ABSTRACT
Background:
Mycobacterium w (Mw), an immunomodulator, resulted in better clinical status in severe coronavirus infectious disease 19 (COVID-19) but no survival benefit in a previous study. Herein, we investigate whether Mw could improve clinical outcomes and survival in COVID-19.
Materials and Methods:
In a multicentric, randomized, double-blind, parallel-group, placebo-controlled trial, we randomized hospitalized subjects with severe COVID-19 to receive either 0.3 mL/day of Mw intradermally or a matching placebo for three consecutive days. The primary outcome was 28-day mortality. The co-primary outcome was the distribution of clinical status assessed on a seven-point ordinal scale ranging from discharged (category 1) to death (category 7) on study days 14, 21, and 28. The key secondary outcomes were the change in sequential organ failure assessment (SOFA) score on days 7 and 14 compared to the baseline, treatment-emergent adverse events, and others.
Results:
We included 273 subjects (136 Mw, 137 placebo). The use of Mw did not improve 28-day survival (Mw vs. placebo, 18 [13.2%] vs. 12 [8.8%], P = 0.259) or the clinical status on days 14 (odds ratio [OR], 1.33; 95% confidence intervals [CI], 0.79-2.3), 21 (OR, 1.49; 95% CI, 0.83-2.7) or 28 (OR, 1.49; 95% CI, 0.79-2.8) between the two study arms. There was no difference in the delta SOFA score or other secondary outcomes between the two groups. We observed higher injection site reactions with Mw.
Conclusion:
Mw did not reduce 28-day mortality or improve clinical status on days 14, 21 and 28 compared to placebo in patients with severe COVID-19. [Trial identifier: CTRI/2020/04/024846]
KEY WORDS: Coronavirus, COVID, Mycobacterium indicus pranii, Mw, mycobacterium w
INTRODUCTION
Coronavirus infectious disease 19 (COVID-19) can cause illness ranging from asymptomatic cases to severe disease, including death. The organ damage in severe COVID-19 is likely due to a dysregulated host immune response.[1,2,3,4] In patients with severe COVID-19, there is a sustained reduction of peripheral lymphocytes.[5,6] Also, there is a suppression of type I and type III interferon, leading to low viral clearance.[2,7] The use of immunosuppressive drugs such as systemic corticosteroids and anti-interleukin 6 receptor monoclonal antibodies used for treating severe COVID-19 could cause a prolonged immunosuppressive state.[8,9,10,11,12] Thus, patients with severe COVID-19 are likely to have a sustained immune-paralytic state after the initial pro-inflammatory state, similar to Gram-negative sepsis.[3] Also, a Th1 predominant response was associated with clinical recovery in COVID-19 infection.[13]
Mycobacterium w (Mw), also known as Mycobacterium indicus pranii, is a non-pathogenic, rapidly growing atypical mycobacterium. Heat-killed Mw administered intradermally is a potent toll-like receptor (TLR)-2 agonist,[14] inhibits TLR-9,[15] and augments the Th1 immune response.[16] Mw has been studied for its immune-modulating properties in patients with sepsis, pulmonary tuberculosis, tuberculous pericarditis, lung cancer, and leprosy.[17,18,19,20,21,22] Elsewhere, Mw alone and in combination with anti-retroviral therapy increased the CD4 T cell count in patients with human immunodeficiency virus (HIV).[23] We have previously shown better clinical outcomes and survival in severe sepsis with Mw.[18,24] Mw (at a dose of 0.3 mL intradermally for three days) in combination with standard care resulted in better clinical status on days 14 and 21 in severe COVID-19 patients than in standard care alone.[25] However, the study had a small sample size, and we found no survival benefits.[25] In another study, Mw resulted in rapid recovery of hospitalized subjects with COVID-19 infection and reduction of inflammatory markers.[18,26] We hypothesized that Mw, by its immunomodulatory mechanism, would result in clinical improvement and reduce mortality in critically ill subjects with COVID-19. In this multicentric study, we evaluate the role of Mw as adjunctive therapy to standard care in critically ill patients with COVID-19.
