The authors have declared that no competing interests exist.
Conceived and designed the experiments: RR BA GHG. Performed the experiments: AM LK RSR ZR SMMK ASMA. Analyzed the data: MAH RR. Contributed reagents/materials/analysis tools: RR BA GHG. Wrote the paper: BA GHG AM RSR KZ PB SB. Contributed overall coordination of the project: ASMA.
‡ These authors are first authors on this work.
Development of new tuberculosis (TB) drugs and alternative treatment strategies are urgently required to control the global spread of TB. Previous results have shown that vitamin D3 (vitD3) and 4-phenyl butyrate (PBA) are potent inducers of the host defense peptide LL-37 that possess anti-mycobacterial effects.
To examine if oral adjunctive therapy with 5,000IU vitD3 or 2x500 mg PBA or PBA+vitD3 to standard chemotherapy would lead to enhanced recovery in sputum smear-positive pulmonary TB patients.
Adult TB patients (n = 288) were enrolled in a randomized, double-blind, placebo-controlled trial conducted in Bangladesh. Primary endpoints included proportions of patients with a negative sputum culture at week 4 and reduction in clinical symptoms at week 8. Clinical assessments and sputum smear microscopy were performed weekly up to week 4, fortnightly up to week 12 and at week 24; TB culture was performed at week 0, 4 and 8; concentrations of LL-37 in cells, 25-hydroxyvitamin D3 (25(OH)D3) in plasma and
At week 4, 71% (46/65) of the patients in the PBA+vitD3-group (
Adjunct therapy with PBA+vitD3 or vitD3 or PBA to standard short-course therapy demonstrated beneficial effects towards clinical recovery and holds potential for host-directed-therapy in the treatment of TB.
clinicaltrials.gov
Tuberculosis (TB) is a global pandemic disease caused by
A series of
There is growing evidence that adjunct host-directed therapies, could serve as novel approaches to improve standard TB treatments [
In this clinical trial, we aimed to test our hypothesis that oral adjunct therapy with PBA and/or vitD3 administrated to patients with active pulmonary TB would increase LL-37 expression in macrophages and other immune cells and eventually increase elimination of
Patients with newly diagnosed sputum smear-positive pulmonary TB were recruited from the National Institute of the Diseases of the Chest and Hospital (NIDCH) in Dhaka, Bangladesh, after providing written informed consent. NIDCH is a government supported research institute and hospital where the majority of the patients are of low socioeconomic status. Inclusion criteria: both males and females (age ≥18 years) with a newly diagnosed sputum smear-positive TB who consented to study enrollment. Exclusion criteria: pregnancy and lactation, relapse TB, HIV infection, hypercalcaemia, regular intake of vitamin D, known concomitant chronic illness such as diabetes, cardiovascular, hepatic and renal diseases and malignancy. Patients with suspicion of prolonged drug abuse were also excluded. Information such as history of contact with active TB cases, duration of illness, BCG vaccination and tuberculin skin test status were recorded.
The study was approved by the Research and Ethical Review Committees at the International Centre for Diarrheal Disease Research, Bangladesh (icddr,b). The trial was registered with ClinicalTrials.gov (registration number NCT01580007) in April 2012 whereas patient recruitment started in December 2010. The reason for not registering the clinical trial before the recruitment of the first patient was that the authors were not aware of journal requirements for prospective registration. Importantly, the delay in trial registration did not have an impact on the study design or on the analysis or presentation of the results (see attached protocol).
The study was a randomized, double blind, placebo controlled 4-arm intervention trial with adjunct therapy with PBA and/or vitD3 for 2 months. TB patients received directly observed treatment, short-course (DOTS) of a 4 fixed-dose-combination (4-FDC) drugs for 2 months followed by 2-FDC for the next 4 months. The 4-FDC consists of Rifampicin 150mg + Isoniazid 75mg + Pyrazinamide 400mg + Ethambutol 275mg/tablet while the 2-FDC consists of Rifampicin 150mg + Isoniazid 75mg/tablet. The company EM-Partners AB (Råå, Sweden) prepared tablets containing the adjunctive study drug (4-phenylbutyrate (PBA) (Tributyrate®) and identical tablets that contained the placebo (tablet bulking agents or excipient). Vitamin D3 (vit D3) (Vigantol oil) and placebo (Miglyol oil) were obtained from Merck KGaA in Darmstadt, Germany through Popular Pharmaceuticals Ltd in Bangladesh. After enrollment, patients were randomized to the following adjunct treatment arms in a 2x2 factorial design and received oral doses of either: (1) placebo PBA and placebo vitD3 or (2) 500 mg twice daily of PBA and placebo vitD3 or (3) placebo PBA and 5000 IU of vitD3 (Cholecalciferol) once daily or (4) PBA combined with vitD3 (PBA+vitD3).
