A One-Year Follow-Up Cohort Study of Ambulatory Patients with SARS-CoV-2 Infection: The Landscape in Mexico

: Background and Objectives : SARS-CoV-2 is the pathogen that causes COVID-19 disease. Although the rate of COVID-19 reinfection is significant, the possible factors associated with this condition remain to be fully elucidated. The aim of the study was to identify clinical and serological factors associated with SARS-CoV-2 reinfection. Materials and Methods : We followed up on 120 patients with mild COVID-19 for one year. Various clinical data were collected


Introduction
In December 2019, several health facilities in Wuhan, China, reported the occurrence of patients with pneumonia of unknown cause, which resembled SARS-CoV or MERS-CoV infections [1].Although the initial cases seemed to be due to zoonotic transmission, it soon became clear that efficient human-to-human transmission was occurring [2].Later, it was described that a novel virus, SARS-CoV-2, is the causative agent of this condition, which was called COVID-19 [3].
As it has been widely described, the clinical presentation of COVID-19 ranges from asymptomatic infection to severe viral pneumonia with respiratory failure and death.While most people infected have a good prognosis and experience mild to moderate respiratory COVID 2024, 4, 848-858.https://doi.org/10.3390/covid4070057https://www.mdpi.com/journal/covidillness, some patients become severely ill, with a significant risk of death.It has also been reported that patients with different underlying medical conditions, as well as nonvaccinated and elderly individuals, are at a higher risk of severe disease and death [4].
Reinfection with SARS-CoV-2 is not unusual and can be caused by the same or by a different virus variant [5][6][7][8].Depending on the population and the criteria used for diagnosing COVID-19 reinfection, the frequency ranges from 6% to 17% in various reports [9][10][11].
According to the National Institute of Health of Mexico (INSP), by the end of 2020, 24.9% of the Mexican population had been infected by SARS-CoV-2 [12].One year later, another serologic surveillance report indicated that 57.9% of the population had been infected [13].By the end of November 2022, the figure rose to 94.4% [14].According to the Mexican Government, as of epidemiological week 37 of 2023, there had been 334,801 registered COVID-19 deaths, with an excess mortality of 638,381 deaths [15].
After the first year of the pandemic, the number of official cases per million inhabitants reported by the United States, Brazil, Peru, and Germany were 4.98, 2.96, 2.34, and 1.76 times higher than those reported by Mexico.However, when examining the official deaths per million inhabitants, these numbers change significantly.In this context, only Peru had more deaths than Mexico (2.20 times more), whereas the United States, Brazil, and Germany had 0.93, 0.72, and 0.56 times fewer deaths than Mexico [16].
In Mexico, the vaccination strategy began during the second wave of COVID-19 at the end of 2020.Initially, the first groups to receive vaccines were the population aged over 60 and healthcare workers.Subsequently, the vaccination program expanded to include the age group 50-59, followed by 40-49, 30-39, 20-29, and then 11-19, with the final group being 5-10.Mexico employed several vaccines in its national vaccination strategy, including those produced by Pfizer/BioNTech, AstraZeneca, CanSino, Sinovac, Abdala, and Sputnik [17].
Since most people who have had a COVID-19 episode produce antibodies against the causal agent, it would be expected that they have a very low risk for reinfection in the near future.However, it has been shown that, in most cases, the levels of neutralizing anti-SARS-CoV-2 antibodies decay rapidly in early convalescence [13].Likewise, the titers of this type of antibody show a significant decline a few months after receiving currently available COVID-19 vaccines [18].Early studies revealed that COVID-19 immune protection lasts about 6-8 weeks [19].For example, a multicentric study from the United Kingdom reported a reinfection rate of 1.9% [20].In India, the reinfection rate before 2022 was 4.5% [21].Until early 2022, SARS-CoV-2 reinfection was rare.However, the emergence of viral variants has led to an increase in reinfections.Additionally, it is important to note that immunity from seasonal coronaviruses is short-term.
Although it is widely known that infections can still occur in individuals with a complete vaccination schedule, it is crucial to study the risk of reinfection, especially in a country like Mexico, where the impact of the pandemic has been profound, and the vaccination strategy was heterogeneous.
In order to gain further insight into the possible factors associated with resistance to SARS-CoV-2 reinfection, we conducted a prospective study of 131 ambulatory patients after their first episode of COVID-19.A secondary objective of this study was to examine the serological response to primary SARS-CoV-2 infection and vaccination.Additionally, the study aimed to determine whether the symptomatology of reinfections differed from that of initial infections.This study began collecting data when almost 25% of the population had been infected with SARS-CoV-2, and the vaccination strategy had just started.Therefore, the context of our population was quite different from that of most other countries.