Methods
Study design: We conducted a multicentre, randomized, double-blind, parallel-group, comparative controlled trial (CTRI/2020/04/024846, registered on 24/04/2020), to evaluate the efficacy of Mw combined with standard therapy in critically ill subjects with COVID-19. The Institute Ethics Committee of each participating centre approved the study protocol. We obtained written informed consent from all the subjects or the next of kin. We conducted the trial according to the principles of the Declaration of Helsinki and the International Conference on Harmonization-Good Clinical Practice guidelines. The protocol is available at http://ctri.nic.in (identifier: CTRI/2020/04/024846). No amendments were made to the protocol after the commencement of the trial. The study was conducted between 15 November 2020 and 31 December 2021, during which the delta variant was prevalent. The last subject was enrolled on 31 August 2021. The final follow-up visit was on 30 September 2021.
Setting: The study was conducted in intensive care units (ICUs) or high dependency units (HDU) of eight tertiary care centres in India.
Patients: We screened consecutive patients aged > 18 years who were positive for severe acute respiratory syndrome (SARS)-CoV-2 RNA on reverse transcriptase-polymerase chain reaction (RT-PCR). We included subjects with a respiratory rate of ≥ 25 breaths/minute and oxygen saturation ≤ 90% on ambient air or requiring supplemental oxygen or mechanical ventilation. Subjects of childbearing age were included if they agreed to take effective contraception measures during the study period. We excluded subjects with any of the following: (1) pregnancy or breastfeeding; (2) prior cardiorespiratory arrest; (3) chronic liver disease; (4) haemodialysis-dependent chronic kidney disease; (5) enrollment in another trial; (6) active malignancy; (7) alanine transaminase and aspartate transaminase > five times upper limit of normal; and (8) subjects unwilling to provide consent. The use of other drugs according to the institutional protocol at each participating centre was allowed. Briefly, the treatments included systemic glucocorticoids, remdesivir, convalescent plasma, tocilizumab, and anticoagulation.
Trial monitoring: An independent data safety monitoring board monitored the trial periodically and evaluated the study data for participant safety, study conduct, and progress. An independent steering committee oversaw the conduct of the entire study.
Randomization: A central team not directly involved in patient care or patient data analysis provided a computer-generated randomization sequence. The randomization was stratified according to the centres. The subjects at each centre received either the investigational drug or a matched placebo in individually numbered packs according to the sequence. The investigators at each participating centre and the subjects were blinded to the treatment allocation. Envelopes were provided to each participating centre for emergency unmasking.
Sample size: We assumed a 15% difference between the two study arms. This required us to enrol 300 subjects with an alpha error of 0.05 and a power of 80% assuming a 5% dropout rate. However, the trial was stopped early because of slow recruitment due to a abatement in cases of COVID-19.
Study procedures: We recorded clinical data for all the subjects on paper case record forms that were subsequently entered into an electronic database and validated by the trial staff at each centre. We assessed the respiratory rate, oxygen supplementation device used (nasal cannula, venturi mask, non-rebreathing mask, high-flow nasal cannula, non-invasive ventilation, or invasive mechanical ventilation), oxygen saturation, the use of concomitant medications, and adverse events during hospitalization. We performed complete blood count, blood glucose, liver and renal function tests, and arterial blood gas analysis each day until day 7, and then on days 14, 21, and 28 if the patient was still hospitalized. We recorded the sequential organ failure assessment (SOFA) score for each day until hospital discharge. We obtained nasopharyngeal or oropharyngeal or endotracheal aspirate for detecting COVID-19 RNA by RT-PCR at days 5, 7, 14, 21, 28, or at the time of hospital discharge.
We evaluated the clinical status of the study participants from day 1 through day 28 or hospital discharge on a seven-point ordinal scale consisting of the following:[27] category 1, not hospitalized with the resumption of normal activities; category 2, not hospitalized but unable to resume normal activities; category 3, hospitalized but not requiring supplemental oxygen; category 4, hospitalized and requiring supplemental oxygen (nasal cannula, venturi mask, or non-rebreathing mask); category 5, hospitalized and requiring nasal high-flow oxygen therapy, non-invasive mechanical ventilation, or both; category 6, hospitalized and requiring invasive mechanical ventilation or extracorporeal membrane oxygenation (ECMO); category 7, death. We recorded the worst score for clinical status every day for hospitalized subjects. We made the final assessment on day 28 in person for a hospitalized subject or telephonically for those discharged before day 28.
Study drug: Each dose of 0.1 ml Mw contains 0.5 × 109 heat-killed Mycobacterium w, 0.9% sodium chloride, and 0.01% thiomersal (as preservative). We used a matching placebo (0.9% sodium chloride, 0.01% thiomersal) as control.