The dose of PBA was chosen based on a recent study from our group where 500 mg PBA given twice daily to healthy volunteers proved to be the optimal oral dose to induce LL-37 and enhance mycobactericidal activity in monocyte-derived macrophages (MDM) obtained from treated individuals [
Independent assistants from the Hospital pharmacy of icddr,b prepared the study medication packs (PBA and placebo tablets; with identical appearance, color and taste), and labeled these tablets with a randomization number corresponding to the computer-generated randomization sequence. Similarly, Popular Pharmaceuticals Ltd. labeled bottles for vitD3 (Vigantol oil and Miglyol placebo oil; with identical appearance, color and taste) with the provided randomization number. To control and balance for the influence of gender a computer-generated stratified block randomization method was used to randomize participants into four groups that would result in equal sample sizes including 4x72 = 288 patients. The randomized block procedure was performed as follows: (1) a block size of four was chosen at two levels: male and female, (2) possible balanced combinations with four subjects were calculated as 24 blocks and (3) blocks were randomly chosen to determine the assignment of all 288 participants. This randomization protocol resulted in 72 participants (36 males and 36 females) in each of the four treatment groups. Treatment allocation was concealed from patients, study investigators and staff.
No published data is available on the culture conversion of drug-sensitive TB patients at week 4 or week 8 in Bangladesh. Data on smear conversion of Bangladeshi TB patients are available from a publication from 1998 [
Primary outcomes of the trial included assessment of both microbiological and clinical endpoints. The microbiological outcome was measured as the proportion of TB patients who became culture negative at week 4, while effect size was assessed for major clinical endpoints (cough remission, reduction in lung involvement in chest x-ray, normalization of fever and weight gain) at week 8. Secondary outcome measures included time to sputum smear conversion, radiological findings, concentrations of 25(OH)D3 in plasma, immunological status measured as the expression of the antimicrobial peptide LL-37 in immune cells and also as killing of intracellular
A data and safety monitoring board (DSMB) was formed that conducted three meetings (before initiation of the study, interim and after completion) to review the results and to advice on the safety to continue or stop the trial after occurrence of a Serious adverse events (SAE) if any. Adverse events (AE) included hypercalcaemia (albumin adjusted plasma calcium >10.5 mg/dL), arthralgia, hepatitis/jaundice, vomiting, anemia, joint pains, body ache, abdominal pain, headache, malaise, itching, dyspepsia, vertigo, and other non-serious AEs, particularly during the first 8 weeks after start of adjunctive treatment. SAE comprised death, hospitalization or life-threatening conditions. The primary safety endpoint was hypercalcaemia (corrected calcium >10.5 mg/dL).
Clinical assessments and sputum microscopy examinations were performed weekly up to week 4, and consecutively at week 6, 8, 10, 12 and 24. Chest radiographs at NIDCH were examined at week 0, 8, 12 and 24. Sputum culture was performed at NIDCH at week 0, 4 and 8 while drug sensitivity testing was performed at icddr,b. Blood samples were collected at week 0, 4, 8, 12 and 24. Hemoglobin, erythrocyte sedimentation rate (ESR), total and differential counts were determined in the whole blood samples. Concentration of 25(OH)D3, calcium, albumin and C-reactive protein (CRP) were measured in plasma at all time points. MDM and non-adherent lymphocytes were separated from whole blood at week 0, 4, 8 and 12 and used for
Acid-fast bacilli (AFB) in patient’s sputum samples were detected with Ziehl-Neelsen staining and direct smear microscopy. To assess
Clinical assessments were performed by the study doctor assisted by a nurse on scheduled visits and were used to calculate numerical clinical scores as previously described [
In addition to the TB score, chest x-ray findings were assessed and scored as previously reported [
Peripheral blood mononuclear cells (PBMC) and plasma were separated from whole blood by Ficoll-PaqueTM PLUS (GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Plasma samples were stored at -20°C for molecular analysis, while isolated PBMCs were washed and resuspended in culture medium (RPMI-1640 supplemented with 10% autologous plasma, 1% L-glutamine, 1% sodium pyruvate and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA)) and plated in two separate 4-well cell culture plates (NUNC, Roskilde, Denmark). One cell culture plate was used for mRNA isolation and analyses of LL-37 peptide, while the other plate was used for assessment of
After three days incubation of PBMCs in 4-well plates, the culture supernatants containing non-adherent cells were removed and centrifuged to collect the clear supernatant or the extracellular fluid (ECF) of PBMCs. Flow cytometry were used to determine that >80% of non-adherent cells in the plates were CD3+ and CD19+ lymphocytes while >90% of adherent cells were monocytes (CD14+ MDM). The cell pellet of non-adherent lymphocytes were treated with 0.1% saponin (Sigma-Aldrich, Steinheim, Germany) for formation of reversible pores in the cell membrane to release their intracellular content, which was collected as intracellular fluid (ICF) after centrifugation. Next, the adherent MDM were harvested using a cell scraper and treated similarly with saponin, the ICF was collected and stored until further analysis. RNAlater (Qiagen GmbH, Hilden, Germany) was added to the MDM cell pellets to prepare for mRNA isolation and subsequent analysis of LL-37 mRNA content.
