Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease-A pilot study.

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9-96.8]; Specificity = 90% [95% CI: 55.5-99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.

In vitro combination with doxycycline plus antifungals against clinical Mucorales pathogens.

PURPOSE: Since systematic antifungals for mucormycosis showed variable MICs depending on strains, effective and safe antifungal therapy was still needed. This study is aimed to evaluate the in vitro activity of doxycycline combined with antifungal therapy against dominant Mucorales pathogens. METHODS: Multidrug susceptibility testing was performed with doxycycline and antifungals, including itraconazole, posaconazole, and amphotericin, in 21 isolates of 8 dominant Mucorales pathogens. RESULTS: The fractional inhibitory concentration index according to M38 showed one Rhizopus arrhizus isolate synergic (∑FICI = 0.375) and other isolates in addition (0.5 < ∑FICI < 4). CONCLUSIONS: Doxycycline was found to have in vitro advantages in combined antifungal treatment over antifungals alone.

Convalescent Adaptive Immunity Is Highly Heterogenous after SARS-CoV-2 Infection.

The optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, we recruited 30 unvaccinated convalescent donors who had previously been infected with COVID-19 and 7 unexposed asymptomatic controls. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG via semiquantitative ELISA, and T-cell immunity against S1 and S2 subunits were studied via IFN-γ enzyme-linked immunosorbent spot (ELISpot) and flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2-positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and the determination of optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Convalescent Adaptive Immunity is Highly Heterogenous after SARS-CoV-2 Infection.

Optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, between April 23, 2020, to May 11, 2020, we recruited 30 COVID-19 unvaccinated convalescent donors and 7 unexposed asymptomatic donors. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG semiquantitative ELISA and T cell immunity against S1 and S2 subunits were studied by IFN-γ Enzyme-Linked Immune absorbent Spot (ELISpot), flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2 positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARSCoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Immune defence to invasive fungal infections: A comprehensive review.

The fungal infections are relatively common in humans that can range from common, mild superficial infections to life-threatening invasive infections. Most of the pathogenic fungi are opportunistic that cause disease under immunocompromised conditions such as HIV infection, cancer, chemotherapy, transplantation and immune suppressive drug users. Efficient detection and treatment of high-risk population remain the highest priority to avert most of the deaths. Majority of invasive infections are caused by Candida, Aspergillus and Cryptococcus species. Lack of effective vaccines, standardised diagnostic tools, efficient antifungal drugs and the emergence of drug-resistant species/strains pose a global threat to control Invasive fungal infections (IFI). A better understanding of the host immune response is one of the major approaches to developing new or improved antifungal strategies to control the IFIs. In this review, we have discussed pathogenesis of medically important fungi, fungal interaction with the host through pattern recognition receptors (PRRs) and the interplay of innate and adaptive immune cells in shaping host immunity to IFI. Further, we emphasized the role of memory cells by offering long-term protection in secondary or subsequent infections. Moreover, we depicted the role of unconventional innate-like immune cells in anti-fungal immunity. We also summarize the available information on the current vaccine strategies, genetic susceptibility to fungal infections, recent co-infections studies and the emergence of drug-resistance, a growing trend throughout the world. Finally, we emphasized the steps to be taken for the control of IFIs.

Genotyping of Mycobacterium leprae for understanding the distribution and transmission of leprosy in endemic provinces of China.

OBJECTIVES: Understanding the nature of Mycobacterium leprae transmission is vital to implement better control strategies for leprosy elimination. The present study expands the knowledge of county-level strain diversity, distribution, and transmission patterns of leprosy in endemic provinces of China. METHODS: We genetically characterized 290 clinical isolates of M. leprae from four endemic provinces using variable number tandem repeats (VNTR) and single nucleotide polymorphisms (SNPs). Attained genetic profiles and cluster consequences were contrasted with geographical and migration features of leprosy at county levels. RESULTS: Considering the allelic variability of 17 VNTR loci by the discriminatory index, (GTA)9, (AT)17, (AT)15, (TA)18, (TTC)21, and (TA)10 are reported to be more highly polymorphic than other loci. The VNTR profile generated the low-density clustering pattern in the counties of Sichuan and Yunnan, whereas clusters have been observed from the isolates from Huayuan (N = 6), Yongding (N = 3), Zixing (N = 3), Chenxi (N = 2) and Zhongfang (N = 2) counties of Hunan, and Zhijin (N = 3), Anlong (N = 2), Zhenning (N = 2), and Xixiu (N = 2) counties of Guizhou. In some clusters, people's social relations have been observed between villages. From the 290 clinical isolates, the most predominantly reported SNP was 3K (278, 95.8%), followed by SNP 1D (10, 3.4%), which are typically observed to be predominant in China. We also detected the novel SNP 3J (2, 0.8%), which has not yet been reported in China. CONCLUSION: The clustering pattern of M. leprae indicates the transmission of leprosy still persists at county levels, suggesting that there is a need to implement better approaches for tracing the close contacts of leprosy patients.

Identification of Human Toll-like Receptor 2-Agonistic Activity in Dihydropyridine-Quinolone Carboxamides.