Intervention: Subjects were randomized to receive a single daily dose of 0.3 ml (in aliquots of 0.1 mL at three different sites) of Mw or a matching placebo intradermally in the deltoid region for three consecutive days. We administered the study drugs within 24 hours of admission to the hospital. We observed the subjects for any adverse effects, local or systemic, that could be associated with the administration of the study drug.
Study outcomes: The primary outcome of the study was 28-day mortality. The co-primary outcome was the distribution of clinical status assessed on the seven-point ordinal scale on days 14, 21, and 28 after randomization. If treatment with Mw improved the outcomes, the distribution of the scores among patients who received Mw would shift more towards lower values of the scale than the distribution of the scores among patients who received a placebo.
The secondary outcomes changed in SOFA score (delta SOFA) on days 7 and 14 compared to the baseline and the maximum SOFA attained during the hospital stay, the proportion of patients with adverse events that occurred on or after the first dose of the study drug for up to 28 days, time to clinical recovery (defined as a reduction by two-points on a seven-point ordinal scale), time to one-point improvement on a seven-point ordinal scale, days on vasopressor drugs, days on mechanical ventilation, time to RT-PCR negativity, and the ICU and the hospital length of stay.
Estimation of serum cytokine levels and analysis of the transcriptomic profile of peripheral blood mononuclear cells (PBMCs): Blinded samples were provided for cytokine analysis (days 0, 7, and 14) and transcriptomics analysis (days 0, and 14) at the coordinating centre (PGIMER, Chandigarh). We measured Th-1, Th-2, and Th-17 cytokines in 50 random subjects (25 in each group) using BD™ Cytometric Bead Array (CBA) kit (Cat no 560484) as per the manufacturer protocol. Additionally, we performed transcriptomic analysis of PBMCs in ten randomly selected subjects (5 in each arm) by RNA sequencing. The RNAseq data was analysed for differentially expressed genes using DSeq2 with an unadjusted P value threshold of 0.05, and a log2 fold change of 1.5 was used for gene expression estimation. The gene set functional enrichment analysis (GO, KEGG, Reactome, DisGeNET, and DO enrichment) was done using the AllEnricher (Detailed methods in supplementary data). The blinded samples were provided for cytokine analysis and transcriptomics analysis.
Statistical analysis: We present the data descriptively as mean and standard deviation (SD), median and interquartile range (IQR), or number and percentage. The difference between the continuous and categorical variables was analysed using the Mann-Whitney U test (or student’s t-test) and the Chi-square test, respectively. We performed all analyses on an intention-to-treat (ITT) basis. All the subjects who were randomized and received at least one study dose were assessed for efficacy and safety. If the subject died before day 14, the day 14 category on the ordinal scale was recorded as “died”; if the subject was discharged before day 14, the category on day 14 was recorded as “not hospitalized”. Similarly, if the subject died before day 7 or day 14, we assumed the highest value for the SOFA (SOFA score of 20). We used the proportional odds model for the ordinal scale data, including treatment as the independent variable and the baseline disease severity (neutrophil-lymphocyte ratio and baseline ordinal score) as a covariate. An odds ratio of less than 1 would suggest treatment with Mw to be superior (lower risk for being in a higher category) to placebo on days 14, 21, and 28. We assumed statistical significance at a P value < 0.05. We performed all statistical analyses using the Statistical Package for Social Sciences (IBM SPSS Statistics, version 23, IBM Corporation, Armonk, NY).
RESULTS
Baseline characteristics of the study subjects: We randomized 273 (n = 136 assigned to Mw arm; n = 137 assigned to placebo arm) subjects [Figure 1]. No patient was excluded from analysis after randomization.
Figure 1.
CONSORT diagram depicting the flow of subjects during the study
The baseline parameters, the severity of illness, and comorbid illnesses in the two groups are described in Table 1. The study population comprised predominantly of men (71.4%, n = 195) with a median (IQR) age of 54 (44-63) years. The median (IQR) time from symptom onset to randomization was 6 (4-7) days and was similar in both the study arms. Dyspnoea, fever, and cough were the frequently reported symptoms. Any comorbid illness was seen in 54% (n = 148) of subjects. Diabetes mellitus, followed by systemic hypertension, were common comorbid illnesses. The median (IQR) PaO2: FiO2 ratio was 112 (76-172) mm Hg and was comparable in the two study arms. The median (IQR) baseline SOFA score was 4 (3-4). The median neutrophil-lymphocyte ratio was 8.4 and was not different between the Mw and the placebo arm. The baseline ordinal scale score was also similar in the two groups. Ninety percent (n = 247) of the study population required oxygen supplementation using a venturi mask or a non-rebreather mask, while the remaining study subjects needed either HFNC or mechanical ventilation at admission. All the subjects received systemic glucocorticoids, remdesivir, and anticoagulation. Fourteen subjects received intravenous tocilizumab, while three subjects were treated with hydroxychloroquine.
