Human Intestinal Defensin 5 Ameliorates the Sensitization of Colonic Cancer Cells to 5-Fluorouracil.

BACKGROUND AND AIM: The increasing dilemma of multidrug-resistant cancer cells in response to currently available chemotherapeutic drugs and their associated side effect(s), calls for the investigation of alternative anticancer advances and molecules. Therefore, the present study aimed to elucidate the combinatorial potential against colon cancer of human defensin 5 in combination with 5-fluorouracil (5-FU), and against 5-FU resistant colon tumor cells. METHODS: The in vivo combinatorial potential of HD-5 with 5-FU was elucidated in terms of tumor morphometrics, apoptosis assay, surface morphology histology of the colon(s), and transcriptional alterations. Changes in membrane dynamics with mucin expression were evaluated by fluorescence microscopy and histochemistry. The in vitro activity of the peptide/drug conjunction was explored by phase contrast microscopy, MTT, LDH assay, and AO/EtBr staining. Chemoresistance to 5-FU was determined by phase contrast microscopy, MTT assay, annexin V-FITC/PI flow cytometry, and MDR-1, Bak, and Bax expression. RESULTS: In vivo decreases in tumor parameters, with a marked increase in apoptosis and neutrophil infiltrations indicated restoration of normal architecture with improved mucin content in the treated colons. This happened with substantial changes in key molecular markers of the intrinsic apoptotic cascade. Membrane dynamics revealed that peptides and chemotherapeutic drugs could bind to cancerous cells by taking advantage of altered levels of membrane fluidity. CONCLUSION: Peptide treatment of drug-resistant Caco-2 cells promotes enhanced 5-FU uptake, in contrast to when cells were treated with 5-FU alone. Hence, HD-5 as an adjunct to 5-FU, exhibited strong cancer cell killing even against 5-FU-resistant tumorigenic cells.

In-vivo expressed mycobacterial transcripts as diagnostic targets for pulmonary tuberculosis.

The nucleic acid amplification tests (NAATs) such as Xpert MTB/RIF have transformed the TB diagnostic field by significantly increasing the case detection. However, newer improved diagnostic assays are still needed to meet the WHO targets to end TB. Present study is based on a novel approach of utilizing the in-vivo expressed specific mycobacterial transcriptomic biomarkers for the diagnosis of pulmonary tuberculosis (PTB). Total 61 subjects were recruited including smear positive (smear+; n = 15), smear negative (smear-; n = 30) PTB patients and disease controls (n = 16). Transcripts of three mycobacterial genes Rv0986, Rv0971c and Rv3121 were analyzed using real time PCR (qRT-PCR) in sputum samples. qRT-PCR with Rv0986, Rv0971c and Rv3121 identified smear + PTB patients with 100 %, 78.6 % and 86.7 % sensitivity respectively. In smear- PTB patients, both Rv0986 and Rv0971c based qRT-PCR resulted in 63 %, sensitivity whereas Rv3121 identified these patients with ∼40 % sensitivity only. The sensitivity of the assay for smear-patients increased to 85 % when combinatorial analysis of qRT-PCR data for all the three genes was used. Thus, in-vivo expressed mycobacterial transcripts have promising potential as biomarkers for PTB diagnosis.

Antibody response to mycobacterial Rpf B protein and its immunodominant peptides in HIV-TB co-infected individuals.

Diagnosis of TB at early stages of HIV infection may lead to timely intervention for improving patient outcome. Antibodies to Mycobacterium tuberculosis recombinant RpfB protein and two immunodominant peptides of Rpf B protein were evaluated in the sera of HIV +TB+, HIV+ and HIV- pulmonary TB patients by ELISA. Serum antibodies from 90 % and 65 % of HIV+TB+ patients reacted to recombinant RpfB protein and synthetic peptide RpfP1 respectively. Overall, this study shows that resuscitation promoting factor B elicits humoral antibody response in HIV+TB+ co-infected individuals and be proposed as a potential biomarker for diagnosis of HIV+TB+ patients, however further longitudinal follow up studies are warranted.

