Influence of Active Tuberculosis on Chemokine and Chemokine Receptor Expression in HIV-Infected Persons

Tuberculosis (TB) is the major opportunistic infection of HIV-1-infected patients in developing countries. Concurrent infection with TB results in immune cells having enhanced susceptibility to HIV-1 infection, which facilitates entry and replication of the virus. Cumulative data from earlier studies indicate that TB provides a milieu of continuous cellular activation and irregularities in cytokine and chemokine circuits that favor viral replication and disease progression. To better understand the interaction of the host with HIV-1 during active tuberculosis, we investigated in vivo expression of the HIV-1 coreceptors, CCR5 and CXCR4, and circulating levels of the inhibitory �-chemokines, macrophage inflammatory protein-1-� (MIP-1�), macrophage inflammatory protein-1-� (MIP-1�), and regulated upon activation T cell expressed and secreted (RANTES), in HIV-positive individuals with and without active pulmonary tuberculosis. We found a significant decrease from normal in the fraction of CD4� T cells expressing CCR5 and CXCR4 in individuals infected with HIV. However, CCR5 and CXCR4 expression did not differ significantly between HIV patients with and without tuberculosis. Higher amounts of MIP-1�, MIP-1�, and RANTES were detected in plasma of HIV-1-positive individuals, particularly those with dual infection, although the increase was not found to be statistically significant

Emergence of new genotypes and lineages of dengue viruses during the 2012–15 epidemics in southern India

Objectives: To genotypically characterize dengue virus (DENV) isolates among dengue-infected children from 2012–13/2014–15 outbreaks in southern India. Methods: Children hospitalized with suspected dengue were tested for dengue RT-PCR targeting Capsid-preMembrane (C-prM) and Envelope (Env) regions. Following virologic confirmation (n = 612), a representative selection of DENV isolates (n = 99) were sequenced for C-prM, aligned using ClustalW and subjected to phylogenetic analysis by maximum-likelihood method in MEGA6. Results: In 2012–13 (n = 113), DENV-3 (44, 38.9%) and DENV-2 (43, 38.1%) predominated; DENV-1 (22, 19.5%) and DENV-4 (1, 0.9%) were less common. The pattern changed in 2014–15 (n = 499), when DENV-1 (329, 65.7%) predominated, followed by DENV-2 (97, 21.2%), DENV-3 (36, 6.7%) and DENV-4 (10, 2.0%). Multiple-serotype co-infections occurred in 2.7% and 5.4% in 2012–13 and 2014–15, respectively. Genotype III (GIII) of DENV-1 predominated (85.7%) in 2012–13, ceding to GI predominance (80.8%) in 2014–15. Among DENV-2, 71.9% (23/32) showed distinct clustering suggesting a new lineage, 'GIVc'. All tested DENV-4 were GIC, whose clustering pattern showed the emergence of two distinct clades. Conclusions: New genotypic/lineage variations in DENV-1 and DENV-2 may have influenced the magnitude and severity of dengue epidemics in southern India during this period. These findings emphasize the role of active surveillance of DENV serotypes/genotypes in aiding outbreak control and vaccine studies. Keywords: Dengue virus, Serotyping, Sequencing, Phylogenetics, Genotypes, Lineage

Molecular surveillance of Dengue Virus (DENV) and its co-infection with Chikungunya Virus (CHIKV) among febrile patients: A comparative study from South Delhi, India

Dengue and Chikungunya are two major arboviral infections transmitted worldwide by the mosquitoes, Aedes aegypti and Ae. albopictus. India suffers enormously with both Dengue and Chikungunya as they pose a great public health challenge. The present study aims to evaluate the prevalence of Dengue Virus (DENV), Chikungunya Virus (CHIKV) and DENV/CHIKV co-infection (by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)/Enzyme Linked Immunosorbent Assay (ELISA), their clinical features, DENV serotypes and CHIKV specific Immunoglobulin G (IgG) within a 7 years gap in the Delhi population. The study sample included clinically suspected febrile patients (?7 days) sera collected during 2017-2018 (n=87) and during 2008-2010 (n=623) from Delhi. Captured ELISA was performed for CHIKV IgG screening and nested PCR was done for DENV serotyping. The percentage prevalence for DENV was significantly higher than CHIKV with 41.38% (n=87) and 16.1% (n=87), respectively; interestingly, DENV/CHIKV co-infection was detected in 10.34% (n=9/87) cases during 2017-2018. Similarly, a high DENV prevalence was observed during 2008-2010 with the prevalence rate of 38.3% (69/180),  34.65% (35/101) and 47.07% (161/342), respectively. DENV 1 and DENV 3 were dominant serotype during 2008-2010 and 2017-2018 respectively. We have noticed a high prevalence (36.67%, 22/60) of the CHIKV IgG antibody in the 2017-2018 samples. Joint pain was more preferential to CHIKV mono-infection and DENV/CHIKV co-infection compared to DENV mono-infection. The present study highlights the need for active surveillance simultaneously for both DENV and CHIKV and to evaluate the role of CHIKV/DENV co-infections in disease severity in the endemic regions.

