Sasheela Ponnampalavanar

Cytokine expression profile of dengue patients at different phases of illness.

Background Dengue is an important medical problem, with symptoms ranging from mild dengue fever to severe forms of the disease, where vascular leakage leads to hypovolemic shock. Cytokines have been implicated to play a role in the progression of severe dengue disease; however, their profile in dengue patients and the synergy that leads to continued plasma leakage is not clearly understood. Herein, we investigated the cytokine kinetics and profiles of dengue patients at different phases of illness to further understand the role of cytokines in dengue disease. Methods and Findings Circulating levels of 29 different types of cytokines were assessed by bead-based ELISA method in dengue patients at the 3 different phases of illness. The association between significant changes in the levels of cytokines and clinical parameters were analyzed. At the febrile phase, IP-10 was significant in dengue patients with and without warning signs. However, MIP-1β was found to be significant in only patients with warning signs at this phase. IP-10 was also significant in both with and without warning signs patients during defervescence. At this phase, MIP-1β and G-CSF were significant in patients without warning signs, whereas MCP-1 was noted to be elevated significantly in patients with warning signs. Significant correlations between the levels of VEGF, RANTES, IL-7, IL-12, PDGF and IL-5 with platelets; VEGF with lymphocytes and neutrophils; G-CSF and IP-10 with atypical lymphocytes and various other cytokines with the liver enzymes were observed in this study. Conclusions The cytokine profile patterns discovered between the different phases of illness indicate an essential role in dengue pathogenesis and with further studies may serve as predictive markers for progression to dengue with warning signs.

Susceptible and protective HLA Class 1 alleles against dengue fever and dengue hemorrhagic fever patients in a Malaysian population.

Background The human leukocyte antigen alleles have been implicated as probable genetic markers in predicting the susceptibility and/or protection to severe manifestations of dengue virus (DENV) infection. In this present study, we aimed to investigate for the first time, the genotype variants of HLA Class 1(-A and -B) of DENV infected patients against healthy individuals in Malaysia. Methodology/Principal Findings This study was carried out with 92 dengue disease patients and 95 healthy controls from three different ethnic groups (Malay, Chinese and Indian) in Malaysia. All patients with clinical and laboratory confirmation of DENV infection were typed for the HLA-A and B loci, using polymerase chain reaction-sequence specific primer techniques. In our total population, a significant increase for HLA-B*53 (P = 0.042, Pc = 1.008) allele and a significant decrease for A*03 (P = 0.015, Pc = 0.18, OR = 5.23, 95% CI = 1.19–23.02) and B*18 (P = 0.017, Pc = 0.408) alleles were noted in DHF patients as compared to healthy donors. We also observed that in the Malay DHF patients, allele B*13 (P = 0.049, Pc = 1.176, OR = 0.18, 95% CI = 0.03–0.90) was present at a significantly higher frequency in this population while allele HLA-B*18 (P = 0.024, Pc = 0.576) was seen to be negatively associated with DHF. Conclusions/Significance These are the first findings on genetic polymorphisms in our population and we conclude that: (1) In our total population, HLA-B*53 probably involve in disease susceptibility, while the HLA-A*03 and HLA-B*18 may confer protection from progression to severe disease; (2) In the Malay population, HLA-B*13 and B*18 are probably associated in disease susceptibility and protection, respectively. These results could furnish as a valuable predictive tool to identify ethnically different individuals at risk and/or protection from severe forms of DENV infection and would provide valuable informations for the design of future dengue vaccine.

Attrition of TCR Vα7.2+ CD161++ MAIT cells in HIV- tuberculosis co-infection is associated with elevated levels of PD-1 expression.