Association of Micronutrients with Tuberculosis Development in HIV Infected Patients.

Human immunodeficiency virus (HIV) infection associated with weakened immune system due to decreased CD4 T cell count favors development of tuberculosis. Effector immune responses are also associated with micronutrient status due to their prominent role in maintaining immune functions. Micronutrient deficiencies are quite common among HIV patients that further result into compromised immunity thus making the conditions even more favorable for mycobacteria to establish disease. So, current study was designed to assess association of different micronutrients with development of TB in HIV patients. Micronutrient levels were measured in asymptomatic HIV patients who were monitored for the development of TB during follow up period (incident TB) within one month to one year and also in symptomatic microbiologically confirmed HIV-TB patients. Among various micronutrients assessed, levels of ferritin were found to be significantly increased (p < 0.05) with significant decreased zinc (p < 0.05) and selenium (p < 0.05) levels in incident TB group as well as in HIV-TB subjects compared to asymptomatic HIV patients who did not develop TB in the follow up period. Importantly, increased levels of ferritin and decreased levels of selenium were significantly associated with development of tuberculosis in HIV patients.

Diagnosis and biomarkers for ocular tuberculosis: From the present into the future.

Tuberculosis is an airborne disease caused by Mycobacterium tuberculosis (Mtb) and can manifest both pulmonary and extrapulmonary disease, including ocular tuberculosis (OTB). Accurate diagnosis and swift optimal treatment initiation for OTB is faced by many challenges combined with the lack of standardized treatment regimens this results in uncertain OTB outcomes. The purpose of this study is to summarize existing diagnostic approaches and recently discovered biomarkers that may contribute to establishing OTB diagnosis, choice of anti-tubercular therapy (ATT) regimen, and treatment monitoring. The keywords ocular tuberculosis, tuberculosis, Mycobacterium, biomarkers, molecular diagnosis, multi-omics, proteomics, genomics, transcriptomics, metabolomics, T-lymphocytes profiling were searched on PubMed and MEDLINE databases. Articles and books published with at least one of the keywords were included and screened for relevance. There was no time limit for study inclusion. More emphasis was placed on recent publications that contributed new information about the pathogenesis, diagnosis, or treatment of OTB. We excluded abstracts and articles that were not written in the English language. References cited within the identified articles were used to further supplement the search. We found 10 studies evaluating the sensitivity and specificity of interferon-gamma release assay (IGRA), and 6 studies evaluating that of tuberculin skin test (TST) in OTB patients. IGRA (Sp = 71-100%, Se = 36-100%) achieves overall better sensitivity and specificity than TST (Sp = 51.1-85.7%; Se = 70.9-98.5%). For nuclear acid amplification tests (NAAT), we found 7 studies on uniplex polymerase chain reaction (PCR) with different Mtb targets, 7 studies on DNA-based multiplex PCR, 1 study on mRNA-based multiplex PCR, 4 studies on loop-mediated isothermal amplification (LAMP) assay with different Mtb targets, 3 studies on GeneXpert assay, 1 study on GeneXpert Ultra assay and 1 study for MTBDRplus assay for OTB. Specificity is overall improved but sensitivity is highly variable for NAATs (excluding uniplex PCR, Sp = 50-100%; Se = 10.5-98%) as compared to IGRA. We also found 3 transcriptomic studies, 6 proteomic studies, 2 studies on stimulation assays, 1 study on intraocular protein analysis and 1 study on T-lymphocyte profiling in OTB patients. All except 1 study evaluated novel, previously undiscovered biomarkers. Only 1 study has been externally validated by a large independent cohort. Future theranostic marker discovery by a multi-omics approach is essential to deepen pathophysiological understanding of OTB. Combined these might result in swift, optimal and personalized treatment regimens to modulate the heterogeneous mechanisms of OTB. Eventually, these studies could improve the current cumbersome diagnosis and management of OTB.