LL-37 peptide levels were measured by ELISA in ICF of MDM and non-adherent lymphocytes and in ECF of PBMC. A standard curve of the ELISA was generated from synthetic LL-37 (Innovagen, Lund, Sweden). Polystyrene microtiter plates (Maxisorp by NUNC, Naperville, IL, USA) were coated with monoclonal anti-LL-37 (5 μg/ml) [
mRNA was extracted from MDM and NAL using the RNeasy Mini kit as described by the manufacturer (Qiagen GmbH). mRNA was reverse-transcribed using Bio-Rad CFX 1000, (Hercules, CA, USA) and cDNA was synthesized using Superscript III First-Strand Synthesis System (Invitrogen, Grand Island, NY, USA). The relative expression of mRNA encoding LL-37 peptide compared to the housekeeping gene 18S rRNA was measured by real-time quantitative RT-PCR using the CFX96 Real-Time PCR Detection Systems (Bio-Rad,) and the 18S rRNA-housekeeping gene kit (Applied Biosystems, Foster City, CA, USA). The sequences of forward and reverse primers for mRNA encoding the LL-37 peptide were 5´-TCACCAGAGGATTGTGACTTCAA-3´ and 5´-TGAGGGTCACTGTCCCCATAC-3´, respectively (Primer Express; Applied Biosystems). The results were analyzed by using a relative standard method [
To determine the capacity of
Plasma levels of 25(OH)D3 was estimated by an electrochemiluminescence immunoassay analyzed on an automated Roche immunoassay analyzer (Cobas e601) using a Vitamin D3 Kit (Roche Diagnostics GmbH, Mannheim, Germany). This method is standardized against standard LC-MS/MS [
Calcium and albumin were measured in plasma by two colorimetric assays, Calcium Gen.2 and ALB plus kit (Roche Diagnostics) respectively, while CRP was determined by an immunoturbidometric method (Roche Diagnostics). All results were obtained using an automated clinical chemistry analyzer (Hitachi 902, Roche diagnostics). Quality control material, Precinorm U and Precipath U (for calcium and albumin) as well as Precinorm Protein and Precipath Protein (for CRP) from Roche diagnostics, were used as internal quality controls. Plasma calcium concentrations were adjusted to plasma albumin. Normal serum calcium (albumin adjusted) range is 8.6 to 10.5 mg/dL. Hypercalcaemia was defined at serum albumin-adjusted Ca >10.5 mg/dL.
Concentration of PBA in plasma could not be measured due to lack of access to an appropriate method.
