Prognostic Value of Neutrophil-to-Lymphocyte Ratio and Vaccination for Negative Conversion Time of Nucleic Acid in Nonsevere COVID-19 Patients Infected by SARS-CoV-2 Omicron Variant

SARS-CoV-2 Omicron variant is significantly different from all the previous variants and has rapidly replaced other variants as the dominant variant across the globe. An easily obtained, inexpensive, and rapid marker is needed to predict the negative conversion time (NCT) of nucleic acid in nonsevere COVID-19 patients infected by the Omicron variant. This retrospective study enrolled 226 patients infected by the Omicron variant between April 23, 2022, and May 16, 2022. The median age of the patients was 61 (interquartile range (IQR), 48–70) years, and 56.2% were male. 84 patients (37.2%) had at least one comorbidity, and 49 patients (21.7%) were classified into the moderate illness group. 145 patients (64.2%) received at least one dose of vaccine, in which 67 patients (29.6%) received a booster dose of vaccine. The median duration of NCT was 8 (IQR, 7–11) days. Univariate Cox analyses found that high NLR (>2.22), aged ≥65 years, vaccination, and moderate illness were significantly related to the NCT of nucleic acid. Multivariate Cox regression analysis showed that high NLR (NLR > 2.22, hazard ratio (HR):0.718, 95% CI: 0.534–0.964, p = 0.028) and vaccination (vaccinated ≥1 dose, HR: 1.536, 95% CI: 1.147–2.058, p = 0.004) were independently associated with NCT of nucleic acid. NLR is a rapid, simple, and useful prognostic factor for predicting NCT of nucleic acid in nonsevere COVID-19 patients with the Omicron variant. In addition, vaccination may also play a valuable role in predicting the NCT of nucleic acid.