Evaluation of immunodominant peptides of in vivo expressed mycobacterial antigens in an ELISA-based diagnostic assay for pulmonary tuberculosis.

Non-sputum-based biomarker assay is urgently required as per WHO's target product pipeline for diagnosis of tuberculosis. Therefore, the current study was designed to evaluate the utility of previously identified proteins, encoded by in vivo expressed mycobacterial transcripts in pulmonary tuberculosis, as diagnostic targets for a serodiagnostic assay. A total of 300 subjects were recruited including smear+, smear- pulmonary tuberculosis (PTB) patients, sarcoidosis patients, lung cancer patients and healthy controls. Proteins encoded by eight in vivo expressed transcripts selected from previous study including those encoded by two topmost expressed and six RD transcripts (Rv0986, Rv0971, Rv1965, Rv1971, Rv2351c, Rv2657c, Rv2674, Rv3121) were analyzed for B-cell epitopes by peptide arrays/bioinformatics. Enzyme-linked immunosorbent assay was used to evaluate the antibody response against the selected peptides in sera from PTB and controls. Overall 12 peptides were selected for serodiagnosis. All the peptides were initially screened for their antibody response. The peptide with highest sensitivity and specificity was further assessed for its serodiagnostic ability in all the study subjects. The mean absorbance values for antibody response to selected peptide were significantly higher (p<0.001) in PTB patients as compared to healthy controls; however, the sensitivity for diagnosis of PTB was 31% for smear+ and 20% for smear- PTB patients. Thus, the peptides encoded by in vivo expressed transcripts elicited a significant antibody response, but are not suitable candidates for serodiagnosis of PTB.

Metabolic switching and cell wall remodelling of Mycobacterium tuberculosis during bone tuberculosis.

OBJECTIVES: Bone tuberculosis (TB) is the third most common types of extrapulmonary tuberculosis. It is critical to understand mycobacterial adaptive strategies within bone lesions to identify mycobacterial factors that may have role in disease pathogenesis. METHODS: Whole genome microarray was used to characterize the in-vivo transcriptome of Mycobacterium tuberculosis (M.tb) within bone TB specimens. Mycobacterial virulent proteins were identified by bioinformatic software. An in vitro osteoblast cell line model was used to study the role of these proteins in bone TB pathogenesis. RESULTS: 914 mycobacterial genes were significantly overexpressed and 1688 were repressed in bone TB specimens. Pathway analysis of differentially expressed genes demonstrated a non-replicative and hypometabolic state of M.tb, reinforcement of the mycobacterial cell wall and induction of DNA damage repair responses, suggesting possible survival strategies of M.tb within bone. Bioinformatics mining of microarray data led to identification of five virulence proteins. The genes encoding these proteins were also upregulated in the in vitro MC3T3 osteoblast cell line model of bone TB. Further, exposure of osteoblast cells to two of these virulence proteins (Rv1046c and Rv3663c) significantly inhibited osteoblast differentiation. CONCLUSION: M.tb alters its transcriptome to establish infection in bone by upregulating certain virulence genes which play a key role in disturbing bone homeostasis.

Anti-staphylococcal responses and their relationship with HLA-DR-DQ polymorphism in granulomatosis with polyangiitis: a preliminary evidence of association with disease outcome.