Table 1.
Baseline demographics, clinical parameters, laboratory investigations, and disease severity at baseline
| Parameter | Mw (n=136) | Placebo (n=137) | Total (n=273) | P-value |
|---|---|---|---|---|
| Demographics | ||||
| Age, in years | 55 (44-63.8) | 52 (45-61) | 54 (44-63) | 0.458 |
| Male sex, No. (%) | 98 (72.1) | 97 (70.8) | 195 (71.4) | 0.894 |
| Clinical parameters | ||||
| Median time from symptom onset to randomization, days | 5 (4-7) | 6 (4-8.5) | 6 (4-7) | 0.175 |
| Symptoms at presentation | ||||
| Fever | 109 (80.1) | 112 (81.8) | 221 (81) | 0.760 |
| Cough | 101 (74.3) | 108 (78.8) | 209 (76.6) | 0.394 |
| Dyspnoea | 114 (83.8) | 113 (82.5) | 227 (83.2) | 0.872 |
| Presence of any comorbid illness, n (%) | 74 (54.4) | 74 (50) | 148 (54.2) | 1.000 |
| Hypertension | 41 (30.1) | 36 (26.3) | 77 (28.2) | 0.503 |
| Diabetes mellitus | 38 (27.9) | 41 (29.9) | 79 (28.9) | 0.790 |
| Thyroid abnormality | 6 (4.4) | 10 (7.3) | 16 (5.9) | 0.441 |
| Coronary artery disease | 9 (6.6) | 6 (4.4) | 15 (5.5) | 0.441 |
| Asthma | 4 (2.9) | 3 (2.2) | 7 (2.6) | 0.722 |
| Two or more comorbid illness, n (%) | 20 (14.6) | 20 (14.7) | 40 (14.7) | 1.000 |
| Respiratory rate, breaths/minute | 26 (25-27) | 26 (25-27) | 26 (25-27) | 0.635 |
| Heart rate, beats/minute | 86 (80-98) | 86 (77-86) | 86 (79-98) | 0.619 |
| PaO2: FiO2 ratio | 125 (75.1-196) | 100 (76-139) | 112 (76-172) | 0.099 |
| Baseline SOFA score | 4 (3-4) | 4 (3-4) | 4 (3-4) | 0.715 |
| Investigations | ||||
| Haemoglobin, g/dL | 13 (11.6-13.8) | 13 (12-14) | 13 (11.6-14) | 0.472 |
| Total leucocyte count,/mL | 9050 (6175-12235) | 9050 (6292-11600) | 9050 (6240-11965) | 0.813 |
| Neutrophil/lymphocyte ratio | 8.5 (4.8-15) | 7.9 (4.3-12.8) | 8.4 (4.6-13.5) | 0.228 |
| Platelet count,/mLx106 | 228 (153-298) | 220 (165-305) | 224 (161-298) | 0.848 |
| Serum creatinine, mg/dL | 0.9 (0.8-1.1) | 0.9 (0.7-1.1) | 0.9 (0.7-1.1) | 0.994 |
| Serum albumin, g/dL | 3.4 (3.1-3.7) | 3.4 (3.1-3.7) | 3.4 (3.1-3.7) | 0.381 |
| Serum bilirubin, mg/dL | 0.6 (0.4-0.7) | 0.5 (0.4-0.7) | 0.5 (0.4-0.7) | 0.032 |
| ALT, U/L | 39 (26.9-63.4) | 46 (26-70) | 42 (26.1-65.8) | 0.607 |
| AST, U/L | 45 (28.9-71.3) | 45 (30-69) | 45 (29.3-70.2) | 0.760 |
| Score on the ordinal scale at baseline, n (%) | 0.429 | |||
| 4- Hospitalized, requiring oxygen supplementation | 120 (88.2) | 127 (92.7) | 247 (90.5) | |
| 5- Hospitalized, requiring high-flow oxygen devices or non-invasive ventilation | 15 (11.1) | 9 (6.6) | 24 (8.8) | |
| 6- Hospitalized, receiving invasive mechanical ventilation or ECMO | 1 (0.7) | 1 (0.7) | 2 (0.7) | |
| Concomitant medication | ||||
| Systemic glucocorticoids | 136 (100) | 137 (100) | 273 (100) | - |
| Anticoagulation | 136 (100) | 137 (100) | 273 (100) | - |
| Remdesivir | 136 (100) | 137 (100) | 273 (100) | |
| Tocilizumab | 8 (5.9) | 6 (4.4) | 14 (5.1) | 0.597 |
| Hydroxychloroquine | 2 (1.5) | 1 (0.7) | 3 (1.1) | 0.622 |
| Antibiotics | 32 (23.5) | 36 (26.3) | 68 (24.9) | 0.675 |