Individuals
In a prospective cohort study with a one-year follow-up, 131 ambulatory COVID-19 patients who attended the Research Center for Health Sciences and Biomedicine (CICSaB) for a SARS-CoV-2 diagnostic in the city of San Luis Potosí, SLP, México, were included.All patients had no history of a previous COVID-19 episode, and their recruitment took place from 4 to 31 January 2021.All patients were asked about the presence of co-morbidities such as systemic arterial hypertension, diabetes mellitus, asthma, COPD, and hypothyroidism.In all cases, a monthly follow-up phone interview was conducted during the time of the study, and at days 21, 60, 90, and 180, additional nasal swabs and blood samples were obtained for SARS-CoV-2 and anti-SARS-CoV-2 antibody detection.If any of the subjects included in the study experienced symptoms of COVID-19 reinfection, at that time, an additional nasal swab and blood sample were obtained.This study was approved by the Ethics Committee of the State of San Luis Potosí, México (SLP/09-2020).

SARS-CoV-2 Detection
Nasal swabs were obtained from both nostrils, and viral RNA was extracted by using the QIAamp Viral RNA Kit (Qiagen N.V., Venlo, Zeeland, Netherlands) in the QIAcube Connect system (Qiagen N.V., Venlo, Zeeland, Netherlands).Then, RT-qPCR with the COVIFLU Kit test (Genes2life, Irapuato, Guanajuato, Mexico) and the QuantStudio 5 Real-Time PCR System (Bio-Rad Lab Inc., Hercules, CA, USA) was performed.The test was considered positive when gene amplification was observed before cycle 38.

Antibody Detection
The presence of anti-SARS-CoV-2 antibodies in serum samples was analyzed by ELISA by using a kit from EUROIMMUN (Perkin-Elmer Co., Waltham, MA, USA), which detects IgG antibodies against the S protein of SARS-CoV-2.

Statistical Analysis
The outcomes of reinfected and non-reinfected and seropositive and seronegative individuals were compared by using the appropriate statistical tests.Categorical variables were analyzed with the chi-square or Fisher exact test.Continuous variables were described using either the arithmetic mean and SD (data with normal distribution) or the median and interquartile range (IQR) (data without normal distribution).The normality of the data was determined using the Shapiro-Wilk test.The comparison of continuous variables was performed using the Student t-test or the Mann-Whitney U test.A p-value less than 0.05 was considered significant.The statistical analysis was performed using SPSS version 28.0.

Basal Clinical and Demographic Characteristics
In January 2021, 131 patients who had a positive molecular test for SARS-CoV-2 were recruited for the study, and 120 of them completed the one-year follow-up.Most of them were female (68.3%), with a median age of 37 years, and 42.5% were overweight (body mass index > 24.9).In addition, 73.3% had at least one co-morbid condition (aside from overweight) (Table 1).The co-morbidities reported by the patients were arterial hypertension (8.3%), diabetes mellitus type 2 (3.3%), and asthma (7.5%).In most cases (70.8%), patients reported previous contact with individuals positive for SARS-CoV-2.Ninety-five percent of the patients reported at least one symptom related to COVID-19 (Table 1).
At the time of their recruitment, 35% of the patients were seropositive for IgG anti-Spike antibodies, while at day 21, 73.5% of them were seropositive, with a median index of 4.25 (IQR, 0.98-6.22).Although the proportion of seropositive individuals remained unchanged at days 60 and 90 (Table 2), the antibody levels significantly decreased at these times (62.6% and 63.3%, respectively, p < 0.05 in both cases).However, at day 180, there was a significant increase in the IgG anti-Spike antibody levels (15.7%, p < 0.05 compared to days 21, 60, and 90, Figure 1).