Chronic nasal carriage of Staphylococcus aureus (S. aureus) is a risk factor for relapse of granulomatosis with polyangiitis (GPA), and genetic susceptibility to infections and autoimmune diseases is majorly affected by HLA genes. Previous studies have shown the association of HLA Class-II genes with GPA susceptibility. Here, we aim to assess immune responses of GPA patients against S. aureus antigens in relation to the HLA-DR-DQ genes polymorphism to determine the disease outcome. A total of 45 GPA patients and 128 healthy controls during 2010-2012 were included in this case-control study. HLA-DRB1/DQB1 allele typing was performed by polymerase chain reaction-sequence-specific primer (PCR-SSP) method. Immune responses against S. aureus antigens were investigated in 20 active vs. remitting GPA (after 6 months of cyclophosphamide and glucocorticoids) patients by Western blot. Statistical analysis was performed using χ2 test and Fisher's exact test. We observed a significant association of DRB1*08, DRB1*16 and DQB1*04 alleles with GPA susceptibility, whereas DRB1*15, DRB1*10 and DQB1*05 alleles were suggested as protective alleles. Among S. aureus antigens, active GPA patients' sera reacted more strongly with 34 and 24 kDa antigens of S. aureus than remitting and healthy control  sera. Furthermore, we observed that the lack of DQB1*06 allele confers complete remission even in the presence of anti-S. aureus antibodies against 24 kDa protein. Our findings suggest that the presence of DQB1*06 allele and S. aureus infection may prolong active disease. Further, our study indicates the potential of using anti-staphylococcal medications for achieving remission in patients having HLA-DQB1*06 allele.

COVID-19 associated variations in liver function parameters: a retrospective study.

BACKGROUND: Characteristics of laboratory findings of COVID-19 patients are of great significance for diagnosis and treatment. Studies that have analysed the variations in hepatic profile in correlation with the inflammatory markers in SARS-CoV-2 are limited. METHODS: We retrospectively analysed liver function tests and inflammatory markers of 170 admitted patients with confirmed COVID-19 in the tertiary care centre, Post Graduate Institute of Medical Education and Research (PGIMER), India, using Roche Cobas Autoanalyzer. RESULTS: Number of patients with normal liver enzyme levels were 63 (41.5%), while with raised levels of any of the liver enzymes were 89 (58.5%), out of which 43 (48.31%) had liver injury which manifested as increased severity in terms of intensive care unit (ICU) requirement (p=0.0005). Significantly raised levels of liver enzymes and liver injury were observed with age (p<0.0001) and in males (p=0.004). Significantly decreased levels of albumin and total proteins and increased levels of total bilirubin (p<0.0001) were seen in patients with abnormal liver enzyme levels and liver injury as compared to patients with normal levels. Significant increase in the levels of alanine transaminase and gamma-glutamyl transferase was seen on the 7th day, CRP and ferritin (p<0.0001) peaks were observed on 2nd and 3rd day respectively. A significant positive correlation was found between the levels of these inflammatory markers and liver function parameters. CONCLUSIONS: More than half of patients admitted to the hospital with SARS-CoV-2 infection had an abnormal liver function which was found to be associated with raised levels of inflammatory markers. Significantly higher proportions of patients with abnormal liver function were elderly and males and were at higher risk of progressing to severe disease.

PM2.5 mediated alterations in the in vitro human granuloma and its effect on reactivation of mycobacteria.

Exposure to particulate matter pollutant PM2.5 diminishes the immune response to mycobacterial antigens relevant to contain the infection in the granuloma, thus leading to reactivation of latent bacilli. The present study was therefore designed based on the hypothesis that exposure to PM2.5 affects the granuloma formation and reactivation of latent mycobacterial bacilli contained in the granuloma. For the sampling of PM2.5, based on initial standardisations, Teflon filter was selected over the quartz filter. Two different approaches were used to study the effect of PM2.5 on the human PBMC granuloma formed by Mycobacterium bovis BCG at multiplicity of infection (MOI) 0.1. In the first approach, granuloma formed in the presence of PM2.5 was loosely packed and ill-defined with significant downregulation of dormancy-associated mycobacterial genes, upregulation of reactivation-associated rpfB gene along with a significant increase in TNFα level without any change in the bacterial load in terms of CFUs. In the second approach, preformed human PBMC granuloma using M. bovis BCG was treated with PM2.5 that resulted in the disruption of granuloma architecture along with downregulation of not only dormancy-associated genes but also reactivation-associated rpfB gene of mycobacterial bacilli recovered from granuloma. However, there was no significant change in the host cytokine levels. Therefore, it can be inferred that PM2.5 can modulate the granuloma formation in vitro as well as mycobacterial gene expression in the granuloma with a possible role in the reactivation of latent bacilli.