All values are presented as median (interquartile range) unless otherwise mentioned ALT: alanine transaminase; AST: aspartate transaminase; ECMO: extracorporeal membrane oxygenation; FiO2: fraction of oxygen in inspired air; PaO2: partial pressure of oxygen in arterial blood; SOFA: sequential organ failure assessment score
Primary outcomes: Thirty subjects (11%) died because of COVID-19. The number of deaths was similar in the two arms (Mw vs. placebo, 18 [13.2%] vs. 12 [8.8%], P = 0.259). The odds of having a low ordinal scale score were also similar between the two study arms on days 14 (OR, 1.33; 95% confidence intervals [CI], 0.79- 2.3, P = 0.285), 21 (OR, 1.49; 95% CI, 0.83-2.7, P = 0.185), and 28 (OR, 1.49; 95% CI, -0.79-2.8, P = 0.214) [Table 2 and Figure 2].
Table 2.
Primary and secondary outcomes
| Parameter | Mw (n=136) | Placebo (n=137) | Total (n=273) | P |
|---|---|---|---|---|
| Primary outcome | ||||
| 28-day mortality | 18 (13.2) | 12 (8.8) | 30 (11) | 0.259 |
| Clinical status (7-point scale) on day 14 | ||||
| 1- Not hospitalized with resumption of normal activities | 89 (65.4) | 99 (72.3) | 188 (68.9) | |
| 2- Not hospitalized, but unable to resume normal activities | 5 (3.7) | 5 (3.6) | 10 (3.7) | |
| 3- Hospitalized, not requiring supplemental oxygen | 6 (4.4) | 5 (3.6) | 11 (4) | |
| 4- Hospitalized, requiring supplemental oxygen | 14 (10.3) | 12 (8.8) | 26 (9.5) | |
| 5- Hospitalized, requiring nasal high-flow oxygen therapy, non-invasive mechanical ventilation, or both | 4 (2.9) | 2 (1.5) | 6 (2.2) | |
| 6- Hospitalized, requiring ECMO (extracorporeal membrane oxygenation), invasive mechanical ventilation, or both | 7 (5.1) | 7 (5.1) | 14 (2.2) | |
| 7- Death | 11 (8.1) | 7 (5.1) | 18 (6.6) | |
| Difference in clinical status distribution vs. placebo, odds ratio (95% CI)* | 1.33 (0.79-2.3) | Reference | 0.285 | |
| Clinical status (7-point scale) on day 21 | ||||
| 1- Not hospitalized with resumption of normal activities | 99 (72.8) | 109 (79.6) | 208 (76.2) | |
| 2- Not hospitalized, but unable to resume normal activities | 3 (2.2) | 4 (2.9) | 7 (2.6) | |
| 3- Hospitalized, not requiring supplemental oxygen | 4 (2.9) | 0 | 4 (1.5) | |
| 4- Hospitalized, requiring supplemental oxygen | 8 (4.5) | 8 | 16 (5.9) | |
| 5- Hospitalized, requiring nasal high-flow oxygen therapy, non-invasive mechanical ventilation, or both | 1 (0.7) | 3 (2.2) | 4 (1.5) | |
| 6- Hospitalized, requiring ECMO (extracorporeal membrane oxygenation), invasive mechanical ventilation, or both | 6 (4.4) | 5 (3.6) | 11 (4) | |
| 7- Death | 15 (11) | 8 (5.8) | 23 (8.4) | |
| Difference in clinical status distribution vs. placebo, odds ratio (95% CI)* | 1.49 (0.83-2.7) | Reference | 0.185 | |
| Clinical status (7-point scale) on day 28 | ||||
| 1- Not hospitalized with resumption of normal activities | 104 (76.5) | 114 (83.2) | 218 (79.9) | |
| 2- Not hospitalized, but unable to resume normal activities | 6 (4.4) | 4 (2.9) | 10 (3.7) | |
| 3- Hospitalized, not requiring supplemental oxygen | 1 (0.7) | 1 (0.7) | 2 (0.7) | |
| 4- Hospitalized, requiring supplemental oxygen | 4 (2.9) | 3 (2.2) | 7 (2.6) | |
| 5- Hospitalized, requiring nasal high-flow oxygen therapy, non-invasive mechanical ventilation, or both | 3 (2.2) | 2 (1.5) | 5 (1.8) | |