Mucosal-associated invariant T (MAIT) cells are evolutionarily conserved antimicrobial MR1-restricted CD8+ T cells co-expressing the semi-invariant TCR Vα7.2, and are numerous in the blood and mucosal tissues of humans. MAIT cells appear to undergo exhaustion in chronic viral infections. However, their role in human immunodeficiency virus type 1 (HIV-1) mono-infection and HIV/tuberculosis (TB) co-infection have seldom been elaborately investigated. We conducted a cross-sectional study to investigate the frequencies and phenotypes of CD161++CD8+ T cells among anti-retroviral therapy (ART)/anti-TB therapy (ATT) treatment-naïve HIV/TB co-infected, ART/TB treated HIV/TB co-infected, ART naïve HIV-infected, ART-treated HIV-infected patients, and HIV negative healthy controls (HCs) by flow cytometry. Our data revealed that the frequency of MAIT cells was severely depleted in HIV mono- and HIV/TB co-infections. Further, PD-1 expression on MAIT cells was significantly increased in HIV mono- and HIV-TB co-infected patients. The frequency of MAIT cells did not show any significant increase despite the initiation of ART and/or ATT. Majority of the MAIT cells in HCs showed a significant increase in CCR6 expression as compared to HIV/TB co-infections. No marked difference was seen with expressions of chemokine co-receptor CCR5 and CD103 among the study groups. Decrease of CCR6 expression appears to explain why HIV-infected patients display weakened mucosal immune responses.

TLR2-induced cytokine responses may characterize HIV-infected patients experiencing mycobacterial immune restoration disease.

Objectives:Most HIV patients who experience Mycobacterium tuberculosis-associated immune restoration disease (TB IRD) display elevated interferon-gamma (IFN&ggr;) responses against mycobacterial antigens, but these can occur without an IRD. Recognition of mycobacteria-associated molecular patterns through toll-like receptors (TLRs) on dendritic cells and monocytes induces cytokine production. Here, we investigate TLR-induced responses in IRD. Design:Peripheral blood mononuclear cells (PBMCs) were collected at approximately weeks 0, 6, 12, 24 and 48 after antiretroviral therapy from five patients experiencing TB IRD, nine matched non-IRD patients and 15 healthy controls. Methods:IFN&ggr; production by PBMC stimulated with protein purified derivative (PPD) was assessed by ELISpot. TLR2 expression on myeloid dendritic cells (mDCs) and monocytes was assessed by flow cytometry. TNF&agr;, IL-12p40 and IL-10 were measured by ELISA in 24-h cultures of PBMC with lipomannan (mycobacteria-derived TLR2 agonist). Results:TLR2 expression on mDC and monocytes was higher in patients than controls at baseline (P < 0.005). TLR2 expression decreased to normal levels on mDC by week 12, but remained higher on monocytes at week 24 (P = 0.02). At week 24, IRD patients showed higher IFN&ggr; responses to PPD (P = 0.02), TLR2 expression on monocytes (P = 0.006) and lipomannan-induced TNF&agr; production (P = 0.016) than non-IRD patients. Lipomannan-induced TNF&agr; and IL-12p40 responses paralleled TB IRD in the patients with high TLR2 expression. IL-10 levels did not associate with IRD. Conclusion:TLR2-induced pro-inflammatory cytokines by dendritic cells or monocytes may contribute to the pathogenesis of mycobacterial IRD.

Cancers in the TREAT Asia HIV Observational Database (TAHOD): a retrospective analysis of risk factors

BackgroundThis retrospective survey describes types of cancers diagnosed in HIV-infected subjects in Asia, and assesses risk factors for cancer in HIV-infected subjects using contemporaneous HIV-infected controls without cancer.MethodsTREAT Asia HIV Observational Database (TAHOD) sites retrospectively reviewed clinic medical records to determine cancer diagnoses since 2000. For each diagnosis, the following data were recorded: date, type, stage, method of diagnosis, demographic data, medical history, and HIV-related information. For risk factor analyses, two HIV-infected control subjects without cancer diagnoses were also selected. Cancers were grouped as AIDS-defining cancers (ADCs), and non-ADCs. Non-ADCs were further categorized as being infection related (NADC-IR) and unrelated (NADC-IUR).ResultsA total of 617 patients were included in this study: 215 cancer cases and 402 controls from 13 sites. The majority of cancer cases were male (71%). The mean age (SD) for cases was 39 (10.6), 46 (11.5) and 44 (13.7) for ADCs, NADC-IURs and NADCs-IR, respectively. The majority (66%) of cancers were ADCs (16% Kaposi sarcoma, 40% non-Hodgkins lymphoma, and 9% cervical cancer). The most common NADCs were lung (6%), breast (5%) and hepatocellular carcinoma and Hodgkins lymphoma (2% each). There were also three (1.4%) cases of leiomyosarcoma reported in this study. In multivariate analyses, individuals with CD4 counts above 200 cells/mm3 were approximately 80% less likely to be diagnosed with an ADC (p < 0.001). Older age (OR: 1.39, p = 0.001) and currently not receiving antiretroviral treatment (OR: 0.29, p = 0.006) were independent predictors of NADCs overall, and similarly for NADCs-IUR. Lower CD4 cell count and higher CDC stage (p = 0.041) were the only independent predictors of NADCs-IR.ConclusionsThe spectrum of cancer diagnoses in the Asia region currently does not appear dissimilar to that observed in non-Asian HIV populations. One interesting finding was the cases of leiomyosarcoma, a smooth-muscle tumour, usually seen in children and young adults with AIDS, yet overall quite rare. Further detailed studies are required to better describe the range of cancers in this region, and to help guide the development of screening programmes.