Immunological aspects of host-pathogen crosstalk in the co-pathogenesis of diabetes and latent tuberculosis.

Introduction: Diabetes is a potent risk factor for the activation of latent tuberculosis and worsens the tuberculosis (TB) treatment outcome. The major reason for mortality and morbidity in diabetic patients is due to their increased susceptibility to TB. Thus, the study was conducted to understand the crosstalk between M. tuberculosis and its host upon latent tuberculosis infection and under hyperglycemic conditions or diabetes. Methods: An animal model was employed to study the relationship between latent tuberculosis and diabetes. BCG immunization was done in mice before infection with M. tuberculosis, and latency was confirmed by bacillary load, histopathological changes in the lungs and gene expression of hspX, tgs1, tgs3 and tgs5. Diabetes was then induced by a single high dose of streptozotocin (150 mg/kg body weight). Host factors, like various cytokines and MMPs (Matrix metalloproteinases), which play an important role in the containment of mycobacterial infection were studied in vivo and in vitro. Results: A murine model of latent TB was developed, which was confirmed by CFU counts (<104 in the lungs and spleen) and granuloma formation in lungs in the latent TB group. Also, the gene expression of hspX, tgs1, and tgs5 was upregulated, and after diabetes induction, blood glucose levels were >200 mg/dl. An in vitro study employing a THP-1 macrophage model of latent and active tuberculosis under normal and high glucose conditions showed that dormant bacilli were better contained in the presence of 5.5 mM glucose concentration as compared with active bacilli. However, the killing and restriction efficiency of macrophages decreased, and CFU counts increased significantly with an increase in glucose concentration. Discussion: The decreased levels of MCP-1, decreased expression of mmp-9, and increased expression of mmp-1 in the latent group at high glucose concentrations could explain the failure of granuloma formation at high glucose conditions.

Distinct interfacial ordering of liquid crystals observed by protein-lipid interactions that enabled the label-free sensing of cytoplasmic protein at the liquid crystal-aqueous interface.

Interfaces formed between a lipid decorated liquid crystal (LC) film and an aqueous phase can mimic the bimolecular membrane where interfacially occurring biological phenomena (e.g., lipid-protein interactions, protein adsorption) can be visually monitored by observing the surface-sensitive orientations of LCs. The ordering behavior of LCs at different phospholipid-based LC interfaces (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and lysophosphatidic acid (LPA)) were investigated to determine the sensing of an important cytoplasmic protein (juxtamembrane of epidermal growth factor receptor (JM-EGFR)). At both DLPC and LPA decorated interfaces, the LC adopts homeotropic ordering, causing a dark optical appearance under crossed polarizers. Interestingly, upon the introduction of JM-EGFR to these LC-aqueous interfaces, the homeotropic orientation of the LC changed to planar (bright optical appearance), suggesting the potential of the designed system for JM-EGFR sensing. The use of different lipid decorated LC-aqueous interfaces results in the emergence of distinct optical patterns. For example, at a DLPC laden interface, elongated bright domains are observed, whereas a uniform bright texture is observed on an LPA laden interface. The DLPC decorated LC-aqueous interface is found to be highly selective for the sensing of JM-EGFR with a detection limit in the nanomolar concentration region (∼ 50 nM). When compared to spectroscopic and other conventional techniques, the LC-based design is simpler, and it allows the simple and label-free optical sensing of JM-EGFR at fluidic interfaces.

Reactivation of latent tuberculosis through modulation of resuscitation promoting factors by diabetes.