Reinfections during Follow-Up
Throughout the one-year follow-up, a total of 46 episodes of acute respiratory tract infection were observed among the participants, with 11 episodes being identified as COVID-19 reinfections.The reinfections occurred between May 2021 and January 2022, during which the predominant circulating virus variants were Delta and Omicron.The median time interval between the first and second infections was 253 days (range: 108-

Reinfections during Follow-Up
Throughout the one-year follow-up, a total of 46 episodes of acute respiratory tract infection were observed among the participants, with 11 episodes being identified as COVID-19 reinfections.The reinfections occurred between May 2021 and January 2022, during which the predominant circulating virus variants were Delta and Omicron.The median time interval between the first and second infections was 253 days (range: 108-370).Only one case of a second reinfection was observed, occurring 128 days after the first reinfection.
In order to identify the possible factors associated with reinfection, we compared the clinical and serological characteristics at recruitment of the non-reinfected (n = 110) and reinfected individuals (n = 10) (Table 3).No demographic characteristics were found to be associated with reinfection (Table 3), and no significant differences in the time of recruitment between patients with and without reinfection (10 and 9 days, respectively; p = 0.474) were detected.Furthermore, the duration and frequency of symptoms during the first infection in the reinfected and non-reinfected groups were similar (Table 3).Comparing the frequency of symptoms between the first infections and the symptoms during reinfections, it was noted that anosmia and dysgeusia were less frequent during reinfections, while conjunctivitis was more frequent (p < 0.05 in all cases, Figure S1).Additionally, significant differences were detected in the seropositivity rate and IgG levels at day 90 when the two groups were compared (p < 0.05 in both cases; refer to Table 4 and Figure 2).Interestingly, a seropositive status at day 90 was associated with protection against reinfection (OR: 0.26, 95% CI: 0.07-0.93,p = 0.038).Equally interesting, no apparent relationship was observed between vaccination status and reinfection or between seropositivity and vaccination at day 90.When the characteristics of these two groups were compared, significant differences were observed in age, the time between recruitment and symptom onset, and the presence of headache, sneezing, anosmia, nasal discharge, and nausea (Table 5, Supplementary Figure S1).Furthermore, the levels of IgG anti-SARS-CoV-2 antibodies at days 21, 60, and 90 were higher in the group of patients who were seropositive at day 90 (Table 5).Patients with headache, sneezing, anosmia, and runny nose had a 3.7-fold likelihood of seropositivity at day 90 (p = 0.009).Finally, at day 180, 76.7% of the participants were vaccinated (Supplementary Table S1), and at this moment, vaccinated subjects showed an 18-fold likelihood of being seropositive (3.55-91.26CI, p < 0.05).Eight out of the ten reinfected patients received at least one dose of the Pfizer-BioNTech COVID-19 vaccine before the reinfection, and no significant differences were observed in the type of vaccine received by non-reinfected and reinfected individuals (Table 4).
This work was conducted during the second year of the pandemic, when variants were significantly different from the original virus.Therefore, one of the main limitations of this study was not following the participants for a second year, when variants such as Alpha, Beta, Delta, Omega, etc., began to be detected.Another limitation of the study was the heterogeneity of the vaccines administered to our population.Despite this, the study provides an understanding of the clinical and serological behavior before the emergence of the variants and in a country with few interventions aimed at reducing transmission at the population level.