Introduction
Since December 2019, coronavirus disease 2019 (COVID- 19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had spread rapidly to many countries [1].SARS-CoV-2 evolves through random mutations, and new mutations can potentially increase or decrease infectiousness and virulence.Te Omicron variants of concern (VOC) (Phylogenetic Assignment of Named Global Outbreak (Pango) lineage designation B.1.1.529) of SARS-CoV-2 emerged in South Africa in November 2021.It is more transmissible than other variants [2].Terefore, the Omicron variant rapidly replaced previous variants as the dominant variant across the globe [3].In late February 2022, a wave of SARS-CoV-2 Omicron variant infection rapidly appeared in Shanghai, China.Phylogenetic features of SARS-CoV-2 viral genomes from 129 patients in Shanghai indicated that all of the new viral genomes were clustered into the SARS-CoV-2 BA.2.2 sublineage.
Te detection of SARS-CoV-2 nucleic acid by reverse transcription polymerase chain reaction (RT-PCR) is still the golden standard for the diagnosis of COVID-19, and it is also of great signifcance for determining discharge and isolation.Te clinical prediction of negative conversion is critical for the proper retesting time, preventing medical waste from repeated nucleic acid tests and unnecessarily prolonged quarantine.Te negative conversion time (NCT) of nucleic acid is also important in terms of viral transmission.Te Omicron variant spreads much faster than the other variants, but the mean length of hospital stays was shorter [4,5].However, there is limited data regarding the potential predictors of NCT in COVID-19 patients with the SARS-CoV-2 Omicron variant.Terefore, we need an easily obtained, inexpensive, and rapid marker to predict the NCT of nucleic acid in nonsevere COVID-19 patients with the SARS-CoV-2 Omicron variant.
Infammation can be caused by infectious diseases, and there is a dysregulated immune response in patients with COVID-19 [6].As an independent predictor of disease deterioration and mortality in COVID-19 patients [7][8][9][10][11][12][13][14], neutrophil-to-lymphocyte ratio (NLR) is closely related to the pathophysiology of COVID-19.Te NLR is calculated as the absolute neutrophil count divided by the lymphocyte count.It is a reliable predictor of COVID-19 progression and can diferentiate between mild/moderate and severe/ critical groups.Higher NLR is associated with higher mortality.Neutrophil to CD4 + lymphocyte ratio and lower levels of CD3 + CD4 + lymphocytes were also useful blood markers in previous studies [15,16].However, the CD4 + lymphocyte test needs fow cytometry which is inconvenient to detect during a large-scale epidemic.As a simple marker of infammation, NLR can be easily obtained from routine blood tests.But the Omicron variant evolves towards being less virulent and is very diferent from the previous variants [17].Te prognostic value of NLR as a predictor of the NCT of the nucleic acid of COVID-19 patients infected by Omicron variant Omicron is unknown.
COVID-19, caused by the SARS-CoV-2 Omicron variant, has become very diferent from that in the early outbreaks in the world.Although the Omicron variant evolves towards less pathogenic, the mortality rate of immunocompromised patients infected with Omicron variant is signifcantly higher than that of nonimmunocompromised patients [18].Vaccinated immunocompromised patients had a poor humoral response.A higher rate of mortality has also been reported in unvaccinated and not fully or efectively vaccinated elderly people [19].Although the vaccines have been widely covered, vaccination rates for older people remain low.Te coverage of the elderly still needs to be improved.Te strict and comprehensive pandemic control strategies can reduce the number of elderly people infected by the Omicron variant so that the mortality rate can be minimized and we can buy time for full vaccination coverage.Like other RNA viruses, SARS-CoV-2 is evolving through random mutations, and new mutations can potentially increase or decrease virulence and infectiousness.Moreover, mutations can increase the ability of the virus to evade adaptive immune responses from past SARS-CoV-2 infections or vaccinations.Vaccination rates were not included in previous studies on the NCT of nucleic acid, and the impact of vaccines on the NCT of nucleic acid is still unknown.
Decisions on infection prevention continue to be broadly guided by strategies based on the NCT of nucleic acid.Terefore, further investigation on the duration of NCT of nucleic acid and factors associated with prolonged negative conversion conducted among larger populations may help to improve the clinical management of COVID-19 patients infected by the SARS-CoV-2 Omicron variant.In the current study, we aimed to assess the prognostic value of NLR and identify the clinical characteristics, including vaccination, that infuence the NCT of nucleic acid in nonsevere COVID-19 patients infected by the SARS-CoV-2 Omicron variant.Referring to the WHO criteria, the severity of the disease is classifed according to the following criteria.Asymptomatic infection: individuals who test positive for SARS-CoV-2 using a nucleic acid amplifcation test but who have no symptoms that are consistent with COVID-19.Mild illness: individuals who have any of the various signs and symptoms of COVID-19 but do not have shortness of breath, dyspnea, or abnormal chest imaging.Moderate Illness: individuals who show evidence of lower respiratory disease during CT examinations and who have an oxygen saturation (SpO 2 ) ≥94% in room air at sea level.Severe Illness: individuals who have SpO 2 <94% in room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO 2 /FiO 2 ) <300 mmHg, a respiratory rate >30 breaths/min, or lung infltrates >50%.Critical Illness: individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction.

Data Collection.
Patients' health information stored in the electronic medical records system was collected.We collected available clinical variables, including demographic characteristics, vaccination status, comorbidities, and ).Te optimal cut-of value for NLR was calculated by X-tile software (Yale University, New Haven, CT, USA) [22].
As time-to-event data, NCT of nucleic acid was the outcome measure and presented with Kaplan-Meier curves.Te logrank test was used for the comparison.To detect the independent predictors of NCT of nucleic acid, univariate and multivariate Cox regression analyses were performed and reported as the hazard ratio (HR) and 95% confdence interval (CI).Data were analyzed using SPSS 25.0 software (SPSS Inc., Chicago, IL).A two-tailed p value <0.05 was considered statistically signifcant.

Results
235 patients who were above the age of 18 years with complete clinical data and blood tests satisfed the inclusion criteria.Among them, 4 patients were excluded from the study for severe illness of COVID-19.Furthermore, 5 patients were excluded because of autoimmune diseases (3 patients) and malignancies under treatment (2 patients).Te study fnally included 226 patients with nonsevere COVID-19 infections.