Statistical analysis was performed using IBM SPSS Statistics 20.0 and Stata 13 (StataCorp, College Station, Texas, USA). Data not normally distributed were log transformed that included CRP, LL-37 peptide expression in MDM and lymphocytes as well as plasma 25(OH)D3 levels. A p-value of ≤0·05 was considered significant. The primary analysis of the outcomes of interest was performed by modified intention-to-treat (ITT) for up to week 12 and per-protocol analysis for week 24. Efficacy was assessed by modified ITT that excluded patients who had a negative sputum culture at baseline. Outcome variables were reported as means, with 95% confidence intervals (CI) or standard deviations when continuous, and categorical variables were reported as numbers with percentages. The Chi-square test was used to compare the proportion of patients who became sputum culture negative at week 4 and 8 compared to placebo. Effect size was estimated as difference in proportion of outcome of interest (sputum culture conversion and clinical endpoints) among treatment groups and strength of effect size was examined using Odds Ratio (OR) generated by multivariable logistic regression model. In the model, placebo was used as reference. A mixed model ANCOVA was used to follow-up outcome values as dependent variable and treatment arm as independent variable, to investigate effectiveness of TB scores, leukocyte and monocyte counts, concentrations of hemoglobin, ESR, CRP, calcium, LL-37 and 25(OH)D3 between the treatment arms and placebo. Least significant difference (LSD) was used for multiple comparison test of means of targeted parameters across the treatment arms. We initially investigated potential treatment and covariate interactions and adjusted for those covariates that were significantly associated (by 5% or more) with the outcomes. The covariates examined in the analyses were age, sex, duration of illness, BCG vaccination status, and history of contact with active TB cases. Analyses of time to sputum smear conversion (at week 0–4, 6, 8, 10, 12) and MDM-mediated killing of
A total of n = 417 sputum smear-positive pulmonary TB patients were screened for eligibility between December 14, 2010, and December 26, 2013. Of these, n = 31 patients did not meet the inclusion criteria, n = 27 patients did not provide consent and n = 71 patients were excluded due to the various reasons described in the flowchart in
PBA, Phenylbutyrate; vitD3, vitamin D3; 1Other reasons for not randomizing include living outside Dhaka, difficult to continue in the trial due to job- and academic activity-related problems. 2At base line two participants in the PBA-group did not receive allocation as they refused to continue in the study just after enrollment. 3There were five dropouts between enrollment and week 8, due to migration to other cities, could not be contacted or refused to continue since they moved from Dhaka to their respective village homes in the country side. 4Excluded from analysis: 28 patients were culture negative at baseline, among them, 3 are included in the above 5 dropouts. 5Excluded from analysis: seven patients had multidrug resistant tuberculosis (MDR TB) unevenly distributed among the treatment arms. 6There were thirty patients who discontinued the intervention between week 12 to 24, due to migration, pilgrimage, sent to jail, could not be contacted via phone or when visits to respective homes were made, refused to come to Dhaka for follow-up visits since they moved to their village homes.
Male patients were slightly higher in numbers than female patients (ratio 1.6:1) in each group (
Features | Placebo (n = 72) | PBA (n = 72) | vitD3 (n = 72) | PBA+vitD3 (n = 72) |
---|---|---|---|---|
Gender (Males) (Number, %) | 44(61%) | 44(61%) | 44(61%) | 45(62·5%) |
History of contacts (Number, %) | ||||
Male | 10(22.7%) | 12(27.3%) | 9(20.5%) | 12(26.7%) |
Female | 13(46.4%) | 8(28.6%) | 10(35.7%) | 12(44.4%) |
BCG given (Number, %) | ||||
Male | 33(75.0%) | 24(54.5%) | 35(79.5%) | 32(77.1%) |
Female | 13(46.4%) | 20(71.4%) | 20(71.4%) | 21(77.8%) |
