Risk factors for severity, viral load, and outcomes in patients with COVID-19

Introduction

In 2019, cases of pneumonia of unknown origin due to a new RNA betacoronavirus, currently severe acute respiratory syndrome coronavirus 2 (SARS CoV2), were identified in Wuhan. The World Health Organization declared an international public health emergency and named it coronavirus disease 2019 (COVID-19)¹. The index case in Mexico was detected in February 2020 in Mexico City. By March, the Ministry of Health confirmed the first death from COVID-19, which occurred at the Hospital Angeles Metropolitano².

SARS-CoV-2 targets nasal and bronchial epithelial cells through the glycosylated spike protein that binds to the angiotensin-converting enzyme receptor 2 (ACE2), this interaction is a potential factor in its infectivity. Transmembrane serine protease Type 2 (TMPRSS2), in the host cell, promotes viral uptake^2,3.

Viral load in the upper respiratory tract appears to reach the highest value at the time of symptom onset⁴. The mean incubation time is 4-7 days after exposure. About 97.5% develop symptoms within the first 11 days⁵.

The average age of hospitalized patients is 47-73 years. Early cohorts at the pandemic had a male preponderance of 60%². Common symptoms include fever, cough, dyspnea, fatigue, myalgias/arthralgias, gastrointestinal symptoms, headache, and rhinorrhea. Anosmia or ageusia may be the only presenting symptom^2,5. About 81% had mild manifestations, 14% severe and 5% had critical manifestations (respiratory failure, septic shock, and/or multiple organ dysfunction)⁶. About 15% of patients will develop severe disease and 5% will require mechanical ventilation⁷. Complications of COVID-19 include multiple organ dysfunction, myocarditis, cardiomyopathy, ventricular arrhythmias, and hemodynamic instability⁸. About 60-90% of hospitalized infected patients have comorbidities; systemic arterial hypertension (SAH), diabetes mellitus 2 (DM2), obesity, cardiovascular disease, lung disease, renal disease, malignancy, liver disease, or autoimmune diseases⁹.

The gold standard for COVID-19 diagnosis is by detection of SARS-CoV-2 RNA by reverse transcription polymerase chain reaction (RT-PCR) of respiratory samples by nasal swab. However, the sensitivity of the tests varies over time in relation to exposure. Due to false-negative rates, clinical, laboratory, and imaging findings may also be used for presumptive diagnosis¹⁰. RT-PCR assays provide information on whether or not SARS-Cov-2 is detected. However, these assays also contain quantitative information on cycle threshold (Ct) values that correlate inversely with viral load. Immunoglobulin M antibodies are detectable 5-day post-infection, with the highest levels during weeks 2-3 of illness, while an Immunoglobulin G response is observed 14 days after symptoms onset^11–13. The most common hematologic abnormality is lymphopenia (< 1.0 × 10⁹/L), present in 83% of hospitalized patients^14,15. Most laboratory characteristics are nonspecific¹⁶.

In a meta-analysis of 703 patients with COVID-19 (553 symptomatic and 150 asymptomatic), it was concluded that there are no differences in viral load between symptomatic and asymptomatic. Asymptomatic subjects may represent a reservoir of infection and contribute significantly to the maintenance of the pandemic¹⁷.

The study aimed to correlate clinical characteristics and outcomes with viral load for SARS-COV2.

Materials and methods

We designed an observational, cross-sectional, and descriptive study that collected data from patients with confirmed COVID-19 from Hospital Angeles Metropolitano during the first wave of contagion in Mexico, from March 13, 2020, to January 12, 2021. Clinical and demographic variables, laboratory studies, as well as viral load and outcomes (discharge home, transfer to COVID-19 hospital, admission or death) were collected. Detection of SARS-Cov-2 was by RT-PCR, reporting it with amplification cycles, being stratified by the laboratory as mild, moderate, and high viral load. Data were collected retrospectively from clinical records. The study was approved by the bioethics committee.

A descriptive statistical analysis was performed using mean and standard deviation. For categorical or dichotomous variables, absolute frequencies and percentages were used. The hypothesis tests for categorical variables were the Chi-square or (Fisher’s exact test) and for linear variables the Student’s t-test for unrelated samples. Relevant variables to discriminate severe cases were used to construct multivariate binary logistic regression models to identify variables independently associated with the presence of severe cases. The strength of association was measured by calculating odd ratios (OR) with a 95% confidence interval (CI). The construction of regression models included a first phase in which the variables identified in the univariate comparison were introduced and subsequently, we used the forward stepwise method to include variables with a value of p = 0.02 in the iterations for the construction of an alternative model. A two-tailed p < 0.05 was considered significant for hypothesis testing. The statistical package IBM Statistics SPSS v21.0 was used for the data analysis.

