmann, Larissa; Fink, Pietra; da Silva, Luana Letícia; Weber, Matheus Nunes; de Almeida, Paula Rodrigues; Fleck, Juliane Deise; Spilki, Fernando Rosado title: Early detection of SARS-CoV-2 P.1 variant in Southern Brazil and reinfection of the same patient by P.2 date: 2021-07-05 journal: Revista do Instituto de Medicina Tropical de Sao Paulo DOI: 10.1590/s1678-9946202163058 sha: a6f721b4e3635d2d68340e25d0b6105c1dcfd0e6 doc_id: 715659 cord_uid: 7evym4gs Multiple variants of the Severe Acute Respiratory Syndrome coronavirus 2 virus (SARS-CoV-2) have been constantly reported across the world. The B.1.1.28 lineage has been evolving in Brazil since February 2020 and originated the P.1 variant of concern (VOC), recently named as the Gamma variant by the newly WHO nomenclature proposal, and P.2 as a variant of interest (VOI). Here we describe an early case of P.1 primary infection in Southern Brazil in late November 2020, soon after the emergence of the variant in Manaus, Northern Brazil. The same male patient was reinfected by another B.1.1.28 variant, namely P.2, in March, 2021. The genomic analysis confirmed genetically significant differences between the two viruses recovered in both infections, the P.1 lineage in the first episode and P.2 in the reinfection. Due the very early detection of P.1, we have also investigated the circulation of P.1 in the same region by differential RT-qPCR, showing that this was an isolated case of P.1 at the time of detection, and this variant has disseminated and became prominent from late January to the end of March, 2021. SARS-CoV-2 recent reports of reinfection have raised critical questions on whether and how well a first infection protects against reinfection. The emergence of the novel SARS-CoV-2 lineages is currently a worldwide concern. The continuous rise of variants is probably related to an increased rate of virus transmission and evasion from the host's immune system. Lately, research groups have reported the emergence of multiple variants of concern (VOC) or of interest (VOI), such as P.1 1 , P.2 2 , N.9 3 and B.1.351 4 in specific Brazilian regions. P.1 (VOC), recently named as Gamma variant by the newly WHO nomenclature proposal 5 as well as P.2 (VOI) variants have evolved from the lineage B.1.1.28 1,2 and harbor E484K (P.1 and P.2) and N501Y (P.1) mutations, which are associated with increased transmissibility or immune evasion 6 . The persistence of protective immunity provided by the coronavirus disease 2019 (COVID- 19) or vaccination is not yet well established and some cases of reinfection have been reported 7, 8 . The B.1.1.28 SARS-CoV-2 lineage has been circulating in Brazil since February 2020; P.2 (alias for B.1.1.28.2) was first detected in Rio de Janeiro 2 harboring the mutation S:E484K, and P.1 (alias of B.1.1.28.1) was first detected in Japanese travelers returning from the Amazonas State and due to the presence of several important mutations in the receptor binding domain -RBD of the viral spike (K417T, E484K, and N501Y) is of particular concern in Brazil. The P.1 variant is widely spread in the country, being associated with local outbreaks of great magnitude due to an enhanced transmissibility. The P.1 lineage more likely appeared in Manaus, Northern Brazil 9 in mid-November, even though, recent studies have shown an earlier possible origin for a P.1 ancestral in mid-August 2020 and for the common P.1 variant in mid-October 10 . The first described cases attributed to P.1 in Southern Brazil so far are from the last week of January 2021. In this study, we report a case of SARS-CoV-2 P.1 infection in a 39-year-old individual from Campo Bom city (latitude 29º40'44" South and at longitude 51º03'10" West), Rio Grande do Sul State the Southernmost State of Brazil, in late November 2020. The same patient acquired a second infection by another variant, the P.2 lineage, in March 2021. Due the very early detection of P.1, we also have investigated the circulation of P.1 in the same geographic region using a differential VOC RT-qPCR. The study was carried out from the time of the first infection (November 2020) to April 2021, since P.1 emerged in Northern Brazil in mid-November 11 and the first cases described in Southern Brazil occurred in late January 2021. Naso-oropharyngeal swab samples from the same patient (LMM38991 and LMM50731) were received at Laboratorio de Microbiologia Molecular of Universidade Feevale, Novo Hamburgo, Rio Grande do Sul State, Brazil, (November 30 th , 2020 and March 11 th , 2021) for SARS-CoV-2 detection. Total nucleic acid was extracted with the commercial MagMAX™ CORE Nucleic Acid Purification Kit (Applied biosystems™, Thermo