Discussion
The frequency of reinfections during the COVID-19 pandemic has raised several questions about the magnitude and lifespan of the immune response against the SARS-CoV-2 virus after the primary infection, as well as the possible role of other risk factors.Despite the duration of COVID-19, at the moment of writing this paper, fewer than ten articles have performed cohort studies of similar populations in other countries.
There are studies in which similarities in the frequency of reinfections have been reported.Among healthcare personnel in nursing homes, a frequency of 2.5% over a 12-month period has been reported [18], and in other studies, it has been reported at 2.2% [19].The frequency varies according to seropositivity, with a higher frequency reported in seronegative people (8.4% vs. 1.9%) [18].Our study showed a higher overall reinfection rate (9.2%) with primary risk factors linked to serologic status at days 60 and 90 and the IgG antibody index at day 90.Despite a higher rate, our study had a longer median time between the first infection and reinfection (253 days), potentially influenced by prevailing SARS-CoV-2 variants.
As expected, the titers of IgM and IgG antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 decreased significantly over a period of approximately six months [21].The same has been reported with anti-nucleocapsid antibodies, showing a decline after 16 weeks, while titers against S-proteins continue to rise and reach a plateau at around six months [22].In our study, antibody waning was observed after day 21, but after that waning, the antibody index remained stable at days 60 and 90.However, due to the vaccination of the study population, it was not possible to establish a clear plateau, as seen in other studies [23].In our study, the seropositivity rate and IgG index at day 90 were not related to vaccination status.
The timing and magnitude of antibody waning after day 21 could be important characteristics of our population.This waning could provide one explanation for the high infection rate and mortality in Mexico.It could demonstrate the presence of immunological differences between populations in their response to SARS-CoV-2.
On the contrary, the increase in the IgG index and seropositivity rate at day 180 was associated with SARS-CoV-2 vaccination.At that point, 81.8% of seropositive patients and 20% of seronegative patients were vaccinated.Seropositive patients at day 180 consistently had higher antibody levels throughout the follow-up period.The seropositive patients at day 180 exhibited a 2.23 times higher antibody index compared to day 90, while there was no significant increase in the antibody index between days 90 and 180 in seronegative patients despite vaccination.This differential response to vaccination could also play a role in the infection rate, in conjunction with the vaccination strategy employed in our country.Additionally, participants who did not mount an adequate immune response to the infection at day 90 also exhibited lower antibody levels in response to vaccination.
Our study found a lower seropositivity rate at day 21 (73.5%) compared to a US cohort (89%) [24], with differences potentially attributed to higher median age, more males, and longer symptom duration in their subjects.Notably, an unexpected association between older age and seropositivity was observed, contradicting the conventional understanding of immune senescence.Despite older age being a known risk factor for severe COVID-19 [25], the intriguing association and the observed resistance to reinfection among seropositive individuals in our study call for further investigation to unravel the underlying factors and implications for COVID-19 outcomes.
We identified several symptoms associated with seropositivity at day 90.Notably, the presence of a headache, sneezing, anosmia, and a runny nose together was strongly associated with seropositivity at day 90, which appeared to be the most significant moment for determining seropositivity against reinfection.Consequently, the presence of these symptoms could serve as predictors of seropositivity for patients who cannot undergo serological testing.
Our cohort study indicated that the main factor related to reinfection could be the initial IgG response to the virus.The response to vaccination could be affected by the initial seropositive response at day 90 of a natural infection.Despite the observed antibody waning in our population, vaccination increases the antibody index but does not provide full protection against reinfections.Also, the symptoms elicited during reinfection could depend on the virus variant.Future studies are expected to provide further insights into differences between natural and vaccine-acquired immunity and the impact of new strains on the risk of reinfection.

Conclusions
During a one-year follow-up period, reinfection was observed in 9.2% of individuals previously infected with the virus.By day 21, 73.5% of patients showed seropositivity, with a median antibody index against the spike protein of 4.25 (IQR = 0.98-6.22).However, there was a significant decrease in IgG index levels by days 60 and 120 during the follow-up period.Notably, at day 90, there were discernible differences in seropositivity and the IgG index between those who experienced reinfection and those who did not within the cohort, suggesting that current seropositivity confers protection against reinfection (OR 0.26, 95%CI 0.07-0.93,p = 0.038).The median time to reinfection was 253 days, with a range of 108 to 370 days.Interestingly, no other variables studied were found to be associated with the risk of reinfection.These findings underscore the importance of monitoring antibody levels and seropositivity status in assessing protection against reinfection over time.

Figure 1 .
Figure 1.A comparison of the IgG anti-S protein index.The results of the positivity rate and antibody index are shown for basal and days 21, 60, 90, and 180.The highest index was observed at day 21.After that day, waning is observed for the index value.The increase observed on day 180 is associated with vaccination (Mann-Whitney U test.* p = 0.01; ** p < 0.000001).

Figure 1 .
Figure 1.A comparison of the IgG anti-S protein index.The results of the positivity rate and antibody index are shown for basal and days 21, 60, 90, and 180.The highest index was observed at day 21.After that day, waning is observed for the index value.The increase observed on day 180 is associated with vaccination (Mann-Whitney U test.* p = 0.01; ** p < 0.000001).

Figure 2 .
Figure 2. IgG index against S protein of SARS−CoV−2.A comparison between median indexes for reinfected (n = 11) and non-reinfected groups (n = 109) during follow-up is shown (+ = positive and

Table 1 .
Demographic data of the patients included in the study at the time of their recruitment.