Using a multiplexed, reporter gene-based, high-throughput screen, we identified 9-fluoro-7-hydroxy-3-methyl-5-oxo-N-(pyridin-3-ylmethyl)-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinoline-6-carboxamide as a TLR2 agonist. Preliminary structure-activity relationship studies on the carboxamide moiety led to the identification of analogues that induce chemokines and cytokines in a TLR2-dependent manner. These results represent new leads for the development of vaccine adjuvants.

Rv2204c, Rv0753c and Rv0009 antigens specific T cell responses in latent and active TB - a flow cytometry-based analysis.

High global prevalence of latent TB infection (LTBI) is a key challenge in distinguishing patients with active pulmonary TB (PTB) from those with LTBI. The functional profile of CD4+ and CD8+ T cell cytokines produced as a response to Mycobacterium tuberculosis antigens vary during the course of tuberculosis (TB) infection. We evaluated antigen-specific CD4+ and CD8+ T cell cytokine response after overnight in vitro stimulation of peripheral blood with mycobacterial antigens ESAT-6, CFP-10, Rv2204c, Rv0753c and Rv0009 by flow cytometry. A significantly higher frequency of antigen-specific CD4+ or CD8+ IFN-γ+ T cells were found in LTBI than in PTB. Among all the antigens used, Rv2204c-specific CD8+ IFN-γ+ displayed the positivity of 72% and 24% in LTBI and PTB respectively. In contrast to IFN-γ, the frequencies of CD4+ or CD8+ secreting TNF-α+ cells were significantly high in PTB compared to LTBI. CD8+TNF-α+ analysis showed 60% positivity in PTB and 13.6% positivity in LTBI against Rv0753c antigen stimulation. We also predicted Rv2204c specific CD8+ T cells secreting IL-10 or IL-4 showed maximum differentiation between LTBI and PTB. In conclusion, altered expression of Rv2204c-specific CD4+IFN-γ+ and CD8+IL-4+ T cells in LTBI and PTB might be a useful biomarker to differentially diagnose LTBI and active TB.

Altered expression of antigen-specific memory and regulatory T-cell subsets differentiate latent and active tuberculosis.

Although one-third of the world population is infected with Mycobacterium tuberculosis, only 5-10% of the infected individuals will develop active tuberculosis (TB) disease and the rest will remain infected with no symptoms, known as latent TB infection (LTBI). Identifying biomarkers that differentiate latent and active TB disease enables effective TB control, as early detection, treatment of active TB and preventive treatment of individuals with LTBI are crucial steps involved in TB control. Here, we have evaluated the frequency of antigen-specific memory and regulatory T (Treg) cells in 15 healthy household contacts (HHC) and 15 pulmonary TB patients (PTB) to identify biomarkers for differential diagnosis of LTBI and active TB. Among all the antigens tested in the present study, early secretory antigenic target-6 (ESAT-6) -specific CD4+ and CD8+ central memory (Tcm) cells showed 93% positivity in HHC and 20% positivity in PTB. The novel test antigens Rv0753c and Rv0009 both displayed 80% and 20% positivity in HHC and PTB, respectively. In contrast to Tcm cells, effector memory T (Tem) cells showed a higher response in PTB than HHC; both ESAT-6 and Rv0009 showed similar positivity of 80% in PTB and 33% in HHC. PTB patients have a higher proportion of circulating antigen-reactive Treg cells (CD4+  CD25+  FoxP3+ ) than LTBI. Rv2204c-specific Treg cells showed maximum positivity of 73% in PTB and 20% in HHC. Collectively, our data conclude that ESAT-6-specific Tcm cells and Rv2204c-specific Treg cells might be useful biomarkers to discriminate LTBI from active TB.

Dynamic IgG antibody response to immunodominant antigens of M. tuberculosis for active TB diagnosis in high endemic settings.

BACKGROUND: Even though various techniques have been developed for rapid diagnosis of tuberculosis (TB), still there is an immense need for a simple, cost effective, highly sensitive and specific test. Hence, one of the possibilities is identification of Mycobacterium tuberculosis specific antibodies in infected serum by using specific antigens. METHODS: We tested 10 recombinant M. tuberculosis antigens to evaluate IgG levels among Healthy control subjects (HCS), Healthy household contacts (HHC) and pulmonary TB patients (PTB) by ELISA. RESULTS: The median IgG levels specific to all the antigens are higher in PTB than HHC and HCS. Amongst single antigens, 38-kDa antigen has showed maximum sensitivity of 50% than any other antigens at 95.5% specificity. Among the two antigen combination, 38-kDa+Rv1860 has showed maximum sensitivity of 66.6% with specificity of 92.2%. The same antigen combination (38-kDa and Rv1860) predominantly identifies smear negative and culture positive TB patients with 68% sensitivity and 92.2% specificity. Most of the antigens have exhibited higher antibody titre in cavitary TB than non cavitary. With regard to latent TB infection (LTBI) identification, Rv1860 has exhibited maximum sensitivity of 53.3% with 95% specificity. CONCLUSIONS: IgG response to combination of recombinant mycobacterial antigens (38-kDa, Rv1860, Rv2204c and Rv0753c) presents good specificity with acceptable level of sensitivity for TB diagnosis.