The evidence of an association between diabetes and latent tuberculosis infection (LTBI) remains limited and inconsistent. Thus, the study aims to delineate the role of diabetes in activation of latent tuberculosis infection. Murine model of latent tuberculosis and diabetes was developed, bacillary load and gene expression of resuscitation promoting factors (rpfA-E) along with histopathological changes in the lungs and spleen were studied. Treatment for LTBI [Rifampicin (RIF) + Isoniazid (INH)] was also given to latently infected mice with or without diabetes for 4 weeks. Diabetes was found to activate latent tuberculosis as the colony forming unit (CFU) counts were observed to be > 104 in lungs and spleen. The gene expression of hspX was downregulated and that of rpfB and rpfD was observed to be upregulated in latently infected mice with diabetes compared to those without diabetes. However, no significant reduction in the CFU counts was observed after 4 weeks of treatment with RIF and INH. Diabetes helps in the progression of LTBI to active disease mainly through altered expression of resuscitation promoting factors rpfB and rpfD, which can serve as important targets to reduce the shared burden of tuberculosis and diabetes.

Corrigendum to (Molecular mechanism of interaction of Mycobacterium tuberculosis with host macrophages under high glucose conditions).

[This corrects the article DOI: 10.1016/j.bbrep.2021.100997.].

Correction to: Combined Analysis of Anti SARS-CoV-2 IgG and IgM Responses in COVID19 Patients in India.

[This corrects the article DOI: 10.1007/s12291-021-00986-x.].

Combined Analysis of Anti SARS-CoV-2 IgG and IgM Responses in COVID19 Patients in India.

Coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a global health problem, India being the second most affected country. The kinetics of antibody response to SARS-CoV-2 in Indian population is not studied yet. To understand serological response in relation to age, gender, time period and severity of disease, Roche Elecsys anti-SARS-CoV-2 test was used which analysed both IgM and IgG. One hundred and three COVID-19 patients were enrolled. Seropositivity was seen in 64% of patients, with 33% at ≤ 7 days, 62% between 8 and 15 days and 81% at ≥ 16 days from the time of admission. Men (65%) showed higher antibody response than women (59%), whereas no difference was observed in seropositivity with respect to age of the patients. Dynamics of antibody responses revealed individual variations. Patients in ICU had higher antibody reactivity with 67% positivity as compared to 60% positivity in non-ICU patients. Kinetics of antibody response during COVID-19 disease varied in relation to gender, age, time period and severity and these factors might play an important role in treatment and control of COVID-19.

Molecular mechanism of interaction of Mycobacterium tuberculosis with host macrophages under high glucose conditions.

Mycobacterium tuberculosis has the potential to escape various cellular defense mechanisms for its survival which include various oxidative stress responses, inhibition of phagosome-lysosomes fusion and alterations in cell death mechanisms of host macrophages that are crucial for its infectivity and dissemination. Diabetic patients are more susceptible to developing tuberculosis because of impairement of innate immunity and prevailing higher glucose levels. Our earlier observations have demonstrated alterations in the protein profile of M. tuberculosis exposed to concurrent high glucose and tuberculosis conditions suggesting a crosstalk between host and pathogen under high glucose conditions. Since high glucose environment plays crucial role in the interaction of mycobacterium with host macrophages which provide a niche for the survival of M. tuberculosis, it is important to understand various interactive mechanisms under such conditions. Initial phagocytosis and containment of M. tuberculosis by macrophages, mode of macrophage cell death, respiratory burst responses, Mycobacterium and lysosomal co-localization were studied in M. tuberculosis H37Rv infected cells in the presence of varied concentrations of glucose in order to mimic diabetes like conditions. It was observed that initial attachment, phagocytosis and later containment were less effective under high glucose conditions in comparison to normal glucose. Mycobacterium infected cells showed more necrosis than apoptosis as cell death mechanism during the course of infection under high glucose concentrations. Co-localization and respiratory burst assay also indicated evasion strategies adopted by M. tuberculosis under such conditions. This study by using THP1 macrophage model of tuberculosis and high glucose conditions showed immune evasion strategies adapted during co-pathogenesis of tuberculosis and diabetes.

Comparison of performances of loop-mediated isothermal amplification, XPERT MTB/RIF and BACTEC MGIT in the diagnosis of childhood tuberculosis.

