Zahra Hasan

Vitamin D accelerates clinical recovery from tuberculosis: results of the SUCCINCT Study [Supplementary Cholecalciferol in recovery from tuberculosis]. A randomized, placebo-controlled, clinical trial of vitamin D supplementation in patients with pulmonary tuberculosis’

BackgroundVitamin D enhances host protective immune responses to Mycobacterium tuberculosis by suppressing Interferon-gamma (IFN-g) and reducing disease associated inflammation in the host. The objectives of this study were to determine whether vitamin D supplementation to patients with tuberculosis (TB) could influence recovery.MethodsTwo hundred and fifty nine patients with pulmonary TB were randomized to receive either 600,000 IU of Intramuscular vitamin D3 or placebo for 2 doses. Assessments were performed at 4, 8 and 12 weeks. Early secreted and T cell activated 6 kDa (ESAT6) and Mycobacterium tuberculosis sonicate (MTBs) antigen induced whole blood stimulated IFN-g responses were measured at 0 and 12 weeks. Statistical comparisons between outcome variables at 0 and 12 weeks were performed using Student’s t-test and Chi2 tests.ResultsAfter 12 weeks, the vitamin D supplemented arm demonstrated significantly greater mean weight gain (kg) + 3.75, (3.16 – 4.34) versus + 2.61 (95% CI 1.99 – 3.23) p 0.009 and lesser residual disease by chest radiograph; number of zones involved 1.35 v/s 1.82 p 0.004 (95% CI 0.15, 0.79) and 50% or greater reduction in cavity size 106 (89.8%) v/s 111 (94.8%), p 0.035. Vitamin D supplementation led to significant increase in MTBs-induced IFN-g secretion in patients with baseline ‘Deficient’ 25-hydroxyvitamin D serum levels (p 0.021).ConclusionsSupplementation with high doses of vitamin D accelerated clinical, radiographic improvement in all TB patients and increased host immune activation in patients with baseline ‘Deficient’ serum vitamin D levels. These results suggest a therapeutic role for vitamin D in the treatment of TB.Trial registrationClinicalTrials.gov; No. NCT01130311; URL: clinicaltrials.gov

Rapid determination of anti-tuberculosis drug resistance from whole-genome sequences

Mycobacterium tuberculosis drug resistance (DR) challenges effective tuberculosis disease control. Current molecular tests examine limited numbers of mutations, and although whole genome sequencing approaches could fully characterise DR, data complexity has restricted their clinical application. A library (1,325 mutations) predictive of DR for 15 anti-tuberculosis drugs was compiled and validated for 11 of them using genomic-phenotypic data from 792 strains. A rapid online ‘TB-Profiler’ tool was developed to report DR and strain-type profiles directly from raw sequences. Using our DR mutation library, in silico diagnostic accuracy was superior to some commercial diagnostics and alternative databases. The library will facilitate sequence-based drug-susceptibility testing.

Cytokine Gene Polymorphisms across Tuberculosis Clinical Spectrum in Pakistani Patients

Background Pakistan ranks 7th globally in terms of tuberculosis (TB) disease burden (incidence 181/100000 pop./yr; prevalence of 329/pop./yr). Reports from different populations show variable associations of TB susceptibility and severity with cytokine gene polymorphisms. Tuberculosis clinical severity is multi-factorial and cytokines play a pivotal role in the modulation of disease severity. We have recently reported that the ratio of two key cytokines (IFNγ and IL10) show significant correlation with the severity spectrum of tuberculosis. The objective of the current study was to analyze the frequency of cytokine gene polymorphisms linked to high and low responder phenotypes (IFNγ +874 T hi→A lo and IL10 −1082 G lo→A hi) in tuberculosis patients. Methods and Findings Study groups were stratified according to disease site as well as disease severity: Pulmonary N = 111 (Minimal, PMN = 19; Moderate, PMD = 63; Advance, PAD = 29); Extra-pulmonary N = 67 (Disseminated DTB = 20, Localized LTB = 47) and compared with healthy controls (TBNA = 188). Genotype analyses were carried out using amplification refractory mutation system-PCR (ARMS-PCR) and stimulated whole blood (WB) culture assay was used for assessing cytokine profiles. Our results suggest that the IFNγ +874 TT genotype and T allele was overrepresented in PMN (p = 0.01) and PMD (p = 0.02). IFNγ +874 TT in combination with IL10 GG lo genotypes showed the highest association (χ2 = 6.66, OR = 6.06, 95% CI = 1.31–28.07, p = 0.01). IFNγ AA lo on the other hand in combination with IL10 GG lo increased the risk of PAD (OR = 5.26; p = 0.005) and DTB (OR = 3.59; p = 0.045). Conclusion These findings are consistent with the role of IL10 in reducing collateral tissue damage and the protective role of IFNγ in limiting disease in the lung.

