Expression of mycolic acid in response to stress and association with differential clinical manifestations of tuberculosis.

Background: Extrapulmonary tuberculosis (EPTB), accounting for 10%-20% of all cases of tuberculosis (TB), is known to be determined by host immunity. However, the contribution of bacterial factors to the development of EPTB has not been studied extensively. Mycolic acids are predominant lipids constituting the cell wall of Mycobacterium tuberculosis, and keto-mycolic acid is involved in the synthesis of foamy macrophages that facilitate persistence of mycobacteria. Hence, the present study was performed to gain an insight into variable expression of mycolic acids in clinical isolates of M. tuberculosis under stress. Methods: Pansusceptible clinical isolates of M. tuberculosis from patients with lymph node TB (LNTB) (n = 10) and pulmonary TB (PTB) (n = 10) were subjected to sodium dodecyl sulfate (SDS) stress, and the expression of mycolic acid and its biosynthetic genes was compared. Any bias arising due to the genotype of the clinical isolates was ruled out by performing single-nucleotide polymorphism cluster grouping (SCG), wherein no significant difference was observed between the SCG of LNTB or PTB isolates. Results: The expression of α-mycolic acid during the exposure to SDS was high in 7/10 (70%) LNTB and 6/10 (60%) PTB isolates. Methoxy mycolic acid showed an increased expression in 7/10 (70%) LNTB isolates and 4/10 (40%) PTB isolates. Increased expression of keto-mycolic acid on exposure with SDS was observed in 8/10 (80%) M. tuberculosis LNTB and 3/10 (30%) PTB isolates. Similarly, the mycolic acid synthesis gene, fas, was upregulated more in LNTB isolates than PTB isolates in vitro and ex vivo. SCG 3a was the most common SCG observed in 40% (8/20) of the isolates, followed by SCG 3b in 30% (6/20) of the isolates. There was no significant difference between the SCG of LNTB or PTB isolates. Conclusion: The higher expression of keto-mycolic acid in LNTB as against PTB isolates may indicate better survival in LNTB isolates in the presence of stress.

Establishment of Elevated Serum Levels of IL-10, IL-8 and TNF-ß as Potential Peripheral Blood Biomarkers in Tubercular Lymphadenitis: A Prospective Observational Cohort Study.

BACKGROUND: Tubercular lymphadenitis (TL) is the most common form of extra-pulmonary tuberculosis (TB) consisting about 15-20% of all TB cases. The currently available diagnostic modalities for (TL), are invasive and involve a high index of suspicion, having limited accuracy. We hypothesized that TL would have a distinct cytokine signature that would distinguish it from pulmonary TB (PTB), peripheral tubercular lymphadenopathy (LNTB), healthy controls (HC), other lymphadenopathies (LAP) and cancerous LAP. To assess this twelve cytokines (Tumor Necrosis Factor (TNF)-α, Interferon (IFN) -γ, Interleukin (IL)-2, IL-12, IL-18, IL-1ß, IL-10, IL-6, IL-4, IL-1Receptor antagonist (IL-1Ra), IL-8 and TNF-ß, which have a role in pathogenesis of tuberculosis, were tested as potential peripheral blood biomarkers to aid the diagnosis of TL when routine investigations prove to be of limited value. METHODS AND FINDINGS: A prospective observational cohort study carried out during 2010-2013. This was a multi-center study with three participating hospitals in Delhi, India where through random sampling cohorts were established. The subjects were above 15 years of age, HIV-negative with no predisposing ailments to TB (n = 338). The discovery cohort (n = 218) had LNTB (n = 50), PTB (n = 84) and HC (n = 84). The independent validation cohort (n = 120) composed of patients with cancerous LAP (n = 35), other LAP (n = 20) as well as with independent PTB (n = 30), LNTB (n = 15) and HC (n = 20). Eight out of twelve cytokines achieved statistical relevance upon evaluation by pairwise and ROC analysis. Further, variable selection using random forest backward elimination revealed six serum biosignatures including IL-12, IL-4, IL-6, IL-10, IL-8 and TNF-ß as optimal for classifying the LNTB status of an individual. For the sake of clinical applicability we further selected a three analyte panel (IL-8, IL-10 and TNF-ß) which was subjected to multinomial modeling in the independent validation cohort which was randomised into training and test cohorts, achieving an overwhelming 95.9% overall classifying accuracy for correctly classifying LNTB cases with a minimal (7%) misclassification error rate in the test cohort. CONCLUSIONS: In our study, a three analyte serum biosignatures and probability equations were established which can guide the physician in their clinical decision making and step wise management of LNTB patients. This set of biomarkers has the potential to be a valuable adjunct to the diagnosis of TL in cases where AFB positivity and granulomatous findings elude the clinician.

Genetic association study suggests a role for SP110 variants in lymph node tuberculosis but not pulmonary tuberculosis in north Indians.

Ethnic specificity is a key determinant in understanding the association of genetic variants with outcome of disease susceptibility. SP110, a component of the nuclear body, has been subjected to association studies with conflicting results. In this study we probed SP110 variants in pulmonary (PTB) and lymph node tuberculosis (LNTB) cases to explore their role in controlling susceptibility to Mycobacterium tuberculosis infection in north Indians. We genotyped 24 SP110 variants in over 140 north Indian tuberculosis cases and 78 ethnicity-matched controls. The SP110 gene variants were available from public databases. The cases and controls were free of any population stratification when subjected to Eigenstrat principal component analysis. Genotyping was carried out using the Sequenom MassARRAY platform. Applying exclusion criteria, 11 single nucleotide polymorphisms (SNPs) of the LNTB panel and 13 SNPs of the PTB panel passed all filters and were analyzed further. No significant association was observed between SP110 variants and PTB. Surprisingly, we discovered evidence of an association of SP110 variants with LNTB, a form of extrapulmonary tuberculosis, at 3 loci, namely, rs6436915, rs1427294, and rs1346311. When permutations analysis (n = 10,000) of allelic p values was undertaken, only rs1427294 passed the test with its p value remaining statistically significant. The C allele of rs1427294 exhibited a 5-fold risk of developing LNTB. No significant haplotypes were observed. In the pilot study presented here, our results provide evidence for the first time that SP110 may be a risk determinant locus in LNTB while confirming a doubtful role of SP110 in PTB in north Indians. In general, the results might indicate a role of SP110 variants in extrapulmonary tuberculosis rather than PTB.