Discussion
Increased NCT of nucleic acid is an independent risk factor for prolonged hospitalizations and quarantine [23].Several studies have evaluated the predictors of NCT infected by other SARS-CoV-2 variants before Omicron.Te Omicron variant is diferent from the previous variant, and there is limited data regarding the potential predictors of negative conversion in the Omicron variant, particularly in high-risk patients.In our study, univariate analysis revealed that clinical characteristics such as the aged (age ≥ 65 years), moderate illness, vaccination, and high NLR were signifcantly related to the NCT of nucleic acid.Comorbidities were not statistically signifcant.Multivariate Cox regression showed that high NLR and vaccination were independently associated with the NCT of nucleic acid.In this study, we reported that NLR might be a simple and useful prognostic factor in the prediction of NCT of nucleic acid in nonsevere COVID-19 patients infected by the SARS-CoV-2 Omicron variant.In addition, we also found that vaccination played a valuable role in predicting the NCT of nucleic acid.
42.0% of the patients in this study were elderly, and more than one-third of the patients (37.2%) had at least one comorbidity.In addition, 49 patients (21.7%) were diagnosed with pneumonia by CT scans and classifed into the moderate illness group.Patients enrolled in this study seem to be more likely to be in the high-risk group.In our cohort, the median duration of NCT of nucleic acid was 8 days, ranging from 2 to 19 days.Tis fnding is markedly shorter compared with previous studies focused on other mutant strains [24][25][26][27][28][29], which reported that the mean NCT of nucleic acid was longer than 10 days.Tis may be attributed to the weaker pathogenicity of the Omicron variant.Te NCT of nucleic acid in this study was similar to the result in another study on Omicron [30], in which the median time was 6 days.More recently, public health guidelines have recommended a shorter period of strict isolation from the onset of symptoms or after the initial positive test.Interestingly, there is still a proportion of patients with NCT of nucleic acid longer than 10 days, and this group of patients, if isolated for a short period, could become a source of infection and thus infect a larger population.
Symptoms such as chest tightness, fever, respiratory symptoms, and digestive symptoms have showed a good performance in predicting the NCT of nucleic acid [31][32][33].International Journal of Clinical Practice But Omicron has an increasing portion of patients with asymptomatic infection; difculties exist in studying the value of symptoms.A longer NCT of nucleic acid has been variously associated with advanced age, disease severity, delayed hospital admission, and comorbidities [25,34,35].It is worth noting that age or comorbidities did not have a signifcant relationship with prolonged NCTof nucleic acid during multivariate Cox regression analysis in our study, which is consistent with the previous study [33].Patients with certain underlying comorbidities are at higher risk of progressing to severe COVID-19 [31].However, the fndings of our study suggest that the importance of such underlying comorbidities may not be signifcant in the duration of disease, which is one of the main treatment outcomes in the group of nonsevere COVID-19 patients.Even though our study population was predominantly elderly, underlying comorbidities did not have a relationship with the NCT of nucleic acid in univariate and multivariate analyses.Tis fnding could be characteristics of asymptomatic or mildly symptomatic patients of COVID-19 infected by the SARS-CoV-2 Omicron variant.However, the results of this study cannot be applied to all patients with COVID-19 infected by the SARS-CoV-2 Omicron variant of varying severity.We demonstrated that NLR is an independent factor associated with NCT of nucleic acid, and high NLR (>2.22) would predict the delayed negative conversion in nonsevere COVID-19 patients with Omicron variant.NLR is an indicator of the systematic infammatory response and has been widely investigated as a reliable predictor of COVID-19 progression [36].SARS-CoV-2-triggered infammation increased NLR, and elevated NLR promoted COVID-19 progression.NLR is signifcantly higher in patients with severe COVID-19 [37,38].Finally, multivariate regression analysis showed that high NLR was independently associated with the NCT of nucleic acid in our study.As expected, the cut-of value of NLR in this study is lower than that for identifying severe/critical patients [39].NLR is a rapid, widely available, inexpensive marker which can be easily obtained from a simple blood test, and it can be widely used in the management of high-risk Omicron infection patients during the pandemic.
In addition, this study also found that vaccination was an independent predictor of NCT of nucleic acid, and patients in the unvaccinated group had remarkably higher risks of delayed NCT of nucleic acid than those in the vaccinated group.Te Omicron variant may evade immunity from previous vaccines or infections more extensively than any other variant, making existing vaccines less efective against the variant [40][41][42].But vaccinated people are likely to have a much lower risk of severe disease from Omicron infection.Te boosters of the COVID-19 vaccination are less efective against symptomatic Omicron infection, but they can provide strong protection against COVID-19-related hospitalization and death [43][44][45].In this study, vaccines help shorten the NCT of nucleic acid in nonsevere COVID-19 patients with Omicron infection.However, only 64.2% of the patients in this study were vaccinated, and 29.6% received a booster dose of the vaccine.Te possible reason for low vaccine coverage in our study is that patients are older and have more underlying diseases.Tese proportions are consistent with those of the whole society in Shanghai.Although overall vaccination coverage now exceeds 90%, vaccination coverage has remained low in elder adults [46].Te next challenge is to take steps to improve vaccine coverage for all people, the elder and vulnerable people in particular.
Tis study has several limitations.As a retrospective study, the study only involved nonsevere COVID-19 cases, so the conclusions cannot be employed for severe and critical Omicron cases.Undoubtedly, NLR is a traditional marker, but it is cheap, convenient, and maybe a better choice.Limited by the sample size, the distinctions between diferent doses of vaccines were not discussed, and large clinical studies are needed to confrm the protective efect of diferent doses and types of vaccines on NCT of nucleic acid.

