Evaluation of ELISA-Based Multiplex Peptides for the Detection of Human Serum Antibodies Induced by Zika Virus Infection across Various Countries.

Zika virus (ZIKV) is a mosquito-borne Flavivirus with a positive-sense RNA genome, which are generally transmitted through the bite of an infected Aedes mosquito. ZIKV infections could be associated with neurological sequelae that, and otherwise produces similar clinical symptoms as other co-circulating pathogens. Past infection with one member of the Flavivirus genus often induces cross-reactive antibodies against other flaviruses. These attributes complicate the ability to differentially diagnose ZIKV infection from other endemic mosquito-borne viruses, making it both a public health issue as well as a diagnostic challenge. We report the results from serological analyses using arbovirus-specific peptides on 339 samples that were previously collected from 6 countries. Overall, we found that our multiplexed peptide-based ELISA was highly efficient for identifying ZIKV antibodies as early as 2 weeks post infection, and that it correlates with microneutralization, plaque reduction neutralization tests (PRNTs) and commercial tests for ZIKV in previously characterized samples. We observed that seropositivity varied by patient cohort, reflecting the sampling period in relation to the 2015-2016 ZIKV outbreak. This work evaluates the accuracy, specificity, and sensitivity of our peptide-based ELISA method for detecting ZIKV antibodies from geographically diverse regions. These findings can contribute to ongoing serological methods development and can be adapted for use in future studies.

Preliminary study on chronic granulomatous disease in Sri Lanka.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency of the phagocytic cells, which results in absent or diminished levels of microbicidal reactive oxygen species. The disease occurs due to germline mutations in the genes encoding the five subunits of NADPH oxidase complex. The present study is a pilot study to understand the clinical and genetic aspects of CGD in Sri Lanka. METHODS: Clinical records of thirteen CGD patients were analysed and compared with similar studies performed in different countries and regions to identify patterns in demographics, clinical manifestations and infectious agents. Genomic DNA and cDNA were analysed in eight patients to identify mutations in CYBB and NCF1 genes, thereby to ascertain the potential X-linked and autosomal recessive (AR) CGD patients. RESULTS: The onset of symptoms in the patient cohort was very early (mean 4.6 months) compared to 20 months in India and 23.9 months in Latin America. Similarly, the age at diagnosis was lower (mean 1.6 years after birth) compared to other studies; 4.5 years in India and 6.1 years in Europe. Pulmonary manifestations were the most common (85%), followed by skin/subcutaneous infections (77%) and lymphadenopathy (62%). The death rate of local patients (38%) was higher than other countries (India 35%, Europe 20%). Majority (77%) were treated for tuberculosis at some point in life. Genetic analysis confirmed six out of eight patients as X-linked CGD cases with mutations in CYBB gene. A novel splice site mutation was identified in P-07 at position c.141+6 which resulted in the deletion of entire exon 2. Two siblings (P-05 and P-06) from consanguineous parents, were identified with AR-CGD based on the homozygous GT deletion mutation in NCF1 gene. CONCLUSIONS: The clinical presentation, manifestations and genetic subtypes in the local cohort, appear to be comparable with global trends. Mycobacterial infections should be investigated and treated with more prominence. Effective treatment options are required to control the high mortality rate.

Gene Expression Profile of Human Cytokines in Response to Burkholderia pseudomallei Infection.

Melioidosis is an underreported infectious disease, caused by the Gram-negative bacterium Burkholderia pseudomallei. Understanding the disease susceptibility and pathogenesis is crucial for developing newer diagnostic and therapeutic strategies for this life-threatening infection. In this study, we aimed to analyze the gene expression levels of important cytokines in melioidosis patients and establish useful correlates with disease biomarkers compared to cases of sepsis infection caused by other pathogens and healthy individuals. A Qiagen common human cytokines array profiling the gene expression of 84 important cytokines by real-time quantitative PCR (RT-qPCR) was used. We analyzed 26 melioidosis cases, 5 healthy controls, and 10 cases of sepsis infection caused by other pathogens. Our results showed consistently upregulated expression of interleukins (IL) interleukin-4 (IL-4), interleukin-17 alpha (IL-17A), IL-23A, and IL-24, interferons (IFN) interferon alpha 1 (IFNA1) and interferon beta 1 (IFNB1), tumor necrosis factor (TNF) superfamily 4 (TNFSF4), transforming growth factor (TGF) superfamily, bone morphogenetic proteins 3 and 6 (BMP3 and BMP6), transforming growth factor beta 1 (TGFB1), and other growth factors, including macrophage colony-stimulating factor (M-CSF), C-fos-induced growth factor (FIGF), and platelet-derived growth factor alpha (PDGFA) polypeptide, in melioidosis patients compared to their expression in other sepsis cases, irrespective of comorbidities, duration of fever/clinical symptoms, and antibiotic treatment. Our findings indicate a dominant Th2- and Th17-type-cytokine response, suggesting that their dysregulation at initial stages of infection may play an important role in disease pathogenesis. IL-1A, interleukin-1 beta (IL-1B), and IL-8 were significantly downregulated in septicemic melioidosis patients compared to their expression in other sepsis cases. These differentially expressed genes may serve as biomarkers for melioidosis diagnosis and targets for therapeutic intervention and may help us understand immune response mechanisms. IMPORTANCE Melioidosis is a life-threatening infectious disease caused by a soil-associated Gram-negative bacterium, B. pseudomallei. Melioidosis is endemic in Southeast Asia and northern Australia; however, the global distribution of B. pseudomallei and the disease burden of melioidosisis are still poorly understood. Melioidosis is difficult to treat, as B. pseudomallei is intrinsically resistant to many antibiotics and requires a long course of antibiotic treatment. The mortality rates remain high in areas of endemicity, with reoccurrence being common. Therefore, it is imperative to diagnose the disease at an early stage and provide vital clinical care to reduce the mortality rate. With limitations in treatment and lack of a vaccine, it is crucial to study the immune response mechanisms to this infection to get a better understanding of disease susceptibility and pathogenesis. Therefore, this study aimed to analyze the gene expression levels of important cytokines to establish useful correlations for diagnostic and therapeutic purposes.

Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines.

UNLABELLED: Tumor necrosis factor alpha (TNF) plays a critical role in the control of Mycobacterium tuberculosis, in part by augmenting T cell responses through promoting macrophage phagolysosomal fusion (thereby optimizing CD4(+) T cell immunity by enhancing antigen presentation) and apoptosis (a process that can lead to cross-priming of CD8(+) T cells). M. tuberculosis can evade antituberculosis (anti-TB) immunity by inhibiting host cell TNF production via expression of specific mycobacterial components. We hypothesized that M. tuberculosis mutants with an increased capacity to induce host cell TNF production (TNF-enhancing mutants) and thus with enhanced immunogenicity can be useful for vaccine development. To identify mycobacterial genes that regulate host cell TNF production, we used a TNF reporter macrophage clone to screen an H37Rv M. tuberculosis cosmid library constructed in M. smegmatis The screen has identified a set of TNF-downregulating mycobacterial genes that, when deleted in H37Rv, generate TNF-enhancing mutants. Analysis of mutants disrupted for a subset of TNF-downregulating genes, annotated to code for triacylglycerol synthases and fatty acyl-coenzyme A (acyl-CoA) synthetase, enzymes that concern lipid biosynthesis and metabolism, has revealed that these strains can promote macrophage phagolysosomal fusion and apoptosis better than wild-type (WT) bacilli. Immunization of mice with the TNF-enhancing M. tuberculosis mutants elicits CD4(+) and CD8(+) T cell responses that are superior to those engendered by WT H37Rv. The results suggest that TNF-upregulating M. tuberculosis genes can be targeted to enhance the immunogenicity of mycobacterial strains that can serve as the substrates for the development of novel anti-TB vaccines. IMPORTANCE: One way to control tuberculosis (TB), which remains a major global public health burden, is by immunization with an effective vaccine. The efficacy of Mycobacterium bovis BCG, the only currently approved TB vaccine, is inconsistent. Tumor necrosis factor alpha (TNF) is a cytokine that plays an important role in controlling TB. M. tuberculosis, the causative agent of TB, can counter this TNF-based defense by decreasing host cell TNF production. This study identified M. tuberculosis genes that can mediate inhibition of TNF production by macrophage (an immune cell critical to the control of TB). We have knocked out a number of these genes to generate M. tuberculosis mutants that can enhance macrophage TNF production. Immunization with these mutants in mice triggered a T cell response stronger than that elicited by the parental bacillus. Since T cell immunity is pivotal in controlling M. tuberculosis, the TNF-enhancing mutants can be used to develop novel TB vaccines.

Polymorphisms of Transporter Associated with Antigen Presentation, Tumor Necrosis Factor-α and Interleukin-10 and their Implications for Protection and Susceptibility to Severe Forms of Dengue Fever in Patients in Sri Lanka.

CONTEXT: To date, a clear understanding of dengue disease pathogenesis remains elusive. Some infected individuals display no symptoms while others develop severe life-threatening forms of the disease. It is widely believed that host genetic factors influence dengue severity. AIMS: This study evaluates the relationship between certain polymorphisms and dengue severity in Sri Lankan patients. SETTINGS AND DESIGN: Polymorphism studies are carried out on genes for; transporter associated with antigen presentation (TAP), promoter of tumor necrosis factor-α (TNF-α), and promoter of interleukin-10 (IL-10). In other populations, TAP1 (333), TAP2 (379), TNF-α (-308), and IL-10 (-1082, -819, -592) have been associated with dengue and a number of different diseases. Data have not been collected previously for these polymorphisms for dengue patients in Sri Lanka. MATERIALS AND METHODS: The polymorphisms were typed by amplification refractory mutation system polymerase chain reaction in 107 dengue hemorrhagic fever (DHF) patients together with 62 healthy controls. STATISTICAL ANALYSIS USED: Pearson's Chi-square contingency table analysis with Yates' correction. RESULTS: Neither the TAP nor the IL-10 polymorphisms considered individually can define dengue disease outcome with regard to severity. However, the genotype combination, IL-10 (-592/-819/-1082) CCA/ATA was significantly associated with development of severe dengue in these patients, suggesting a risk factor to developing DHF. Also, identified is the genotype combination IL-10 (-592/-819/-1082) ATA/ATG which suggested a possibility for protection from DHF. The TNF-α (-308) GG genotype was also significantly associated with severe dengue, suggesting a significant risk factor. CONCLUSIONS: The results reported here are specific to the Sri Lankan population. Comparisons with previous reports imply that data may vary from population to population.