| 6- Hospitalized, requiring ECMO (extracorporeal membrane oxygenation), invasive mechanical ventilation, or both | 0 | 1 (0.7) | 1 (0.4) | |
| 7- Death | 18 (13.2) | 12 (8.8) | 30 (11) | |
| Difference in clinical status distribution vs. placebo, odds ratio (95% CI)* | 1.49 (0.79-2.8) | Reference | 0.214 | |
| Secondary outcomes | ||||
| Delta SOFA score at day 7 | 0 (0-1) | 0 (0-2) | 0 (0-2) | 0.099 |
| Delta SOFA score at day 14 | 0 (0-2) | 0 (0-2) | 0 (0-2) | 0.170 |
| Maximum SOFA score | 4 (3-5) | 4 (3-5) | 4 (3-5) | 0.928 |
| Time to reduction by one-point on seven-point ordinal scale, in days | 5 (3-10) | 5 (3-9) | 5 (3-9) | 0.730 |
| Time to reduction by two-point on seven-point ordinal scale, in days | 7 (5-13) | 7 (5-12) | 7 (5-12.8) | 0.934 |
| Days on vasopressor drug#, in days | 3 (2-5) | 4 (1.8-13) | 3 (2-5.3) | 0.689 |
| Days on mechanical ventilation#, in days | 6.5 (3.5-20.3) | 8.5 (2.3-20.8) | 7.5 (2.8-20.3) | 1.000 |
| ICU length of stay, in days | 7 (5-12) | 8 (5-11) | 8 (5-11 | 0.888 |
| Hospital length of stay, in days | 8 (7-14) | 9 (7-13) | 9 (7-13.3) | 0.773 |
| Time to PCR negativity, days | 9 (4.5-14) | 9 (7-14) | 9 (6-14) | 0.455 |
| Adverse events | ||||
| $Injection site reaction | 19 (14) | 4 (2.9) | 23 (8.4) | 0.001 |
*The odds and p-value for the Mycobacterium w (Mw) treatment arm comparison were estimated using the proportional odds assumption after adjustment for baseline disease severity (baseline ordinal scale and neutrophil-lymphocyte ratio) All values are described as median (interquartile range) or number (percentage) unless specified #includes invasive and non-invasive ventilation $Six subjects in the Mw arm developed local site abscess that spontaneously drained and caused ulceration CI: confidence interval; ICU: intensive care unit; ECMO: extracorporeal membrane oxygenation; SOFA: sequential organ failure assessment score
Figure 2.
Plots depicting the ordinal outcomes in the Mw and the placebo arm on days 7 through 28. We found no difference in the odds of having a lower ordinal scale score between the two groups
Secondary outcomes: There was no difference in the median delta SOFA score at day 7 and day 14 in the Mw arm compared to the placebo. Also, there was no difference in the maximum SOFA score during hospitalization between the two study arms [Table 2]. We also did not observe any difference in the time to clinical improvement (a two-point improvement on an ordinal scale), days spent on a mechanical ventilator, days on vasopressor therapy, or time to achieve a negative RT-PCR [Table 2]. We did not find any difference in the ICU and the hospital length of stay between the two groups [Table 2].
Adverse events: The use of Mw was associated with a significantly higher incidence of local site reaction than placebo (19 [14%] vs. 4 (2.9%), P = 0.001). Six subjects had local site abscess formation that spontaneously drained and caused ulceration. There were no safety concerns associated with the study drug on organ function, vital signs, and laboratory parameters. No death was attributed to the study drug.