Mycobacterial antigen driven activation of CD14++ CD16-monocytes is a predictor of tuberculosis-associated immune reconstitution inflammatory syndrome

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14++CD16− monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment

Dynamics of T-Lymphocyte Activation Related to Paradoxical Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome in Persons With Advanced HIV

Most persons living with HIV (PLWH) experience a significant restoration of their immunity associated with successful inhibition of viral replication after antiretroviral therapy (ART) initiation. Nevertheless, with the robust quantitative and qualitative restoration of CD4(+) T-lymphocytes, a fraction of patients co-infected with tuberculosis develop immune reconstitution inflammatory syndrome (TB-IRIS), a dysregulated inflammatory response that can be associated with significant tissue damage. Several studies underscored the role of adaptive immune cells in IRIS pathogenesis, but to what degree T lymphocyte activation contributes to TB-IRIS development remains largely elusive. Here, we sought to dissect the phenotypic landscape of T lymphocyte activation in PLWH coinfected with TB inititating ART, focusing on characterization of the profiles linked to development of TB-IRIS. We confirmed previous observations demonstrating that TB-IRIS individuals display pronounced CD4(+) lymphopenia prior to ART initiation. Additionally, we found an ART-induced increase in T lymphocyte activation, proliferation and cytotoxicity among TB-IRIS patients. Importantly, we demonstrate that TB-IRIS subjects display higher frequencies of cytotoxic CD8(+) T lymphocytes which is not affected by ART. Moreover, These patients exhibit higher levels of activated (HLA-DR(+)) and profilerative (Ki-67(+)) CD4(+) T cells after ART commencenment than their Non-IRIS counterparts. Our network analysis reveal significant negative correlations between Total CD4(+) T cells counts and the frequencies of Cytotoxic CD8(+) T cells in our study population which could suggest the existance of compensatory mechanisms for Mtb-infected cells elimination in the face of severe CD4(+) T cell lymphopenia. We also investigated the correlation between T lymphocyte activation profiles and the abundance of several inflammatory molecules in plasma. We applied unsupervised machine learning techniques to predict and diagnose TB-IRIS before and during ART. Our analyses suggest that CD4(+) T cell activation markers are good TB-IRIS predictors, whereas the combination of CD4(+) and CD8(+) T cells markers are better at diagnosing TB-IRIS patients during IRIS events Overall, our findings contribute to a more refined understanding of immunological mechanisms in TB-IRIS pathogenesis that may assist in new diagnostic tools and more targeted patient management

Optimization of Flow-Cytometry Based Assay for Measuring Neutralizing Antibody Responses against Each of the Four Dengue Virus Serotypes

Dengue is an important public health problem worldwide, with India contributing nearly a third of global dengue disease burden. The measurement of neutralizing antibody responses is critical for understanding dengue pathophysiology, vaccine development and evaluation. Historically, dengue virus neutralization titers were measured using plaque reduction neutralization tests (PRNTs), which were later adapted to focus reduction neutralization tests (FRNTs). Given the slow and laborious nature of both these assays, there has been interest in adapting a high-throughput flow cytometry based neutralization assay. However, flow cytometry based assays typically underestimate neutralization titers, and in situations where the titers are low they can even fail to detect neutralization activity. In this study, by evaluating graded numbers of input Vero cell numbers and viral inoculum, we optimized the flow cytometry based neutralization assay in such a way that it is sensitive and scores titers that are in concordance with focus reduction neutralization tests for each of the four dengue virus serotypes (p < 0.0001). Given that dengue is a global public health concern, and several research groups are making efforts to understand its pathophysiology and accelerate vaccine development and evaluation both in India and worldwide, our findings have timely significance for facilitating these efforts

Incomplete immunological recovery following anti-tuberculosis treatment in HIV-infected individuals with active tuberculosis

Background & objectives: Mycobacterium tuberculosis infection has been shown to result in increased HIV replication and disease progression in HIV-infected individuals through increased immune activation. The objective of this study was to correlate plasma levels of immune activation markers with the presence of tuberculosis (TB) in HIV-infected and uninfected individuals, and to study the changes following anti-tuberculosis treatment. Methods: Plasma markers of immune activation - neopterin, beta-2-microglobulin (β2M) and soluble tumour necrosis factor alpha receptor type I (sTNFα-RI) were measured by ELISA in 42 HIV positive TB patients (HIV+TB+) undergoing a six-month course of TB chemotherapy. Thirty seven HIV+ persons without active TB, 38 TB patients without HIV infection, and 62 healthy volunteers served as controls. Results: Plasma levels of all three markers were elevated in HIV+ individuals, more so in those with active TB. When HIV+ individuals were further categorized based on CD4+ T cell counts, HIV+TB+ patients with CD4+ T cells counts < 200 cells/μl were found to have the highest levels at baseline with a steep fall in neopterin and sTNFα-RI during treatment, but in most instances the levels did not drop to normal. β2M levels remained persistently high despite completing TB treatment. Interpretation & conclusions: The fi ndings of the study suggest that both HIV and TB act synergistically to activate the host immune system. Although ATT was effective in clearing M. tuberculosis infection, a high proportion of HIV+ TB patients continued to have levels well above the normal range, indicating that underlying immune activation persists despite TB treatment. None of the markers were specifi c enough to be used to assess cure of TB