Conclusions
In summary, the current study shows that NLR may be a rapid, simple, and useful prognostic factor for predicting NCT of nucleic acid in nonsevere COVID-19 patients with the SARS-CoV-2 Omicron variant, and it can be used extensively in the

Figure 1 :
Figure 1: Negative conversion curves estimating the cumulative probability of negative conversion of nucleic acid in total nonsevere COVID-19 patients infected by the SARS-CoV-2 Omicron variant.

2
International Journal of Clinical Practice laboratory tests.Comorbidities included hypertension, diabetes, cerebro-cardiovascular diseases, chronic respiratory diseases (asthma, chronic obstructive pulmonary disease, or interstitial lung disease), and chronic kidney disease.Laboratory tests comprised white leukocyte count, neutrophil count, lymphocyte count, monocyte count, platelet count, hemoglobin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum creatinine, and D-dimer.Te derived hematological indicator was NLR.Te blood test data collected were the results of the frst examination after admission before the negative conversion of nucleic acid tests.2.3.NCT of Nucleic Acid.Strict pandemic control strategies were taken in Shanghai, and nucleic acid detections were carried out regularly among Shanghai residents.If the nucleic acid test was positive, they would be transferred to the shelter hospitals or designated tertiary hospitals.Na- [20,21]ngeal swab specimens were collected from each patient every day during hospitalization.RNA was extracted from the samples and then underwent RT-PCR by DIAN DIAGNOSTICS Group Co., Ltd., Shanghai, China, which has a laboratory that provided nucleic acid testing and analysis for Lingang shelter hospital.A cycle threshold (Ct) value (N gene and ORF gene) of 40 or more was considered a negative test.Te standard of negative conversion was two successive negative nucleic acid tests at minimum 24-hour sampling intervals.Te frst nucleic acid negative date of two consecutive nucleic acid negative dates after admission was defned as the negative date of nucleic acid.Te primary outcome of this study is the NCT of nucleic acid, which was calculated as the number of days between the date of nucleic acid positive in community screening before admission and the negative date of nucleic acid after admission[20,21].

Table 2 :
Univariable and multivariate Cox regression analyses for risk factors associated with the NCT of nucleic acid in nonsevere COVID-19 patients infected by the SARS-CoV-2 Omicron variant.