Extensively Drug-Resistant Tuberculosis, Pakistan

Frequency of extensively drug-resistant tuberculosis in Pakistan increased from 1.5% in 2006 to 4.5% in 2009 (p<0.01). To understand the epidemiology, we genotyped selected strains by using spoligotyping, mycobacterial interspersed repetitive units–variable number of tandem repeats, and IS6110 restriction fragment length polymorphism analysis.

Spoligotyping of Mycobacterium tuberculosis isolates from Pakistan reveals predominance of Central Asian Strain 1 and Beijing isolates.

ABSTRACT The estimated incidence of tuberculosis in Pakistan is 181 per 100,000; however, there is limited information on Mycobacterium tuberculosis genotypes circulating in the country. We studied 314 M. tuberculosis clinical isolates; of these, 197 (63%) isolates grouped into 22 different clusters, while 119 (37%) had unique spoligotypes. Eighty-nine percent of the isolates were pulmonary (Pul), and 11% were extrapulmonary (E-Pul). We identified Central Asian Strain (CAS), Beijing, T1, Latin American-Mediterranean, and East African-Indian genogroups. Beijing strains, reportedly the most prevalent spoligotype worldwide, constituted 6% of our strain population. The CAS1 strain comprised 121 (39%) of the study isolates. No difference was observed between clustered isolates from cases of Pul and E-Pul tuberculosis. However, E-Pul isolates included a greater number of unique spoligotypes than Pul isolates (P = 0.005). The overall percentage of drug resistance was 54%, and that of MDR strains was 40%. While CAS1 strains were not associated with drug resistance, the relative risk of MDR was significant in Beijing strains compared to the non-Beijing groups (95% confidence interval, 1.2 to 8.9). The fact that the predominant strain, CAS1, is not associated with drug resistance is encouraging and suggests that an effective tuberculosis control program should be able to limit the high incidence of disease in this region.

Whole Genome Sequencing Based Characterization of Extensively Drug- Resistant Mycobacterium tuberculosis Isolates from Pakistan

Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91–94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.

Population-based resistance of Mycobacterium tuberculosis isolates to pyrazinamide and fluoroquinolones: results from a multicountry surveillance project

Summary Background Pyrazinamide and fluoroquinolones are essential antituberculosis drugs in new rifampicin-sparing regimens. However, little information about the extent of resistance to these drugs at the population level is available. Methods In a molecular epidemiology analysis, we used population-based surveys from Azerbaijan, Bangladesh, Belarus, Pakistan, and South Africa to investigate resistance to pyrazinamide and fluoroquinolones among patients with tuberculosis. Resistance to pyrazinamide was assessed by gene sequencing with the detection of resistance-conferring mutations in the pncA gene, and susceptibility testing to fluoroquinolones was conducted using the MGIT system. Findings Pyrazinamide resistance was assessed in 4972 patients. Levels of resistance varied substantially in the surveyed settings (3·0–42·1%). In all settings, pyrazinamide resistance was significantly associated with rifampicin resistance. Among 5015 patients who underwent susceptibility testing to fluoroquinolones, proportions of resistance ranged from 1·0–16·6% for ofloxacin, to 0·5–12·4% for levofloxacin, and 0·9–14·6% for moxifloxacin when tested at 0·5 μg/mL. High levels of ofloxacin resistance were detected in Pakistan. Resistance to moxifloxacin and gatifloxacin when tested at 2 μg/mL was low in all countries. Interpretation Although pyrazinamide resistance was significantly associated with rifampicin resistance, this drug may still be effective in 19–63% of patients with rifampicin-resistant tuberculosis. Even though the high level of resistance to ofloxacin found in Pakistan is worrisome because it might be the expression of extensive and unregulated use of fluoroquinolones in some parts of Asia, the negligible levels of resistance to fourth-generation fluoroquinolones documented in all survey sites is an encouraging finding. Rational use of this class of antibiotics should therefore be ensured to preserve its effectiveness. Funding Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.

CCL2 responses to Mycobacterium tuberculosis are associated with disease severity in tuberculosis.

