Detectable Serum Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load (RNAemia) Is Closely Correlated With Drastically Elevated Interleukin 6 Level in Critically Ill Patients With Coronavirus Disease 2019

Abstract

Background

Although the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in respiratory specimens has been widely used to diagnose coronavirus disease 2019 (COVID-19), it is undeniable that serum SARS-CoV-2 nucleic acid (RNAemia) could be detected in a fraction of COVID-19 patients. However, it is not clear whether testing for RNAemia is correlated with the occurrence of cytokine storms or with the specific class of patients.

Methods

This study enrolled 48 patients with COVID-19 admitted to the General Hospital of Central Theater Command, People’s Liberation Army, a designated hospital in Wuhan, China. The patients were divided into 3 groups according to the Diagnosis and Treatment of New Coronavirus Pneumonia (sixth edition) guidelines issued by the National Health Commission of China. Clinical and laboratory data were collected, and the serum viral load and interleukin 6 (IL-6) level were determined.

Results

Analysis of clinical characteristics of 48 cases of COVID-19 showed that RNAemia was diagnosed only in the critically ill group and seemed to reflect the severity of the disease. Furthermore, the level of the inflammatory cytokine IL-6 in critically ill patients increased significantly, almost 10 times that in other patients. More importantly, the extremely high IL-6 level was closely correlated with the detection of RNAemia (R = 0.902).

Conclusions

Detectable serum SARS-CoV-2 RNA (RNAemia) in patients with COVID-19 was associated with elevated IL-6 concentration and poor prognosis. Because elevated IL-6 may be part of a larger cytokine storm that could worsen outcome, IL-6 could be a potential therapeutic target for critically ill patients with an excessive inflammatory response.

Coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been described by the World Health Organization (WHO) as a public health emergency of international concern [1–3]. As of 12 April 2020, > 1 600 000 cases of COVID-19 have been reported globally to the WHO, with > 99 000 deaths.

SARS-CoV-2 and severe acute respiratory syndrome coronavirus (SARS-CoV) likely use the same angiotensin-converting enzyme 2 (ACE2) as the entry receptor [4–6], but the clinical manifestations of the 2 diseases are different. After infection, patients with COVID-19 may develop mild, moderate, or severe symptoms. Patients with mild disease may be asymptomatic. Patients with moderate disease may exhibit symptoms of fever, nonproductive cough, dyspnea, myalgia, fatigue, and radiographic evidence of pneumonia; most of them appear to have a good prognosis. In contrast, some patients may develop severe pneumonia, acute respiratory distress syndrome, or multiple organ failure [7–10]. Importantly, in critically ill patients, SARS-CoV-2 infection is also associated with an inflammatory cytokine storm [11, 12], which is mainly characterized by elevated plasma concentration of interleukin 6 (IL-6). Several recent COVID-19 clinical studies have shown that the level of IL-6 in the severe group was higher than that in the moderate group [11, 13–15], suggesting that IL-6 can be used as a biomarker for severity assessment. However, how to quantitatively correlate IL-6 levels with critically ill patients is still unknown.

Real-time reverse-transcription polymerase chain reaction (RT-PCR) assay with primers and probes targeting the nucleocapsid protein (N) and open reading frame 1ab (ORF1ab) genes of SARS-CoV-2 from throat swab samples has been widely used in the diagnosis of COVID-19 patients. A recent study showed that the viral load in respiratory specimens of symptomatic patients was similar to that of asymptomatic patients [16], implying that the viral load in respiratory specimens may not objectively reflect the disease severity. Serum SARS-CoV-2 viral RNA (termed RNAemia by the authors in a recent study) was detected in 15% of the COVID-19 patients [8], but the relevant characterizations are still lacking. In particular, it is unclear whether RNAemia can be considered as a prognostic indicator, especially for severe or critically ill patients.

In this study, we systematically quantified the serum SARS-CoV-2 viral load (RNAemia) of various patient groups and characterized the relationship between RNAemia, IL-6 level, and disease severity.

MATERIALS AND METHODS

Data Collection

This retrospective study was approved by the Ethics Committee of the General Hospital of Central Theater Command. Oral consent was obtained from patients. Pharyngeal swab samples collected at the General Hospital of Central Theater Command from 1 to 19 February 2020 confirmed 48 enrolled COVID-19 patients by real-time RT-PCR assay. Immediately after admission, the patients’ specimens for IL-6 and viral RNA levels were obtained. Their medical records were collected, including epidemiology, demographics, clinical manifestations, radiological characteristics, laboratory data, and outcome data. All data were checked by a team of trained physicians.

Laboratory Examination

Serum samples and throat swabs were collected from all patients and RNA was extracted. RT-PCR assay was used to determine the viral load by using a SARS-CoV-2 nucleic acid detection kit (Daan Gene Ltd, Guangzhou, China; catalog number DA0930-DA0932). Two target genes were amplified and tested simultaneously, namely ORF1ab and N. According to the manufacturer’s procedures, a cycle threshold (Ct) value of < 40 was defined as a positive result, and a Ct value of ≥ 40 was defined as a negative result. Specimens, including sputum or alveolar lavatory fluid, blood, urine, and feces, were cultured to identify pathogenic bacteria or fungi that may be associated with SARS-CoV-2 infection. The level of inflammatory cytokine IL-6 was measured using a kit from Roche Ltd (Mannheim, Germany; catalog number 05109442190).