Cytokine and transcriptomic analysis: We measured Th-1, Th-2, and Th-17 cytokines in 50 random subjects (25 in each arm). Serum IL-17A levels were significantly higher in the Mw arm at baseline, days 7 and 14 [Table 3]. The use of Mw resulted in a significant increase in IL-10 on day 7 compared to the placebo arm. The IL-6 decreased at days 7 and 14 in both study arms; however, the quantum of fall tended to be higher in the Mw arm.
Table 3.
Trends of cytokine levels in subjects with severe COVID-19 in the two study arms
| Cytokines | Placebo (n=25) | Mw (n=25) | ||||
|---|---|---|---|---|---|---|
|
|
|
|||||
| Day 0 | Day 7 | Day 14 | Day 0 | Day 7 | Day 14 | |
| IL-2 | 0.6±0.9 | 0.8±1.1 | 0.3±0.6 | 0.6±0.7 | 1.2±2.9 | 0.4±0.7 |
| IL-4 | 2.8±4.6 | 2.4±4.1 | 0.9±2.2 | 1.8±2.9 | 2.6±6.9 | 47.1±229.2 |
| IL-6 | 156.3±342.6 | 113.5±258.7a | 50.6±98.9a | 360.9±1204 | 197.2±423.8a | 33.4±62.5a |
| IL-10 | 8.3±15.9 | 4.4±7a,c | 2.9±5.3a | 4.9±4.7 | 7.4±9.9a,c | 1.7±2.6a |
| IL-17A | 10.8±12.8b | 15.3±23.6b | 8.4±11b | 34.7±56.6b | 26±62.2b | 13.9±54.4b |
| IFN-g | 2.2±2.6 | 2.3±2.3 | 1.8±2.7 | 2.3±2.3 | 3.3±5.7 | 1.6±2.8 |
| TNF-a | 1.4±2.2 | 1.3±1.6 | 0.8±1.8 | 0.9±1.9 | 2.1±5.8 | 0.6±1.9 |
a: significantly different within subjects but not between subjects; b: significantly different between subjects; c: significantly between and within subjects All values are presented as mean±standard deviation in pg/mL IL: interleukin; IFN- γ: interferon-gamma; TNF- α: tumour necrosis factor-alpha
We randomly selected 10 subjects (5 in each arm) to study the change in gene expression profile from baseline to day 14. There was a variable gene expression at baseline [Table 4]. However, post-treatment, there was an upregulation of most genes involved in Th1, Th2 and Th-17 pathways in the Mw arm, while there was a downregulation of most genes in the placebo arm.
Table 4.
Classification of the genes based on their clustering with immune pathways TH1, TH2, and TH17. The gene symbols and corresponding expression (mean for all samples) are provided for Day 0 and Day 14 for both groups. The log2 fold change has been reported with the P value for the compared conditions of Day 0 Vs Day 14 results
| TH Type | Gene Symbol | Placebo (n=5) | Mw (n=5) | ||||||
|---|---|---|---|---|---|---|---|---|---|
|
|
|
||||||||
| Day_0 Mean | Day_14 Mean | Log2FoldChange | p value A | Day_0 Mean | Day_14 Mean | Log2Foldchange | p Value B | ||
| TH1 | IL12RB2 | - | - | - | - | - | - | - | - |
| TH1 | FGF2 | 42.6 | 2.2 | -6.609 | 2.63E-05 | - | - | - | - |
| TH1 | IL21-AS1 | 1.2 | 0.2 | -8.208 | 0.000245 | 0.6 | 7.2 | 5.213 | 0.006569 |
| TH17 | CA12 | - | - | - | - | - | - | - | - |
| TH17 | MCAM | 3.8 | 5.2 | -5.442 | 0.000783 | - | - | - | - |
| TH17 | TNFSF13B | 126.4 | 52.8 | -3.085 | 0.002151 | 0.2 | 4.4 | 4.46 | 0.004685 |
| TH17 | ZC2HC1A | - | - | - | - | 0.2 | 3 | 4.687 | 0.007105 |
| TH17 | HLF | - | - | - | - | 0.2 | 4.4 | 5.317 | 0.003302 |
| TH17 | CCR6 | - | - | - | - | - | - | - | - |
| TH17 | PALLD | 19.8 | 19.2 | -4.839 | 8.75E-05 | 0.6 | 5.6 | 5.351 | 0.005577 |
| TH17 | SLC4A10 | - | - | - | - | 0.4 | 10.8 | 6.319 | 0.000579 |
| TH17 | NTRK2 | - | - | - | - | 0.6 | 5.2 | 4.415 | 0.007107 |
| TH17 | KIT | - | - | - | - | 3.2 | 10.8 | 3.383 | 0.003424 |
| TH17 | KIF5C | - | - | - | - | 1.2 | 9.6 | 4.256 | 0.004151 |
| TH2 | CDH1 | - | - | - | - | 0.6 | 4.2 | 4.685 | 0.007892 |
| TH2 | HOXA1 | - | - | - | - | - | - | - | - |
| TH2 | TRIM2 | 1.6 | 7.6 | -6.014 | 0.000838 | 0.6 | 4.4 | 5.348 | 0.007161 |
| TH2 | AKAP12 | - | - | - | - | 0.6 | 8.6 | 5.383 | 0.006994 |