Background Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNγ, TNFα and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. Methodology We investigated live M. tuberculosis- and M. bovis BCG- induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNγ, IL10 and CCL3, and mRNA expression of CCL2, TNFα, CCL3 and CXCL8 were determined. Results M. tuberculosis- and BCG- induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis –induced IFNγ was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNγ was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFα mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. Conclusions The increased CCL2 and TNFα in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

Relationship between Circulating Levels of IFN-γ, IL-10, CXCL9 and CCL2 in Pulmonary and Extrapulmonary Tuberculosis is Dependent on Disease Severity

Protection against Mycobacterium tuberculosis infection is dependent on T cell and macrophage activation regulated by cytokines. Cytokines and chemokines produced at disease sites may be released into circulation. Data available on circulating cytokines in tuberculosis (TB) is mostly on pulmonary TB (PTB) with limited information on extrapulmonary disease (EPul‐TB). We measured interferon‐gamma (IFN‐γ), interkeukin‐10 (IL‐10), CXCL9 and CCL2 in sera of patients (n = 80) including; PTB (n = 42), EPul‐TB (n = 38) and BCG vaccinated healthy endemic controls (EC, n = 42). EPul‐TB patients comprised those with less severe (LNTB) or severe (SevTB) disease. Serum IFN‐γ, IL‐10 and CXCL9 levels were significantly greater while CCL2 was reduced in TB patients as compared with EC. IFN‐γ was significantly greater in PTB as compared with LNTB (P = 0.002) and SevTB (P = 0.029). CXCL9 was greater in PTB as compared with LNTB (P = 0.009). In contrast, CCL2 levels were reduced in PTB as compared with LNTB (P = 0.021) and SevTB (P = 0.024). A Spearman’s rank correlation analysis determined a positive association between IFN‐γ and IL‐10 (rho = 0.473, P = 0.002) and IFN‐γ and CXCL9 (rho = 0.403, P = 0.008) in the PTB group. However, in SevTB, only IFN‐γ and CXCL9 were positively associated (rho = 0.529, P = 0.016). Systemic levels of cytokines are reflective of local responses at disease sites. Therefore, our data suggests that in PTB increased IFN‐γ and CXCL9 balanced by IL‐10 may result in a more effective cell mediated response in the host. However, elevated inflammatory chemokines CXCL9 and CCL2 in severe EPul‐TB without concomitant down modulatory cytokines may exacerbate disease related pathology and hamper restriction of M. tuberculosis infection.

Elevated ex vivo monocyte chemotactic protein-1 (CCL2) in pulmonary as compared with extra-pulmonary tuberculosis.

BackgroundTuberculosis causes 3 million deaths annually. The most common site of tuberculosis is pulmonary however; extra-pulmonary forms of the disease also remain prevalent. Restriction of Mycobacterium tuberculosis depends on effective recruitment and subsequent activation of T lymphocytes, mononuclear and polymorphonuclear cells to the site of infection. Tumor necrosis factor (TNF)-α is essential for granuloma formation and is a potent activator of monocyte chemotactic protein (MCP-1, CCL2). CCL2 is essential for recruitment of monocytes and T cells and has been shown to play a role in protection against tuberculosis. Interleukin -8 (CXCL8) is a potent activator of neutrophils. Increased levels of CCL2, CXCL8 and TNFα are reported in tuberculosis but their significance in different forms of tuberculosis is as yet unclear. We have used an ex vivo assay to investigate differences in immune parameters in patients with either pulmonary or extra-pulmonary tuberculosis.MethodsSerum levels of CCL2, CXCL8 and TNFα were measured in patients with pulmonary tuberculosis (N = 12), extra-pulmonary tuberculosis (N = 8) and BCG-vaccinated healthy volunteers (N = 12). Whole blood cells were stimulated with non-pathogenic Mycobacterium bovis bacille-Calmette Guerin (BCG) vaccine strain or bacterial lipopolysaccharide (LPS) and cyto/chemokines were monitored in supernatants.ResultsCirculating serum levels of CXCL8 and TNFα were raised in all tuberculosis patients, while CCL2 levels were not. There was no difference in spontaneous cytokine secretion from whole blood cells between patients and controls. M. bovis BCG-induced ex vivo CCL2 secretion was significantly greater in pulmonary as compared with both extra-pulmonary tuberculosis patients and endemic controls. In response to LPS stimulation, patients with pulmonary tuberculosis showed increased CCL2 and TNFα responses as compared with the extra-pulmonary group. BCG-, and LPS-induced CXCL8 secretion was comparable between patients and controls.ConclusionCCL2 is activated by TNFα and is essential for recruitment of monocytes and T cells to the site of mycobacterial infection. Increased CCL2 activation in pulmonary tuberculosis may result in a stronger cellular response as compared with extra-pulmonary tuberculosis patients, and this may contribute to the localization of infection to the pulmonary site.