Plasma interleukin-18 levels are a biomarker of innate immune responses that predict and characterize tuberculosis-associated immune reconstitution inflammatory syndrome.

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is a substantial problem in HIV/TB coinfected patients commencing antiretroviral therapy (ART). The immunopathogenesis of TB-IRIS includes increased production of proinflammatory chemokines and cytokines, including interleukin-18, which is a signature cytokine of the nucleotide-binding domain and leucine-rich repeat pyrin containing protein-3 inflammasome. We compared plasma levels of interleukin-18 and other biomarkers of monocyte/macrophage activation in the prediction and characterization of TB-IRIS. Methods: Biomarkers were assayed pre-ART and during TB-IRIS, or equivalent time-point, in a case–control study of Malaysian HIV patients with paradoxical or unmasking TB-IRIS (n = 15), TB no IRIS (n = 14), and no TB or IRIS (n = 15). Findings for interleukin-18 were verified in another cohort of patients with paradoxical TB-IRIS (n = 26) and their controls (n = 22) from India. Results: Interleukin-18 was higher in TB-IRIS patients pre-ART and during the event in both Malaysian patients (P < 0.0001) and Indian patients (P < 0.01). CXCL10 was higher pre-ART (P < 0.001), mainly in paradoxical TB-IRIS patients, and during TB-IRIS (P < 0.001), whereas CXCL8 was only higher during TB-IRIS (P < 0.001). Soluble(s) CD14 was increased in all patients with HIV/TB coinfection pre-ART and during TB-IRIS or equivalent time-point, compared with patients without TB. In contrast, interferon-&ggr; was lower before and during TB-IRIS. By receiver operating curve analysis, CXCL10, and/or interleukin-18 pre-ART were predictive of TB-IRIS. Conclusion: Plasma interleukin-18 levels pre-ART are candidate biomarkers for predicting paradoxical and unmasking TB-IRIS and should be investigated for risk stratification and elucidation of disease pathogenesis.

Molecular diversity of HIV-1 and surveillance of transmitted drug resistance variants among treatment Naïve patients, 5 years after active introduction of HAART in Kuala Lumpur, Malaysia.

Expansion of antiretroviral treatment programs have led to the growing concern for the development of antiretroviral drug resistance. The aims were to assess the prevalence of drug resistant HIV‐1 variants and to identify circulating subtypes among HAART‐naïve patients. Plasma specimens from N = 100 HIV+ HAART‐naïve adult were collected between March 2008 and August 2010 and viral RNA were extracted for nested PCR and sequenced. PR‐RT sequences were protein aligned and checked for transmitted drug resistance mutations. Phylogenetic reconstruction and recombination analysis were performed to determine the genotypes. Based on the WHO consensus guidelines, none of the recruited patients had any transmitted drug resistance mutations. When analyzed against the Stanford guidelines, 35% of patients had at least one reported mutation that may reduce drug susceptibility to PI (24%), NRTI (5%), and NNRTI (14%). The commonly detected mutation that may affect current first line therapy was V179D (3%), which may lead to reduced susceptibility to NNRTI. The predominant circulating HIV‐1 genotypes were CRF01_AE (51%) and CRF33_01B (17%). The prevalence of unique recombinant forms (URF) was 7%; five distinct recombinant structures involving CRF01_AE and subtype B′ were observed, among them a cluster of three isolates that could form a novel circulating recombinant form (CRF) candidate. Transmitted drug resistance prevalence among HAART‐naïve patients was low in this cohort of patients in Kuala Lumpur despite introduction of HAART 5 years ago. Owing to the high genetic diversity, continued molecular surveillance can identify the persistent emergence of HIV‐1 URF and novel CRF with significant epidemiological impact. J. Med. Virol. 86:38–44, 2014.