Statistical Analysis

According to the Diagnosis and Treatment of New Coronavirus Pneumonia (sixth edition) guidelines issued by the National Health Commission of China, COVID-19 patients were classified. There was no mild case in the enrolled patients. Classification variables were described as frequency ratios or percentages, and the significance was detected by the χ ² or Fisher exact test. The quantitative variables of parameters were expressed as mean ± standard deviation, and significance was tested by t test. For items that did not conform to the normal distribution, the significance was determined by the Kruskal-Wallis test. SPSS statistical software (Macintosh version 26.0, IBM SPSS, Armonk, New York) and R program were used for statistical analysis.

For testing the differences of the patients with negative and positive quantitative PCR results, multiresponse permutation procedure was used with Bray-Curtis method. The null hypothesis was that there was no difference among the groups in a Monte Carlo randomization procedure with 999 permutations. P values < .05 were considered statistically significant.

RESULTS

Characteristics of COVID-19 Patients Enrolled in This Study

A total of 48 patients with laboratory-confirmed SARS-CoV-2 infection were enrolled in the study. According to the Diagnosis and Treatment of New Coronavirus Pneumonia (sixth edition) guidelines issued by the National Health Commission of China, the COVID-19 patients were categorized into 3 groups: 21 moderate cases (43.7%), 10 severe cases (20.8%), and 17 critically ill cases (35.4%). Severe patients also met at least 1 of the following conditions: (1) shortness of breath with respiratory rate ≥ 30 breaths/minute; (2) oxygen saturation at resting state ≤ 93%; or (3) partial pressure of oxygen/fraction of inspired oxygen ratio ≤ 300 mm Hg. The admission of critically ill patients must meet at least 1 of the following additional conditions: (1) respiratory failure requiring mechanical ventilation; (2) shock; or (3) multiple organ failure requiring transfer to the intensive care unit. Three patients in the critical group died after disease onset.

As shown in Table 1, the enrolled COVID-19 patients consisted of 31 males (77.1%) and 17 females (22.9%). The critically ill patients (mean age, 79.6 ± 12.6 years) were older than the severe patients (mean age, 63.9 ± 15.2 years) and the moderate patients (mean age, 52.8 ± 14.2 years) without statistical significance, and the majority of severely ill patients were male (88.2%), displaying an age- and male-dependent severity. Notably, several underlying diseases were implicated in the COVID-19 patients, among whom high-risk factors included diabetes (12 [25%]), hypertension (23 [49.7%]), and heart disease (8 [16.7%]). Mixed fungal infection was found in 27.1% of patients, and bacterial infection did not seem to be common (2.1%).

Serum SARS-CoV-2 Nucleic Acid Is Only Detectable in Critically Ill Patients

We examined the SARS-CoV-2 viral load in patient serum using real-time RT-PCR. As shown in Figure 1, 5 of 48 cases (10.4%) were confirmed as positive (the numbers of patients testing positive for the N and ORF1ab genes were 3 and 5, respectively), which was similar to a previous study [8]. Interestingly, although all patients’ pharyngeal swab samples tested positive, serum samples from the moderate or severe group did not show a positive result. In contrast, all 5 positive results were from critically ill patients, 2 of whom died after the onset of COVID-19 (Supplementary Table 1). Respiratory failure was the leading cause of all deaths. The underlying diseases were found in 1 death with positive RNA (hypertension) and 1 death without positive RNA (hypertension and diabetes). Other RNA-positive patients had no comorbidities.

Since inflammatory cytokine storms frequently occur in critically ill COVID-19 patients, we then investigated whether any laboratory parameters were associated with RNAemia and had an impact on the severity of COVID-19.

Sharply Increased IL-6 Level Is Strongly Associated With COVID-19 Severity

As shown in Table 2, the absolute count of peripheral blood lymphocytes was lower in severe patients than in moderate patients, and even lower in critically ill patients, which was consistent with recent reports [17, 18], whereas the absolute count of neutrophils was higher in critically ill patients. The procalcitonin level in critically ill patients was higher than that in the other 2 groups and similar to other clinical observation [19], indicating a significantly increased inflammatory response in these patients. Remarkably, sharply increased IL-6 level was observed in critically ill patients, which was almost 10 times that of severe patients, and all deaths exhibited extremely high IL-6 values (Supplementary Table 1), suggesting that IL-6 might be an important biomarker to judge the poor prognosis of COVID-19 patients. The extremely high level of IL-6 is a hallmark and important driving force of cytokine storm [20], which may cause multiple organ dysfunction in critically ill patients [12]. Consistently, the parameters that reflect organ dysfunction, including troponin T, serum creatinine, and blood urea nitrogen, were higher in critically ill patients compared with the other 2 groups.