Age, years (Mean±SD) | 26.7±8.1 | 26.8±7.3 | 28.1±9.9 | 26.8±6.9 |
Weight, kg (Mean±SD) | ||||
Male | 46.3±6.6 | 48.9±6.5 | 47.9±8.4 | 46.6±6.6 |
Female | 39.8±7.8 | 37.0±5.7 | 38.4±7.6 | 39.8±7.8 |
Tuberculin skin test done | 7(2.4%) | 12(4.2%) | 8(2.8%) | 9(3.1%) |
Duration of illness, days (Mean±SD) | ||||
Male | 50.9±26.8 | 48.8±20.6 | 55.1±2 6.5 | 53.6±28.7 |
Female | 51.9±23.5 | 53.6±19.9 | 51.9±26.5 | 47.2±22.9 |
ESR, mm 1st hr (Mean±SD) | 60.2±34.9 | 56.9±32.5 | 54.0±31.1 | 56.8 |
Hb, gm/dl (Mean±SD) | 11.6±1.6 | 11.5±1.7 | 11.3±1.9 | 11.7±1.8 |
WBC, 1x103/cmm (Mean±SD) | 10.56±2.63 | 9.99±2.16 | 10.93±2.88 | 11.36±3.21 |
Vitamin D nmol/L | 28.1±16.2 | 23.8±14.8 | 28.0±17.5 | 26.8±16.3 |
Deficient, <30 nmol/L | 40(62.5%) | 41(70.7%) | 46(74.2%) | 39(60.0%) |
Insufficient, 30–50 nmol/L | 19(29.7%) | 13(22.4%) | 8(12.9%) | 20(30.8%) |
Sufficient, >50 nmol/L | 5(7.8%) | 4(6.9%) | 8(12.9) | 6(9.2%) |
Data is presented as mean ± standard deviation or number with percentage in parentheses. BCG,
There were no differences in the occurrence and distribution of most of the AEs between the study arms (
Types of adverse effects | Placebo n = 72 | PBA n = 69 | vitD3 n = 71 | PBA+vitD3 n = 72 |
---|---|---|---|---|
Anemia | 42(58.3%) | 46(64.7%) | 42(59.2%) | 46(63.9%) |
Anorexia | 33(45.8%) | 25(36.2%) | 28(39.4%) | 31(43.1%) |
Joint pain | 20(27.8%) | 18(26.1%) | 26(36.6%) | 17(23.6%) |
Chest pain | 16(22.2%) | 10(15.5%) | 15(21.1%) | 12(16.7%) |
Nausea and or vomiting | 13(18.1%) |
5(7.2%) |
4(5.6%) |
5(6.9%) |
Body ache | 6(8.3%) | 4(5.8%) | 4(5.6%) | 7(9.7%) |
General weakness | 4(5.6%) | 2(2.9%) | 7(9.9%) | 8(11.1%) |
Itching or skin irritation | 3(4.2%) | 2(2.9%) | 1(1.4%) | 2(2.8%) |
Dyspepsia | 3(4.2%) | 4(5.8%) | 1(1.4%) | 3(4.2%) |
Vertigo | 1(1.4%) | 3(4.3%) | 2(2.8%) | 2(2.8%) |
Abdominal pain | 2(2.8%) | 3(4.3%) | 1(1.4%) | 4(5.6%) |
a,bDifferent superscripts in a row show significant difference between the groups. Significance
There were four cases of SAEs that included hospitalization; one patient had severe vomiting with breathing difficulties, two patients experienced severe reactions of anti-TB treatment including elevated levels of serum glutamic pyruvic transaminase (SGPT), and one patient had severe loin pain due to urinary tract infection (
Patient | Sex | Symptoms of serious adverse events | Treatment arms |
---|---|---|---|
001 | Female | Nausea, profuse vomiting, and breathing difficulties | PBA+vitD3 |
002 | Female | Severe abdominal pain, nausea, vomiting, left loin pain | Placebo |
003 | Female | Body ache, vomiting, generalized weakness, elevated SGPT | PBA |
004 | Male | Nausea, vomiting and low grade fever, elevated SGPT | PBA |
Note. SGPT, serum glutamic pyruvic transaminase
Excluding 28 culture negative cases and 7 MDR-TB cases, the number of patients who completed week 4 was 249, which constituted the modified ITT population for analysis of primary endpoint 1. Chi-square test was applied to compare the proportion of patients between intervention groups and placebo. The proportion of patients being culture-negative at week 4 were higher in the PBA+vitD3-group (71%; 46/65) (
The odds ratio of sputum culture conversion was estimated by using multivariable logistic regression adjusting for age and sex. The odds of sputum culture being negative at week 4 was 3.42 times higher in the PBA+vitD3-group (95% Confidence interval (CI), 1.64–7.15,
Points show the age- and sex-adjusted odds ratio (OR) values, and vertical lines delineate 95% confidence intervals. Adjusted OR is shown for four treatment groups (PBA, vitD3 and PBA+vitD3) at week 4 and 8 vs. placebo group. (A) The odds of sputum culture being negative at week 4 was 3.42 times higher in the PBA+vitD3-group (95% Confidence interval (CI), 1.64–7.15) and 2.20 times higher in vitD3-group (95% CI, 1.07–4.51) compared to the placebo-group. (B) The odds of sputum culture being negative at week 8 was 7.26 times higher in the vitD3-group (95% CI, 0.06–25.5), 2.62 times higher in PBA+vitD3-group (95% CI, 0.64–10.72) and 1.36 times higher in the PBA-group (95% CI, 0.40–4.59) compared to the placebo-group.
