विभिन्न इंफ्लेमेटरी मार्करों का सापेक्ष महत्व और कोविड-19 में मृत्यु दर के लिए उनके महत्वपूर्ण थ्रेशोल्ड।

The most correct classification of mortality was by D-dimer level of more than 302.5 ng/mL but that too did not exceed 80% (Table 6). The correct classification of survivals by the levels less than these cut-offs was even lower at nearly 70%. The survival was best detected (88.2%) by CPK values less than 206.5 μg/L but this cut-off correctly classified mortality in only 44.0% cases (Table 6). Considering a correct classification of both deaths and survivals together, the best overall accuracy was 82.8% by CPK with the optimal cut-off 206.5 μg/L, followed by IL-6 with cut-off 45.4 pg/mL. LDH with cut-off 403.9 U/L also had overall accuracy of 82.0%. No marker was able to correctly classify deaths and survivals in more than 83% cases, and most of the correct classifications were for survivals and not many for mortality. Other markers had lower performance. The overall accuracy was least (61.6%) by CRP at the optimal cut-off of 12.4 mg/L.

Extremely high levels of these markers were also seen, though rare, and turned out to be associated with high mortality. There were 130 (1.4%) cases with LDH level 1000 U/L or more and 93 (71.5%) of them died. Similarly, 113 (0.9%) cases had D-dimer level 10,000 ng/mL or more and 59 (52.2%) died, 97 (0.8%) cases with IL-6 level at least 1,000 pg/mL and 62 (63.9%) died. Only 4 (0.1%) cases had ferritin level 10,000 μg/L or more and 2 (50.0%) died. Thus, extremely high levels of these markers had high mortality.

Logistic Regression

Steeply increasing mortality with increasing levels of some markers violates the assumption of linearity for valid logistic regression results when the levels are considered as the continuous variables. The trend in Figure 2 is a testimony for this. We tried log transformation (Supplement) but that too did not help because the mortality was exceedingly high for some levels (Figure S1). As a remedy, we used quintile categories for each marker without considering the ingredient to obviate the need for linearity.15 The results of multivariable logistic regression are in Table 7. The highest adjusted odds ratio (aOR), with Q1 as the reference category, adjusted for other markers, was 8.40 for Q2 levels of troponin-I, which could be an aberration due to the lack of a trend. For stable trend, the highest aOR was 7.62 for Q5 of IL-6 followed by 6.04 for Q5 levels of D-dimer and the lowest was 0.81 for Q4 of CPK, though not statistically significant (P = 0.326). These ORs indicate that the risk of mortality increased to as much as 7 times when the levels of IL-6 and D-dimer reached the top quintile. At the stricter 1% level of significance, CRP, ferritin, CPK, and ALC did not provide statistically significant aOR for any quintile category. Q5 (> 433 U/L) was significant for LDH, and Q3, Q4 and Q5 for D-Dimer (>192 ng/mL) and all quintiles after the first for IL-6 (> 4.5 pg/mL). Except for Q4, troponin-I levels more than 0.002 ng/mL also had significant aOR for mortality. The multivariable logistic regression, which considered all the markers together for both mortality and survival, could correctly classify 91.7% cases in all, including survivals, but only 23.2% deaths were correctly classified. This indicates minor association of these markers with overall deaths and indicates that other factors also contributed to the mortality.

DISCUSSION

Several meta-analyses2, 4, 5, 16, 17 and individual studies18, 19 have concluded that elevated levels of inflammatory markers are associated with severity and mortality in COVID-19 cases. This is obvious because the elevated (low of ALC) levels of these markers indicate greater tissue damage that leads to deterioration of the patient condition. We could not find specific information in the literature on the relative importance of various markers as a risk factor for mortality and on the critical thresholds. Also, while previous studies, including meta-analyses, have had relatively small sample sizes, we have analysed data from more than 10,000 patients for 5 of the 8 markers and a substantially large minimum sample of 4566 for CPK. Thus, our results are likely to offer better reliability.

Our search did not find any article that describes the level of inflammatory markers in COVID-19 patients in such detail. The typical gamma distribution of values of D-dimer, LDH, CPK, and ALC, and its special case exponential distribution of the CRP, ferritin, IL-6, and troponin values could help anticipate the pattern of their level in future COVID-19 patients and to apply the right statistical method for their analysis when an exact parametric analysis is required.

Quantile values presented by us for admitted COVID-19 patients describe, among others, the bottom 20% values and the top 20% values (Table 1). These have not been reported earlier. Elevated values of D-dimer, ferritin, CPK, and troponin-I, and low values of ALC were seen in less than one-half of the cases but CRP, IL-6, and LDH were elevated in 60%-70% cases (Table 2). The median, for example 19.1 pg/mL for IL-6, tells that one half of the admitted patients had levels less than this value and the other half the higher levels. An interesting finding from our analysis is the low correlation between the values of the most markers (Table 3), indicating they generally operate independently of one another. However, the correlation between ferritin and LDH was 0.427, which is an exception. Similarly, there was a correlation 0.313 between D-dimer and troponin. Such a correlation between these two markers has also been observed in acute myocardial infarction.20

Our major concern in this communication is to investigate the relative importance of various inflammatory markers in predicting mortality and to identify the critical thresholds. While these markers are known as sensitive indicators of severity, there appears to be a lack of work on these aspects. The elevated (or low levels of ALC) level of these markers in the early phase of the disease can alert the clinicians regarding the possibility of deterioration of the patient, and their relative importance can help in better and more evidence-based clinical decision-making.