RNAemia Is Closely Associated With IL-6 Level in Critically Ill Patients With COVID-19

In a recent study, RNAemia was linked to COVID-19 [8]. Our data strongly suggest that both RNAemia and IL-6 concentration ≥ 100 pg/mL were exclusively observed in critically ill patients, which prompted us to further study the relationship between them. As shown in Figure 2A, patients with RNAemia exhibited a much higher IL-6 level compared with other patients. We then checked the IL-6 value in each patient with RNAemia. Strikingly, all of their IL-6 values exceeded 100 pg/mL (Supplementary Table 1). To further confirm the relationship between them, we first analyzed the IL-6 values of critically ill patients. Notably, mortality appeared to be associated with an IL-6 value of ≥ 100 pg/mL, since all deaths in this study were in this group (Supplementary Table 1). We therefore defined an IL-6 value of ≥ 100 pg/mL as high and the rest as low. In the critically ill group, patients with high IL-6 accounted for 35.3% (Figure 2B). As shown in Table 3, the incidence of RNAemia was closely correlated with high IL-6 level in critically ill patients (R = 0.902). These data demonstrated that RNAemia was related to a poor prognosis. Indeed, compared with patients without RNAemia, all patients with RNAemia had a higher risk of multiple organ damage (Figure 3).

DISCUSSION

Although the detection of throat swab rather than serum SARS-CoV-2 viral load is widely used for COVID-19 diagnosis, an undeniable fact is that serum SARS-CoV-2 nucleic acid (RNAemia) is only detectable in some patients [8]. However, it is not clear under what circumstances it can be detected and whether the incidence of RNAemia is related to certain types of patients. In this study, we investigated the distribution of RNAemia-positive cases in each patient group and found that these cases were only confirmed in critically ill patients, which indicates that RNAemia is not a casual event. Moreover, the laboratory data analysis strongly suggests that the level of the inflammatory cytokine IL-6 in critically ill patients was significantly elevated. More importantly, the extremely high IL-6 level was closely correlated with the incidence of RNAemia and mortality. Based on our observations and literature reports, older men with underlying diseases, high IL-6 levels, and detectable RNAemia are more likely to have adverse clinical outcomes. Our work may provide clues for developing new COVID-19 diagnostic strategies and therapeutic targets.

Although recent studies have shown that IL-6 level is increased in patients with severe disease [14], its level in critically ill COVID-19 patients is still unknown. IL-6 is one of the main proinflammatory factors that contribute to the formation of cytokine storms, which largely enhance the vascular permeability and impair organ function. This observation might help explain why RNAemia can be detected only in patients with an extremely high level of IL-6. We still cannot rule out the possibility that the SARS-CoV-2 virus population explodes in a short period, which in turn triggers a cytokine storm characterized by increased levels of cytokines such as IL-6. Therefore, the combination of the IL-6 level and serum viral RNA Ct value may be regarded as an effective marker for standard clinical measures to predict impending adverse outcomes with high accuracy. We must point out that due to the shortage of detection kits, results of other cytokines cannot be obtained and those investigations are warranted in future studies.

Host-oriented therapies should be selected because of the high mortality rate in critically ill patients with COVID-19. IL-6 functions as a critical mediator of respiratory failure, shock, and multiorgan dysfunction [21]. Whether IL-6 can be a therapeutic target for critically ill patients is a direction worth studying. Notably, IL-6R monoclonal antibody (tocilizumab)–directed COVID-19 therapy has been used in a clinical trial in China (ChiCTR2000029765), and has recently been incorporated into COVID-19 management guidelines generated in China and Italy. Our data strongly supported this notion, but the efficacy of IL-6 monoclonal antibody–directed therapy remains to be fully evaluated. Our observations also indicate that the combination of antiviral and anti-inflammatory treatments may be important for critically ill COVID-19 patients. Therefore, baricitinib, a powerful inhibitor of the IL6/JAK/STAT signaling pathway and clathrin-mediated endocytosis, may be effective against the consequences of elevated cytokine levels and SARS-CoV-2 infection in patients with severe COVID-19 [22, 23].

Supplementary Data

Supplementary materials are available at Clinical 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.

Notes

Author contributions. F. L. and J. D. conceived and designed the study. F. L., X. C., Y. F., and D. M. contributed to the literature search. X. C., B. Z., Q. H., Y. L., B. Y., and J. D. contributed to data collection. X. C., Y. Q., Y. C., and F. L. contributed to data analysis. F. L. and X. C. contributed to data interpretation. Y. Q. and X. C. contributed to the figures. F. L., X. C., and J. X. contributed to the writing of the report.

Acknowledgments. The authors thank Louise T. Chow (University of Alabama at Birmingham) for her suggestions and critical reading of this manuscript.

Financial support. This work was supported by the National Natural Science Foundation of China (grant number 91859206); the Medical Science Advancement Program (Basic Medical Science) of Wuhan University (grant number TFJC2018003); and the Fundamental Research Funds for the Central Universities (grant number 2042019kf1018).

Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

References

Author notes