PpiA antigen specific immune response is a potential biomarker for latent tuberculosis infection.

One third of the world's population is estimated to harbour latent tuberculosis infection (LTBI). Around 10% of them have the life time risk of developing active tuberculosis (PTB). Currently there is no gold standard test for identifying LTBI. Therefore identification of specific markers for LTBI will help as to develop a test specific for LTBI. Earlier, in our immunoproteomic analysis, we found that peptidyl-prolyl cis-trans isomerase A (PpiA) protein-containing fractions induced significantly higher interferon-gamma (IFN-γ) response in LTBI than in PTB. Immunological characterisation of recombinant PpiA protein was carried out in the current study. We have studied 10 cytokines and 2 chemokine responses against PpiA and standard antigens such as early secretory antigenic target-6 (ESAT-6) and culture filtrate antigen-10 (CFP-10). In healthy household contacts (HHC), all the tested antigens induced significantly higher levels of IFN-γ and Interlukin-8 (IL-8) compared with those in PTB. PpiA-specific IL-12p40 response was significantly increased in HHC compared with that in PTB. PpiA antigen-specific IFN-γ and IL-12p40 both showed 86% positivity in HHC, whereas in PTB, they showed 20% and 38% positivity, respectively. In terms of IFN-γ/TNF-α ratio, PpiA displayed 86% (30/35) positivity in HHC and 18% (7/39) positivity in PTB. In summary we found that PpiA-specific IFN-γ and IFN-γ/TNF-α ratio response were specific biomarkers for LTBI identification.

Evaluation of cytokine and chemokine response elicited by Rv2204c and Rv0753c to detect latent tuberculosis infection.

Latent TB infection (LTBI) is one of the major contributing factors for the high incidence of TB in India that in turn significantly contributes to the pool of active TB. Hence, identification and treatment of LTBI is of utmost importance. Currently, no specific diagnostic test is available for LTBI. Earlier, in our immunoproteomic analysis, we identified Rv2204c and Rv0753c protein-containing fractions induced significantly higher interferon-gamma (IFN-γ) response in LTBI than in active TB. In this study, we evaluated cytokine and chemokine response against M. tuberculosis antigens for improving LTBI identification. Two M. tb proteins Rv2204c and Rv0753c were cloned, over expressed in E. coli and purified by affinity chromatography. Antigen-specific immune response was evaluated in 39 pulmonary TB patients (PTB) and 35 healthy house-hold contacts (HHC). After whole blood culture for 6 days, the secretion of cytokines and chemokines were quantified in culture supernatants using Enzyme Linked Immune Sorbent Assay (ELISA). Antigen specific cytokines such as interferon gamma (IFN-γ), interleukin-6 (IL-6), IL-8, IL-12p40 and chemokines like monocyte chemotactic proteins MCP-1, MCP-2 were significantly higher in HHC than PTB. In contrast to other cytokines, tumor necrosis factor-alpha (TNF)-α response was significantly increased in PTB compared with HHC. Both Rv2204c and Rv0753c antigen specific IFN-γ response showed 86% positivity in HHC; whereas in PTB, these antigens showed 18% and 21% positivity respectively. Rv2204c antigen-specific IFN-γ/TNF-α response displayed maximum positivity of 91% in HHC and minimum positivity of 10% (4/39) in PTB. Rv2204c and Rv0753c specific IFN-γ and IFN-γ/TNF-α responses showed the most promising accuracy in identifying LTBI.

IFN-γ/TNF-α ratio in response to immuno proteomically identified human T-cell antigens of Mycobacterium tuberculosis - The most suitable surrogate biomarker for latent TB infection.

The enormous reservoir of latent TB infection (LTBI) poses a major hurdle for global TB control. The existing Tuberculin skin test (TST) and IFN-γ release assays (IGRAs) are found to be suboptimal for LTBI diagnosis. Previously we had taken an immunoproteomic approach and identified 10 protein fractions (contains 16 proteins), which are solely recognized by LTBI. In a cohort of 40 pulmonary TB patients (PTB) and 35 healthy household contacts (HHC), IFN-γ and TNF-α response were measured against 16 antigens by using 1:10 diluted whole blood assay. Among all the antigens, IFN-γ response to Rv2626c has shown positivity of 88.57% in HHC and 7.5% in PTB group. IFN-γ response to combination of Rv2626c + Rv3716c has demonstrated 100% positivity in HHC and 17.5% positivity in PTB respectively. Compared to individual cytokines (i.e. IFN-γ and TNF-α), ratio of IFN-γ/TNF-α has shown promising results for diagnosis of LTBI. IFN-γ/TNF-α ratio against Rv3716c and TrxC has exhibited a positivity of 94.29% in HHC and 5% in PTB group. Accession of Rv2626c and Rv3716c may improve the diagnostic performance of existing QFT-GIT. Independent of QFT-GIT assay, ratio of IFN-γ/TNF-α in response to either Rv3716c or TrxC may acts as suitable surrogate biomarker for LTBI.