Fisher Scientific, Waltham, MA, USA) kit, using the automated equipment KingFisher™ Duo Prime (Thermo Fisher Scientific™). RT-qPCR for SARS-CoV-2 cDNA detection targeting the viral E gene was performed according to the Charité Institute, Berlin, Germany, protocols 12 and was carried out using AgPath-ID One-Step RT-PCR Reagents (Thermo Fisher Scientific™). A differential RT-qPCR for the P1 lineage 9 that detects the ORF 1b deletion (NSP6: S106 del, G107 del, F108 del) was performed. This deletion is a genetic signature of the VOCs P.1, B.1.1.7and B.1.351. The differential RT-qPCR was carried out using AgPath-ID One-Step RT-PCR Reagents (Thermo Fisher Scientific™); primers and probes were used following the same protocol as described by Naveca et al. 9 . A total of 302 samples was tested (Ct values for the E gene ranging between 10 and 20) randomly distributed over 22 weeks that started on November 2 nd , 2020 until April 4 th , 2021. New SARS-CoV-2 cases, hospitalizations and deaths in the neighboring regions were compared with the P.1 lineage study results. This study was approved by the National Committee of Research Ethics and the Institutional Ethical Review Board of the Universidade Feevale (protocol Nº 33202820.7.1001.5348), following Brazilian regulations and international ethical standards. RNA was extracted from naso-oropharyngeal swab samples and reverse transcription reaction was performed using the SuperScript IV reverse transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA). Preparation of the whole viral genome library was performed using the QIAseq SARS-CoV-2 Primer Panel paired for library enrichment and QIAseq FX DNA Library UDI Kit, according to the manufacturer instructions (QIAGEN, Hilden, Germany). Sequencing was implemented in an Illumina MiSeq platform using MiSeq Reagent Kit v3 (600-cycle) from Illumina Inc. (Foster city, CA, USA). All procedures were conducted in a laminar flow to minimize the risk of contamination. The FASTQ reads were imported to Geneious Prime, trimmed (BBDuk 37.25), and mapped against the reference sequence hCoV-19/Wuhan/WIV04/2019 (EPI_ISL_402124) available in the EpiCoV database from GISAID 13 . A total of 97 Brazilian SARS-CoV-2 complete genomes and the reference sequence (EPI_ISL_402124) (>29 kb) were retrieved from the GISAID database and aligned with the sequences generated herein. Sequence alignment was performed using the Clustal Omega software and the reference sequence from Wuhan was applied as the outgroup. The Maximum Likelihood phylogenetic analysis under the General Time Reversible model, allowing for a proportion of invariable sites and substitution rates were inferred empirically in IQ-TREE v2.1.2 web server 14 especially for maximum-likelihood (ML, applying 200 replicates and 1,000 bootstraps. A 39-years-old male patient, presenting with comorbidities (chronic cardiovascular disease and diabetes mellitus) reported two clinical episodes of COVID-19. The first one was on November 30 th , 2020, and the second one on March 11 th , 2021. During the first infection period, the patient's symptomatology was not reported. However, the patient claimed to have had contact with his brother, who had previously tested positive for SARS-CoV-2. He had also visited his SARS-CoV-2 infected father at the hospital in a room shared with other COVID-19 diagnosed patients and, despite the efforts, it was not possible to know if the patient has had previous contact with any traveler from Manaus, since the region is highly known for trade and tourism. In the second infectious episode, the patient experienced dyspnea, fatigue and respiratory distress and an oxygen saturation < 95% as a clinical sign. The second infection evolved with complications, and the patient was admitted to an Intensive Care Unit (ICU) and intubated due to a severe loss of pulmonary capacity. The patient unfortunately died on March 19 th , 2021, 12 days after the onset of symptoms. Both diagnostic RT-qPCR assays were positive, presenting a Ct value of 30.07 (LMM38991) and 18.83 (LMM50731). The differential RT-qPCR retrospective study resulted in 226 negative samples and 122 positive samples for the P.1 variant. Except for the reinfected patient (who was not analyzed according to the CT value), the circulation of P.1 in Rio Grande do Sul State was evidenced in our sampling from January 27 th , 2021 on ( Figure 1A) . Furthermore, the peak of P.1 lineage detections coincides with the peak of SARS-CoV-2 new cases ( Figure 1B) , hospitalizations ( Figure 1C ) and deaths ( Figure 1D ) in the neighboring region. Two high-quality SARS-CoV-2 whole-genome sequences, named LMM38991/2020 and LMM50731/2021 (EPI_ISL_1630158 and EPI_ISL_1629809) were recovered from the same patient, corresponding to the first infection and the reinfection, respectively. Consensus sequences LMM38991/2020 and LMM50731/2021 presented a mean coverage of 1,405x and 1,263x. The sequences were first classified using the Pangolin COVID-19 Lineage Assigner tool 15 indicating the presence of two discordant SARS-CoV-2 lineages: the P.1 lineage in the primo-infection (LMM38991/2020; Gisaid access EPI_ISL_ EPI_ISL_1630158) and the P.2 lineage (LMM50731/2021; Gisaid access EPI_ISL_1629809) in reinfection. The phylogenetic analysis confirmed previous results. LMM38991/2020 was clearly clustered with the P. This study described a COVID-19 primo-infection caused by a P.1 lineage followed by a reinfection by a P.2 lineage in a three-month interval. A male, 39-years-old patient with history of comorbidities, presented two clinical episodes of COVID-19. Few reinfection cases were described worldwide, especially with detection of different SARS-CoV-2 lineages 16 . In addition, there are apparently more cases reporting asymptomatic/mild disease during reinfection episodes 17 , although some cases showed a more severe illness in the second episode 16,18 as the one described here. These evidences are reinforced by the apparent low viral quantification determined by RT-qPCR in the primoinfection. Viral immune evasion or limited and transitory protective immunity might be the cause of this reinfection as observed in the last cases to date 7 , especially emerging lineages reinfections (P.1 and P.2) that might also reflect the ability of S:K484 virus to escape from anti-SARS-CoV-2 neutralizing antibodies. Although P.1 and P.2 variants have been related to antibody evasion in patients previously immunized by non-mutated lineages 6 in the case described herein, the patient may have produced antibodies that included the S:484K site, but apparently these antibodies did not prevent a second infection by the P.2 lineage. The P.1 lineage has most probably emerged in Brazil in middle November 2020 in Manaus, Northern region 9,11 . However, recent studies have shown an earlier possible origin for an ancestral P.1 in mid-August 2020 and for the nowadays common P.1 variant in mid-October 10 . Our findings showed that the P.1 lineage was present in Southern Brazil soon after, in late November. Later, other early detections of the P.1 lineage were described in other Brazilian States, such as Bahia, Salvador city 19 . According to our retrospective analysis, based on differential qRT-PCR, it is important to note that despite the case reported herein, the P.1 lineage did not spread in a first moment, as observed in other Brazilian cities and also in other countries. In Argentina, the first P.1 case was reported on February 8 th , 2021, but the variant consistently spread between March and April, 2021 20 . Expressive number of positive P.1 cases were found only at the end of January in Southern Brazil, that replaced the parental lineages thereafter. In summary, this study reported and characterized an early primary COVID-19 caused by the P.1 lineage followed by a reinfection episode, in Southern, Brazil some months later. The P.1 lineage is spreading rapidly across Brazil 21 and after its establishment, has been related to an exponential increment of transmissibility and hospitalization rates 11 as observed in our data. It is important to understand the new lineages origin, but since the patient made several personal contacts, including some with close family members that were also infected with SARS-CoV-2, there was a higher risk of infection. Despite our efforts, due the high number of infected contacts that our patient had prior to and during the first infection, it was impossible to establish the source of infection. Nevertheless, reinfection studies are essential to understand whether these are isolated or widely distributed cases 17 . These cases have attracted considerable attention since they indicate that SARS-CoV-2 infections do not uniformly confer protective immunity for infected individuals. We gratefully acknowledge the following Authors from the Originating laboratories responsible for obtaining the specimens, as well as the Submitting laboratories where the genome data were generated and shared via GISAID, on which this research is based. All Submitters of data may be contacted directly via www.gisaid.org. Authors are sorted alphabetically. 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Detection of SARS-CoV-2 lineage P.1 in patients from a region with exponentially increasing hospitalisation rate The authors have no conflict of interests to declare.