| TH2 | ATP1A4 | - | - | - | - | 0.4 | 5.4 | 5.185 | 0.005564 |
| TH2 | GPR85 | - | - | - | - | 1828 | 644.8 | -2.129 | 0.000138 |
| TH2 | IGHM | - | - | - | - | 0.6 | 7.4 | 5.454 | 0.002575 |
| TH2 | PDE4DIP | - | - | - | - | 0 | 3.8 | 6.154 | 0.000986 |
| TH2 | AHI1-DT | - | - | - | - | 5.2 | 13.4 | 3.311 | 0.002711 |
DISCUSSION
Adjunctive Mycobacterium w did not reduce 28-day mortality or improve clinical status on days 14, 21 and 28 than standard of care alone in critically ill patients with severe COVID-19. We found upregulation of genes involved in Th-1, Th-2 and Th-17 with Mw. On the contrary, there was downregulation of Th-1, Th-2 and Th-17 genes in the placebo arm.
The results of the present study are different from our previous study,[25] where we found improvement in clinical status with Mw. While Mw was ineffective in the current study, there could be other reasons for Mw failure. One important cause could be the improvement in the overall care of COVID-19 subjects with time and standardization of treatment. This was highlighted by a mortality rate of 11% in the current study compared to 21% mortality in our previous study.[25] The current study was also unable to detect a small difference in mortality. Finally, the lack of efficacy could also be due to a change in the strain of COVID-19. The ARMY-2 included subjects infected with the delta variant compared to ARMY-1, which included the initial strains of COVID-19.
COVID-19 evades innate immunity at multiple levels by impeding the production of type I and III interferons.[7] There is also a reduction in the peripheral T cells with severe disease.[5,6] This causes a state of immune suppression. Simultaneously, unabated viral replication causes a hyperinflammatory response in critically ill patients with SARS-CoV-2.[1,2] Thus, there is both a state of hyper-immune response and an immune-suppressive state in SARS-CoV-2.[3] In the current study, we found high IL-6 levels at baseline that were reduced with treatment in both arms. However, the magnitude of the fall was higher in the Mw arm. Further, Mw arm had higher IL-10 levels on day 7 compared to the placebo arm. Mw also resulted in the upregulation of genes involved in the innate and adaptive immune response on day 14. The findings support the role of Mw in modulating immunity by suppressing the overexpressed inflammatory cytokines while at the same time inducing adaptive immune response for effective clearing of the virus. However, despite these differences at the molecular level, the use of Mw did not improve clinical outcomes.
Finally, our study is not without limitations. We could not achieve the target sample size of 300 subjects due to the reduction in COVID-19 cases. Also, our assumption of a 15% difference in mortality in the two groups was not met, making the current trial underpowered to detect a small difference. We did not measure the cytokine levels in all our subjects, which could have enabled us to better understand the mechanism of action of Mw in COVID-19. The genetic expression was different in the two arms at baseline and thus could affect the interpretation of the results.
In conclusion, the use of an immunomodulator Mycobacterium w in addition to standard care did not result in better survival or clinical status compared to standard care alone.
Financial support and sponsorship
Council of Scientific and Industrial Research-New Millennium Indian Technology Leadership Initiative No. 5/258/93/2020-NMITLI of the manuscript.
Conflicts of interest
There are no conflicts of interest.
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