Results

Data from 325 patients with an average age of 51 ± 17 years were included in the study. Of which 52.9% were male and 45.8% female. Detection of SARS-Cov-2 was by RT-PCR, from a nasopharyngeal swab, taken at the first medical contact, within 3-10 days of symptoms onset. A viral load by amplification curve estimate 28 ± 6.8 cycles. They were classified as mild viral load estimate > 30 Ct, moderate 21-35 Ct, and high < 25 Ct in 29.2, 41.8 and 28.3%, respectively. Demographic characteristics, comorbidities, and symptoms at admission are detailed in Table 1.

At the outcome analysis, it was determined that 42.9% of the patients were in conditions to be discharged, the remaining 57.1% were severe cases, of which 45% were admitted, 51.1% were transferred to another center and 3.3% died. The association between viral load estimation and outcomes is summarized in Table 2. When comparing the characteristics between severe and outpatient cases, statistically significant differences were found, especially due to the presence of comorbidities and alterations in laboratory studies. All comparisons are shown in Table 3. Due to the stratification finding of in the laboratory reports of a moderate viral load of 21-35 Ct, a cutoff value of 25 Ct was considered for the rest of the comparisons.

Multivariate analysis revealed that the independent risk factors associated with severity were age over 65 years OR = 1.38 (95% CI 3.05-11.4, p < 0.01); dyspnea OR = 5.91 (3.05-11.44, p < 0.001); vomiting OR = 3.8 (1.29-11.4, p = 0.015), viral load > 25 OR = 3.07 (1.35-6.9, p = 0.007), a protective effect was found due to the presence of headache OR = 0.32 (0.22-0.58, p < 0.001); odynophagia 0.41 (0.22-0.78, p = 0.006), and anosmia/hyposmia OR = 0.05 (0.015-0.23, p < 0.001). Model A and B (Table 4 and Table 5).

Discussion

At the beginning of the COVID-19 pandemic in Mexico, different containment measures were implemented, and new work and logistics plans for medical care were established. Some institutions, hospitals, and some temporary units were designated to be 100% care centers for COVID-19 cases, others 100% free of COVID-19 cases, and some that worked in a hybrid manner. At the beginning of the pandemic, the Hospital Ángeles Metropolitano provided its services to COVID-19 cases, subsequently different work plans were delegated within Grupo Ángeles to safely provide service to non-COVID-19 cases, so the cases that warranted hospital admission due to SARS-CoV2 infection were transferred to the hospitals assigned for COVID-19, those who did not merit admission were discharged home with alarm and isolation data. For this reason, most of the patients in our study were evaluated in the emergency department where their outcome was determined: discharge, admission, transfer, or death. The patients in our study were the first ones to be evaluated at the hospital since the beginning of the pandemic, so they are patients infected with the first variant of COVID-19 and without SARS-CoV2 vaccine.

We introduce an epidemiological study that analyzes demographic characteristics, which, in terms of age and sex, the characteristics are similar to what was published in the Mexican population. During the first stage of the pandemic, a predominance of the male sex was found, with an average age between 40 and 60 years. The most frequent comorbidities were SAH, DM2, and lung disease. With the exception of obesity, which predominates in most published studies, the majority of our population was only overweight. The most frequent symptoms referred to admission were cough, fever, fatigue, myalgias/arthralgias, headache, and dyspnea in successive order, which also coincides with what is reported in the literature. In our study, a significant difference was found in the presence of dyspnea and vomiting, as well as age over 65 years for severe cases. Unlike the presence of headache, odynophagia, anosmia, or hyposmia, which were significant in the outpatient’s group, so the presence of these symptoms in the initial assessment could suggest the course of a mild or moderate condition.

There are several proposed theories of the mechanisms for anosmia, inflammation of the epithelium of the nasal mucosa, apoptosis of olfactory cells, changes in olfactory cilia, damage to the olfactory bulb, olfactory neurons, and totipotent cells of the olfactory epithelium¹⁸. One of the mechanisms of anosmia is due to the ACE2, which is a receptor for COVID-19, and could be present in large quantities in the olfactory tract. The union of the virus with these receptors causes the release of cytokines and greater inflammation of the epithelium¹⁹.

Sven Saussez et al. tried to answer why anosmia seems to be less prevalent in severe cases than in mild-to-moderate cases. In a European study of 417 patients with mild-to-moderate COVID-19, 86% reported anosmia and 88% dysgeusia. In a series of 2013 patients with the same conditions, 87% reported anosmia. In Saussez et al. study, of 86 outpatients with mild-to-moderate symptoms of COVID-19 associated with subjective anosmia, psychophysical measurements of smell were performed “Snifin Sticks,” they found 47.7% anosmic, 14% hyposmic and 38.3% without olfactory alteration²⁰. Romero-Sánchez et al., In a series of 841 patients with severe COVID-19 with in-hospital management, 57.4% developed some neurological symptoms and only 5% presented anosmia²¹.