Prophylactic platelet transfusion plus supportive care versus supportive care alone in adults with dengue and thrombocytopenia: a multicentre, open-label, randomised, superiority trial

BACKGROUND Dengue is the commonest vector-borne infection worldwide. It is often associated with thrombocytopenia, and prophylactic platelet transfusion is widely used despite the dearth of robust evidence. We aimed to assess the efficacy and safety of prophylactic platelet transfusion in the prevention of bleeding in adults with dengue and thrombocytopenia. METHODS We did an open-label, randomised, superiority trial in five hospitals in Singapore and Malaysia. We recruited patients aged at least 21 years who had laboratory-confirmed dengue (confirmed or probable) and thrombocytopenia (≤20 000 platelets per μL), without persistent mild bleeding or any severe bleeding. Patients were assigned (1:1), with randomly permuted block sizes of four or six and stratified by centre, to receive prophylactic platelet transfusion in addition to supportive care (transfusion group) or supportive care alone (control group). In the transfusion group, 4 units of pooled platelets were given each day when platelet count was 20 000 per μL or lower; supportive care consisted of bed rest, fluid therapy, and fever and pain medications. The primary endpoint was clinical bleeding (excluding petechiae) by study day 7 or hospital discharge (whichever was earlier), analysed by intention to treat. Safety outcomes were analysed according to the actual treatment received. This study was registered with ClinicalTrials.gov, number NCT01030211, and is completed. FINDINGS Between April 29, 2010, and Dec 9, 2014, we randomly assigned 372 patients to the transfusion group (n=188) or the control group (n=184). The intention-to-treat analysis included 187 patients in the transfusion group (one patient was withdrawn immediately) and 182 in the control group (one was withdrawn immediately and one did not have confirmed or probable dengue). Clinical bleeding by day 7 or hospital discharge occurred in 40 (21%) patients in the transfusion group and 48 (26%) patients in the control group (risk difference -4·98% [95% CI -15·08 to 5·34]; relative risk 0·81 [95% CI 0·56 to 1·17]; p=0·16). 13 adverse events occurred in the transfusion group and two occurred in the control group (5·81% [-4·42 to 16·01]; 6·26 [1·43 to 27·34]; p=0·0064). Adverse events that were possibly, probably, or definitely related to transfusion included three cases of urticaria, one maculopapular rash, one pruritus, and one chest pain, as well as one case each of anaphylaxis, transfusion-related acute lung injury, and fluid overload that resulted in serious adverse events. No death was reported. INTERPRETATION In adult patients with dengue and thrombocytopenia, prophylactic platelet transfusion was not superior to supportive care in preventing bleeding, and might be associated with adverse events. FUNDING National Medical Research Council, Singapore.

HIV and aging: Insights from the Asia Pacific HIV Observational Database (APHOD)

The proportion of people living with HIV/AIDS in the ageing population (> 50 years old) is increasing. We aimed to explore the relationship between older age and treatment outcomes in HIV‐positive persons from the Asia Pacific region.

Concurrent loss of co-stimulatory molecules and functional cytokine secretion attributes leads to proliferative senescence of CD8+ T cells in HIV/TB co-infection

The role of T-cell immunosenescence and functional CD8(+) T-cell responses in HIV/TB co-infection is unclear. We examined and correlated surrogate markers of HIV disease progression with immune activation, immunosenescence and differentiation using T-cell pools of HIV/TB co-infected, HIV-infected and healthy controls. Our investigations showed increased plasma viremia and reduced CD4/CD8 T-cell ratio in HIV/TB co-infected subjects relative to HIV-infected, and also a closer association with changes in the expression of CD38, a cyclic ADP ribose hydrolase and CD57, which were consistently expressed on late-senescent CD8(+) T cells. Up-regulation of CD57 and CD38 were directly proportional to lack of co-stimulatory markers on CD8(+) T cells, besides diminished expression of CD127 (IL-7Rα) on CD57(+)CD4(+) T cells. Notably, intracellular IFN-γ, perforin and granzyme B levels in HIV-specific CD8(+) T cells of HIV/TB co-infected subjects were diminished. Intracellular CD57 levels in HIV gag p24-specific CD8(+) T cells were significantly increased in HIV/TB co-infection. We suggest that HIV-TB co-infection contributes to senescence associated with chronic immune activation, which could be due to functional insufficiency of CD8(+) T cells.