The odds ratio of major clinical endpoints was estimated by using multivariable logistic regression adjusting for age and sex. There were 249 patients who completed week 8, which formed the modified ITT population for analysis of primary endpoint 2. Only patients in the PBA-group had higher recovery from fever at week 2 compared to placebo (
Fever | Cough | Weight gain | Chest x-ray | |
---|---|---|---|---|
Week 2 | Adjusted OR (95% CI) | Adjusted OR (95% CI) | Adjusted OR (95% CI) | Adjusted OR (95% CI) |
Placebo | Reference | Reference | Reference | |
PBA | 1.93(0.93–4.03) | 1.25(0.47–3.36) | 0.79(0.38–1.63) | |
vitD3 | 1.21(0.58–2.53) | 0.31(0.08–1.19) | 0.94(0.46–1.90) | |
PBA+vitD3 | 1.30(0.63–2.68) | 0.60(0.20–1.81) | 0.67(0.33–1.36) | |
Placebo | Reference | Reference | Reference | |
PBA | 1.14(0.55–2.33) | 1.47(0.66–3.29) | 0.68(0.33–1.39) | |
vitD3 | 0.97(0.46–1.89) | 0.94(0.41–2.17) | 0.46(0.23–0.94) | |
PBA+vitD3 | 1.39(0.69–2.81) | 0.58(0.24–1.42) | 0.52(0.26–1.05) | |
Placebo | Reference | Reference | Reference | Reference |
PBA | 1.05(0.49–2.22) | 1.56(0.75–3.20) | 0.96(0.47–1.98) | 1.50(0.65–3.44) |
vitD3 | 1.37(0.65–2.92) | 1.65(0.82–3.66) | 0.93(0.46–1.89) | 0.87(0.35–2.18) |
PBA+vitD3 | 1.57(0.74–3.35) | 1.32(0.66–2.66) | 0.80(0.39–1.61) | 1.55(0.67–3.56) |
Note. The decrease in fever to normal temperature, disappearance of cough, reduction of lung involvement (as judged by chest x-ray) and increase in weight at various intervals were considered as clinical endpoints.
Comparison of time to sputum smear conversion (sputum smear becoming negative) for the different intervention groups compared to placebo was performed using Log Rank test. No significant differences were obtained between the placebo and intervention groups (log rank 0.228,
The log rank analysis showed no significant differences between the placebo and the intervention groups (log rank 0.228,
The standard classification proposed by the Institute of Medicine (IOM) was followed as sufficient vitamin D status, >50 nmol/L; insufficient status, 30–50 nmol/L; and deficient status, below 30 nmol/L [
The groups receiving vitD3 supplementation (vitD3 and PBA+vitD3-groups) exhibited significantly higher concentrations of plasma 25-hydroxyvitamin D3 at week 4, 8 and 12 intervals compared to placebo after initiation of therapy (p<0.000 for all).
No cases of hypercalcemia were obtained after adjunct therapy; however, prevalence of hypocalcemia was common in all groups. Hypocalcemia was defined as concentration of adjusted plasma calcium <8.6 mg/dL (
Intervals | Placebo n = 64 | PBA n = 58 | vitD3 n = 62 | PBA+vitD3 n = 65 | |
---|---|---|---|---|---|
CRP, μg/dL | Week 0 | 32.14±0.37 | 25.06±0.37 | 26.18±0.37 | 34.51±0.37 |
Week 4 | 10.67±0.53 | 7.36±0.53 | 8.61±0.53 | 9.91±0.54 | |
Week 8 | 5.80±0.60 | 4.88±0.60 | 5.68±0.60 | 5.82±0.60 | |
Week 12 | 3.12±0.58 | 2.98±0.57 | 3.06±0.57 | 3.06±0.57 | |
Adjusted Ca, mg/dL | Week 0 | 8.65±0.59 | 8.65±0.56 | 8.82±0.55 | 8.67±0.58 |
Week 4 | 8.61±0.57 | 8.56±0.61 | 8.74±0.69 | 8.59±0.65 | |
Week 8 | 8.46±0.67 | 8.37±0.61 | 8.57±0.61 | 8.54±0.59 | |
Week 12 | 8.50±0.57 | 8.55±0.66 | 8.69±0.70 | 8.61±0.57 | |
Hypocalcaemia | Week 0 | 29 (45.3%) | 25 (41.7%) | 16 (26.2%) | 31 (47.7%) |