Such findings may suggest that in patients with efficient local immunity, virus replication in the nasal and olfactory mucosa may provoke a local inflammatory reaction and a pattern of otorhinolaryngological symptoms, unlike severe patients, where such symptoms were less frequent. Thus, patients with a mild form of infection have stronger local immunity and therefore higher rates of local symptoms. Moreover, the severe forms of infection, or weaker local immunity, fewer otorhinolaryngological, and more respiratory symptoms.

It should be considered that in our study, age was also significant, being higher in severe cases, in older patients the olfactory function decreases. Therefore, it may be more difficult to detect deterioration in olfactory function, either due to age or by the severity of other non-otorhinolaryngologic symptoms.

Several studies have been carried out comparing viral load in symptomatic and non-symptomatic patients, in which there was no significant difference, thus understanding that transmissibility is the same in both cases.

Our hypothesis was that evaluating the SARS-Cov-2 viral load through Ct analysis of an initial sample could be a valuable clinical tool to identify patients at higher risk of intubation and death. In our study, the viral load was significant for severe cases, since those with > 25 were associated with the need for hospitalization, transfer to intensive care units, intubation, and even higher mortality. This differs from a retrospective study in New York where the relationship between Ct and severity was demonstrated, where the value of Ct < 25 was independently associated with in-hospital mortality and the need for intubation. In this study, Magleby et al. stratified the Ct value as follows: high Ct < 25, medium Ct 25-30, and low Ct > 30²². In a Mexican population study where 21,110 confirmed cases of COVID-19 were included, they found that 53% had a high viral load, hospitalization was more frequent in subjects with a medium viral load and deaths were more prevalent in those with a high viral load²³. In a Mexican pediatric population study, 176 positive tests were identified, of which Ct values were observed with a median of 31.9-34.01, 13.1% presented very high viral load, 9.1% high, 14.2% medium and 63.6 % low, no relationship with sex or age was found²⁴.

With the findings in our study, we could deduce that severe cases may have less risk of transmissibility than outpatient cases; however, they continue to be highly contagious. High viral load Ct < 25 was not associated with increased severity. Determining the Ct number in positive PCR tests could help at the clinical practice when interpreted in the right context.

By carrying out the study before vaccination and generation of other COVID-19 variants, it was possible to perform a homogeneous clinical evaluation, allowing adequate statistical analysis with clinical characteristics. Among the weaknesses of the study, the PCR was performed in the first medical evaluation, so it could be different stages of the disease.

We propose that our findings are due to the role of innate immunity as a first line of antiviral defense. Innate immune responses limit viral entry, translation, replication, and assembly, help identify and eliminate infected cells, and coordinate and accelerate the development of adaptive immunity. Cell surface, endosomal, and cytosolic pattern recognition receptors respond to pathogen-associated molecular patterns to trigger inflammatory responses and programmed cell death that limit viral infection and promote clearance. Excessive immune activation can cause systemic inflammation, which adds to that caused by obesity or smoking damage, causing serious diseases, highlighting the balance that must be achieved. Hence, these mechanisms could influence the viral load value obtained, as well as the different clinical conditions. So we know that, the protective factors are the elevation of anti-inflammatory cytokines, antibody response and T-cell activation, as well as the low expression of ACE2. Unlike risk factors with increased pro inflammatory cytokines, increased ACE2 level, and TMPRSS2 expression²⁵.

Conclusion

The presence of anosmia/hyposmia, odynophagia, and headache was more frequent in non-severe cases, which suggests better local immunity. Patients with more severe symptoms were associated with older age > 65 years, dyspnea, vomiting, and viral load Ct > 25. The findings on viral load may suggest less risk of viral transmissibility in patients under in-hospital management compared to outpatients. Further studies are needed to establish the correlation of viral load with severity and outcomes. Innate immunity, as well as adaptive immunity, plays a fundamental role in the evolution of SARS-Cov2 infection.

Funding

The authors declare that they have not received funding.

Conflicts of interest

The authors declare no conflicts of interest.

Ethical disclosures

Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.

Confidentiality of data. The authors declare that no patient data appear in this article. Furthermore, they have acknowledged and followed the recommendations as per the SAGER guidelines depending on the type and nature of the study.

Right to privacy and informed consent. The authors declare that no patient data appear in this article.

Use of artificial intelligence for generating text. The authors declare that they have not used any type of generative artificial intelligence for the writing of this manuscript nor for the creation of images, graphics, tables, or their corresponding captions.