AIM: Key to the successful management of paediatric pulmonary tuberculosis (PTB) lies in the early detection and proper treatment. We evaluated the performances of modern diagnostic tests: loop-mediated isothermal amplification (LAMP-IS6110), Xpert MTB/RIF (Cepheid) and mycobacteria growth indicator tube (BACTEC MGIT 960 culture) against a modified version of international consensus diagnostic definition (i.e. composite reference standard (CRS)). METHODS: A cross-sectional analytical study was conducted in a tertiary care hospital in North India from July 2016 to December 2017 involving 100 children <14 years with suspected PTB. Respiratory specimens (sputum, gastric lavage and/or bronchoalveolar lavage) were collected and subjected to LAMP-IS6110, Xpert MTB/RIF and BACTEC MGIT 960 culture assay. RESULTS: Fifty-five children had confirmed and probable TB according to the CRS (prevalence = 58.5%). The sensitivity of BACTEC MGIT 960 culture, Xpert MTB/RIF and LAMP-IS6110 assay was 14%, 9.1% and 10.91%, respectively, when compared against the predefined CRS. The specificity for all these tests was 100%. When compared with BACTEC MGIT 960 culture as the gold standard, the LAMP-IS6110 assay and Xpert MTB/RIF assay had the sensitivity of 85.71% (95% CI: 42.13-99.64%) and 71.43% (95% CI: 29.04-96.33%), respectively. The specificity of both assays was 100%. CONCLUSIONS: We noted that LAMP-IS6110 performed better than Xpert MTB/RIF (Cepheid) in terms of sensitivity when compared against BACTEC MGIT 960 culture as reference standard, though specificity of both the tests was comparable. The diagnostic performance of BACTEC MGIT 960 culture was better than LAMP-IS6110 and Xpert MTB/RIF in paediatric PTB, when compared against CRS.

Differential diagnosis of disseminated Mycobacterium avium and Mycobacterium tuberculosis infection in HIV patients using duplex PCR.

Background: Disseminated Mycobacterium avium complex (MAC) and Mycobacterium tuberculosis infections have almost similar clinical presentations but require different therapeutic management. Materials & methods: A duplex PCR was designed based on the sequence variation between the genes encoding catalase-peroxidase (KatG) of M. avium complex and M. tuberculosis, so as to discriminate MAC, M. tuberculosis and mixed mycobacterial (MAC + M. tuberculosis) infections in HIV patients. Results: An accurate, single-step differential diagnosis of disseminated mycobacterial infections in HIV patients was achieved with specific detection of a single band each for M. avium (120 bp) and M. tuberculosis (90 bp) and two bands for the mixed (120 and 90 bp) infections. Conclusion:katG gene-based duplex PCR can facilitate quick differential diagnosis of disseminated MAC and M. tuberculosis infections in HIV patients.

Proteomic profile of vitreous in patients with tubercular uveitis.

OBJECTIVE: To elucidate disease-specific host protein profile in vitreous fluid of patients with intraocular inflammation due to tubercular uveitis (TBU). METHODS: Vitreous samples from 13 patients with TBU (group A), 7 with non-TBU (group B) and 9 with no uveitis (group C) were analysed by shotgun proteomics using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were subjected to pathway analysis using WEB-based Gene SeT Analysis Toolkit software. RESULTS: Compared to control groups (B + C combined), group A (TBU) displayed 32 (11 upregulated, 21 downregulated) DEPs, which revealed an upregulation of coagulation cascades, complement and classic pathways, and downregulation of metabolism of carbohydrates, gluconeogenesis, glucose metabolism and glycolysis/gluconeogenesis pathways. When compared to group B (non-TBU) alone, TBU displayed 58 DEPs (21 upregulated, 37 downregulated), with an upregulation of apoptosis, KRAS signaling, diabetes pathways, classic pathways, and downregulation of MTORC1 signaling, glycolysis/gluconeogenesis, and glucose metabolism. CONCLUSION: This differential protein profile provides novel insights into the molecular mechanisms of TBU and a baseline to explore vitreous biomarkers to differentiate TBU from non-TBU, warranting future studies to identify and validate them as a diagnostic tool in TBU. The enriched pathways generate interesting hypotheses and drive further research.