Week 4 | 31 (48.4%) | 28 (46.7%) | 27 (44.3%) | 31 (47.7%) | |
Week 8 | 33 (51.6%) | 38 (63.3%) | 34 (55.7%) | 32 (49.2%) | |
Week 12 | 31 (48.4%) | 26 (43.3%) | 22 (36.1%) | 32 (49.2%) | |
Hemoglobin, g/dL | Week 0 | 11.62±1.60 | 11.68±1.72 | 11.03±1.80 | 11.76±1.85 |
Week 4 | 12.08±1.80 | 12.36±1.77 | 11.95±1.84 | 12.16±2.91 | |
Week 8 | 12.41±1.58 | 12.36±1.89 | 12.29±1.89 | 12.29±1.81 | |
Week 24 |
13.41±1.71 | 13.53±1.92 | 13.16±1.96 | 13.29±1.87 | |
ESR, mm 1st hr | Week 0 | 60.4±35.3 | 57.4±31.9 | 49.8±29.2 | 57.8±34.5 |
Week 4 | 40.0±27.2 | 37.7±28.9 | 41.3±25.4 | 41.6±30.3 | |
Week 8 | 36.3±22.2 | 35.4±27.0 | 33.5±22.0 | 32.5±20.1 | |
Week 24 |
23.0±18.0 | 20.9±15.6 | 21.1±17.1 | 25.2±17.4 | |
Total leukocyte, | Week 0 | 10.59±2.66 | 9.85±2.67 | 10.87±2.66 | 11.33±2.66 |
1x103/cmm | Week 4 | 9.55±2.54 | 9.26±2.55 | 9.89±2.54 | 9.54±2.54 |
Week 8 | 8.99±2.20 | 8.85±2.21 | 8.50±2.20 | 8.41±2.21 | |
Week 24 |
8.24±2.17 | 7.80±2.18 | 8.56±2.18 | 7.88±2.18 | |
Lymphocytes % | Week 0 | 23.65±7.88 | 24.85±7.92 | 24.18±7.90 | 24.36±7.90 |
Week 4 | 25.81±8.93 | 28.73±8.97 | 28.11±8.94 | 28.43±8.94 | |
Week 8 | 28.73±9.16 | 29.43±9.20 | 30.92±9.17 | 30.14±9.17 | |
Week 24 |
34.78±12.04 | 33.47±12.08 | 33.78±12.04 | 35.29±12.04 | |
Neutrophils % | Week 0 | 70.30±8.78 | 68.41±8.82 | 69.34±8.79 | 69.81±8.80 |
Week 4 | 67.54±10.13 | 63.80±10.18 | 64.65±10.15 | 63.83±10.15 | |
Week 8 | 63.70±10.10 | 63.05±10.14 | 61.35±10.12 | 61.74±10.12 | |
Week 24 |
55.39±12.37 | 56.54±12.42 | 55.70±12.38 | 54.86±12.39 | |
Monocyte, % | Week 0 | 3.34±2.38 | 3.90±3.43 | 3.39±2.37 | 3.57±2.31 |
Week 4 | 3.51±2.38 | 3.93±2.79 | 3.50±2.33 | 3.46±2.45 | |
Week 8 | 3.52±2.36 | 3.98±3.17 | 4.32±2.54 | 4.21±2.96 | |
Week 24 |
5.62±3.87a | 5.31±2.81 | 4.93±2.84 | 4.46±2.38b | |
Body weight, Kg | Week 0 | 43.7±7.4 | 44.4±9.1 | 44.2±9.4 | 44.1±7.7 |
Week 4 | 44.2±7.3 | 45.1±9.2 | 45.2±8.9 | 44.9±8.0 | |
Week 8 | 44.9±7.6 | 45.8±9.3 | 46.3±9.5 | 45.6±8.1 | |
Week 12 | 45.6±7.7 | 46.5±9.3 | 47.0±9.5 | 46.3±7.9 |
Data is presented as mean ± standard deviation or numbers with percentage in parentheses. ESR, erythrocyte sedimentation rate; CRP, C-reactive protein. In a row, different superscripts show significant difference between the groups at a given time point.
#At week 24, sample size in four groups were: placebo, n = 54; PBA, n = 49, vitD3, n = 49; PBA+vitD3, n = 54. From 13 patients, hematological reports were not available. Hypocalcemia was defined as concentration of adjusted plasma calcium <8.5 mg/dL. Statistical analyses were done by ANCOVA adjusting for covariates. There were interaction effects of age, sex, contact history and BCG status on ESR and monocyte counts and for the rest of the data interactions were obtained with age and sex only. P is significant when
ANCOVA model was applied to compare the difference in means of LL-37 peptide/mRNA concentrations of the 3 intervention groups with that of the placebo at various time intervals. Concentration of LL-37 peptide was measured in 3 different cell types, thus analysis was performed for each cell type. Age, sex, contact history and BCG status were used as covariates for analyzing LL-37 peptide in lymphocytes and PBMC, and LL-37 mRNA in MDM, while only age and contact history were adjusted for LL-37 peptide in MDM.