In a systematic review of 28 studies (n = 4663), Zhang et al.16 observed increased CRP in 73.6% patients, increased IL-6 in 53.1%, and increased LDH in 46.2%. We observed elevated LDH in more (56.7% - Table 2) cases but elevated CRP and IL-6 in nearly the same percentage of cases as reported by them. The meta-analysis of 7 studies in the same paper by Zhang et al. found increased CRP and increased LDH significantly associated with severity. They also studied other markers, but not the ones we analysed in this report.

The meta-analysis of 56 studies (n = 8719) by Ji et al.17 reported the weighted mean difference of CRP, IL-6, and other markers between severe and non-severe cases, and deceased and surviving patients. They concluded that the disease severity is associated with higher levels of inflammatory markers. In a review of 72 studies, Tjendra et al.5 studied multiple markers and reported that 'markedly' elevated levels were associated with the severity of disease. Loomba et al.4 analysed 10 studies (n = 1584) and reported significant differences in the levels of various markers between the patients who survived and who did not2 Another meta-analysis which included 23 studies (n = 4313) found significantly higher levels of CRP and IL-6, among others, in severe patients.

In a pooled analysis of 9 studies (n = 1532), Henry et al.18, reported that elevated LDH levels were associated with a 6-fold increase in odds of developing severe disease and a 16-fold increase in odds of mortality. Their report suggests that, on average, nearly 90% of severe patients had elevated LDH against only nearly 32% in non-severe patients. In a study of 923 patients in China, Zhang et al.19 concluded that ALC levels remaining low after the first week following symptoms onset are highly predictive of in-hospital death.

In an IL-6-based mortality risk model21 on patients in Spain found IL-6, CRP, LDH, ferritin, and D-dimer had AUROC > 0.70 and described them as ‘predictive’ of mortality. The study by Marimuthu et al.21 in India reported highest AUROC = 0.740 for IL-6 among the five markers they studied. These studies are similar to ours but were based only on 611 and 221 patients, respectively. The threshold of 0.70 for AUROC chosen by them seems too low to us for ‘prediction’ of mortality. We also observed AUROC > 0.70 for all the markers except CPK and ALC, but the AUROC must be at least 0.80 to have ‘good’ inherent validity14. With this criterion, we found only LDH and troponin as a good indicator of mortality with AUROC exceeding 0.80. D-dimer and IL-6 were close, with AUROC 0.797 and 0.793, respectively.

The optimal cut-off revealed by the model proposed by Laguna Goya et al.21 had 88% sensitivity and 89% specificity for mortality which is promising, but they did not provide details of how these values were obtained. Marimuthu et al.22 reported that an IL-6 level > 60.5 pg/mL and D-dimer level > 0.5 μg/mL ‘predicted’ in-hospital mortality with sensitivities of 80% and 76.7%, respectively. However, the specificities were lower, at 65% and 60%, respectively.

Our quintile-based multivariable logistic model correctly classified only 23.2% deaths but was excellent in correctly classifying 98.4% of survivals. The low accuracy of mortality may have happened because this model considered all the markers together with no fixed threshold and optimized the classification of both the deaths and survivals.

All the studies, including ours, overwhelmingly suggest that the elevated levels of the inflammatory markers (or low levels of ALC) in COVID-19 patients are significantly associated with death. To us this is obvious. No study seems to have commented on which marker is more important than others as a risk of mortality and what are the critical thresholds. We conducted three types of analyses to investigate the association of different levels with mortality. First, assessing mortality at the conventional normal cut-offs; second, determining the optimal cut-offs that correctly classify both survival and mortality simultaneously; and third ,identifying the critical thresholds associated with a high risk of mortality.

The use of the term ‘predictivity’ by some authors18,19,21 seems misplaced because the studies were based on records where deaths already occurred with several factors playing their role. These were not prospective studies designed to determine mortality at different levels of the markers. Correct classification of deaths and survival in a retrospective study cannot be interpreted as predictive. Predictivity has causal overtones whereas this study focuses solely on association. Thus, we avoid the term predictivity and discuss only the association that could at best be called a significant risk factor. In our case, even a significant association does not necessarily translate into the significant risk of mortality because we have a huge sample size that makes it easy to obtain statistical significance even at a strict level of 1%.

The ROC analyses show that the best overall accuracy for correct classification of mortality and survival was achieved by CPK at the optimal cut-off of 206.5 U/L; however, even this did not exceed 83% (Table 6). Whilst this result substantially included correct survivals, it also misclassified many deaths. Thus, a marker that can reliably identify the mortality as well as survival is elusive. Among those studied, our results suggest that the levels of IL-6, CPK, LDH, CPK, and troponin-I have relatively more importance for correctly classifying both mortality and survival. The performance of D-dimer was moderate and the role of CRP, ferritin, and ALC was relatively poor. Quintile analysis (Figure 2) and the logistic results (Table 7) show that among the individual markers, the risk of mortality steeply increased with the levels of IL-6 and D-dimer, and not so much with the elevated levels of other markers we studied. LDH became a substantial risk when it reached its highest quintile (> 433 U/L), but troponin, IL-6, and D-dimer posed higher risks at a relatively lower level (> 0.012 ng/mL, 4.5 pg/mL, and 192 ng/mL, respectively). These include values at the upper end of the conventional normal levels, which are thresholds that require special attention in COVID-19 patients. CPK, CRP and ALC seemed to have a poor association with mortality while ferritin expressed a modest risk.