A significant increase in LL-37 peptide concentrations in MDM was found in the PBA-group compared to placebo at week 12 (
Concentration of LL-37 mRNA in MDM increased in the PBA+vitD3-group (
Complete sets of macrophages were obtained from 244 TB patients at week 0, 4, 8 and 12. Within each group, the capacity of MDM to kill intra-cellular
Data are expressed as viability of
The composite TB score (
Standard deviation is shown as vertical bar. Comparisons of intervention arms are made with the placebo arm with statistically significant differences being shown in asterisks. The PBA-group demonstrated significantly lower TB scores than the placebo group at week 2, 4, 8, 10 and 12. At week 10 all three intervention groups showed lower scores than the placebo group. Multivariate regression analysis was utilized for comparison of mean effect of clinical scores in the different intervention groups.
At week 0, 8, 12 and 24, the composite TB score also included chest x-ray data. The TB score data were analyzed by ANCOVA after adjusting for age and sex. The complete data set for longitudinal analysis of chest x-ray was available for 211 out of the total of 219 TB patients. Reasons for missing results included chest x-ray plates that were insufficient or unavailable to the study physician. Here, the PBA-group showed significantly lower TB score compared to the placebo-group at week-8 (
Patients were stratified into vitD3-supplemented (vitD3 and PBA+vitD3) and non-vitD3 supplemented groups (PBA and placebo). The proportions of patients being culture-negative at week 4 and week 8 were higher in the vitD3-supplemented-group compared to non-vitD3-group (week 4, 66%; 84/127 vs. 44%; 54/122; p = 0.001) (week 8, 96.9%; 123/127 vs. 90.2%; 110/122;
In this clinical trial, we show positive effects of a novel ‘host-directed therapy’ by inducing the production of antimicrobial peptides (AMPs). Our hypothesis was that oral adjunct therapy with PBA alone or in combination with vitD3 to TB patients would increase the expression of the AMP LL-37, a marker for multiple AMPs, in alveolar macrophages and pulmonary epithelium and eventually accelerate elimination of
In similar clinical trials, the primary endpoint is usually culture conversion assessed at several time points after initiation of TB therapy. Although there are no published data on sputum culture conversion in Bangladesh, the smear conversion rate of Bangladeshi TB patients is estimated as fairly good ranging from 13 to 22 days (K Zaman, personal communication). Martineau
A significant effect of adjunctive therapy with PBA could be demonstrated using a composite TB score including resolution of symptoms [
The role of 1,25(OH)2D3 as an inducer of LL-37 expression has previously been well-established [
Altogether, the findings of reduced TB scores, increased LL-37 concentration in MDM and increased MDM-mediated killing of
Our study confirms that there were no safety concerns regarding the daily oral doses of vitD3 and PBA that were given to the study patients. It is important to note that 91% of the Bangladeshi TB patients demonstrated low vitD3 status and 26–48% patients demonstrated hypocalcaemia at baseline. Treatment with vitD3 increased plasma levels of 25(OH)D3 in both vitD3 and PBA+vitD3 arms. However, there were no cases of hypercalcaemia, although hypocalcaemia persisted in patients who had low calcium at baseline. We found that the 2-month dose of vitD3 used in the trial enabled almost 100% patients to attain sufficient vitamin D status (>50 nmol/L). PBA is an FDA approved drug for urea cycle disorders [
The limitations of this study were that negative
The therapeutic potential of LL-37 in various infections has been demonstrated by
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The authors express their gratitude to the TB patients who participated in the clinical trial. The authors also express their sincere thanks to Saiful Islam, Shamim Hossain, Dr. Sabrina Jabeen and Dr Barkatullah (NIDCH) for their support in patient enrollment and collecting data. We acknowledge the kind assistance provided by the staff at the DOTS centre as well as in the National Tuberculosis Reference Laboratory at NIDCH for their efforts in the implementation of the study.
1,25-dihydroxyvitamin D3
25-Hydroxyvitamin D3
4 drug fixed-dose-combination
Adverse events
Bacillus Calmette–Guérin
colony forming units
C-reactive protein
vitamin D External Quality Assessment Scheme
Directly Observed Treatment, Short-Course
Drug susceptibility tests
extracellular fluid
erythrocyte sedimentation rate
International Centre for Diarrheal Disease Research, Bangladesh
intracellular fluid
Lowenstein-Jensen
monocyte-derived macrophages
multidrug-resistant tuberculosis
multiplicity of infection
National Institute of the Diseases of the Chest and Hospital
4-phenylbutyrate
peripheral blood mononuclear cells
Serious adverse events
tuberculosis
vitamin D3