The efficacy of pre-erythrocytic stage malaria antigens or vaccine platforms is routinely assessed in murine models challenged with Plasmodium sporozoites. Relative liver-stage parasite burden is ...quantified using reverse transcription quantitative PCR (RTqPCR), which relies on constitutively expressed endogenous control reference genes. However, the stability of host-reference gene expression for RTqPCR analysis following Plasmodium challenge and immunization has not been systematically evaluated. Herein, we evaluated the stability of expression of twelve common RTqPCR reference genes in a murine model of Plasmodium yoelii sporozoite challenge and DNA-adenovirus IV 'Prime-Target' immunization. Significant changes in expression for six of twelve reference genes were shown by one-way ANOVA, when comparing gene expression levels among challenge, immunized, and naïve mice groups. These changes were attributed to parasite challenge or immunization when comparing group means using post-hoc Bonferroni corrected multiple comparison testing. Succinate dehydrogenase (SDHA) and TATA-binding protein (TBP) were identified as stable host-reference genes suitable for relative RTqPCR data normalisation, using the RefFinder package. We defined a robust threshold of 'partial-protection' with these genes and developed a strategy to simultaneously quantify matched host parasite burden and cytokine responses following immunisation or challenge. This is the first report systematically identifying reliable host reference genes for RTqPCR analysis following Plasmodium sporozoite challenge. A robust RTqPCR protocol incorporating reliable reference genes which enables simultaneous analysis of host whole-liver cytokine responses and parasite burden will significantly standardise and enhance results between international malaria vaccine efficacy studies.
Whole-blood-derived transcriptional profiling is widely used in biomarker discovery, immunological research, and therapeutic development. Traditional molecular and high-throughput transcriptomic ...platforms, including molecular assays with quantitative PCR (qPCR) and RNA-sequencing (RNA-seq), are dependent upon high-quality and intact RNA. However, collecting high-quality RNA from field studies in remote tropical locations can be challenging due to resource restrictions and logistics of post-collection processing. The current study tested the relative performance of the two most widely used whole-blood RNA collection systems, PAXgene® and Tempus™, in optimal laboratory conditions as well as suboptimal conditions in tropical field sites, including the effects of extended storage times and high storage temperatures. We found that Tempus™ tubes maintained a slightly higher RNA quantity and integrity relative to PAXgene® tubes at suboptimal tropical conditions. Both PAXgene® and Tempus™ tubes gave similar RNA purity (A260/A280). Additionally, Tempus™ tubes preferentially maintained the stability of mRNA transcripts for two reference genes tested, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), even when RNA quality decreased with storage length and temperature. Both tube types preserved the rRNA transcript 18S ribosomal RNA (18S) equally. Our results suggest that Tempus™ blood RNA collection tubes are preferable to PAXgene® for whole-blood collection in suboptimal tropical conditions for RNA-based studies in resource-limited settings.
Chronic kidney disease of unknown etiology (CKDu) imposes a substantial burden on public health in Sri Lankan agricultural communities. High seroprevalences of hantavirus have been reported in CKDu ...patients in several locations of Sri Lanka. We carried out a cross-sectional study followed by an unmatched case-control comparison in two geographically distinct areas of Sri Lanka, Girandurukotte (CKDu endemic) and Kandy (CKDu non-endemic) to determine whether exposure to hantaviruses is a potential risk factor in patients with kidney disease. An indirect immunofluorescent antibody assay using two antigens, Thailand orthohantavirus-infected and recombinant N protein-expressing Vero E6 cells, were used for serodiagnosis. Participants' demographic and other socio-economic data were collected through a structured questionnaire. Fifty kidney disease patients and 270 controls from Kandy and 104 kidney disease patients and 242 controls from Girandurukotte were examined. Seropositivities were 50% and 17.4% in kidney patients and controls, respectively, in Girandurukotte, and they were 18% and 7% in Kandy. The odds of exposure to hantaviruses were higher for kidney disease patients than for controls in both Girandurukotte (OR:3.66, 95% CI:2.01 to 6.64) and Kandy (OR:2.64, 95% CI:1.07 to 6.54) in binary logistic regression models. According to statistical analysis, individuals exposed to hantaviruses had a higher risk of developing renal impairment. Therefore, hantavirus infection might be an important risk factor for development of kidney disease in Sri Lanka.
Extranodal natural killer/T-cell lymphoma (NKTCL) is an aggressive malignancy that has been etiologically linked to Epstein-Barr virus (EBV) infection, with EBV gene transcripts identified in almost ...all cases. However, the humoral immune response to EBV in NKTCL patients has not been well characterized. We examined the antibody response to EBV in plasma samples from 51 NKTCL cases and 154 controls from Hong Kong and Taiwan who were part of the multi-center, hospital-based AsiaLymph case-control study. The EBV-directed serological response was characterized using a protein microarray that measured IgG and IgA antibodies against 202 protein sequences representing the entire EBV proteome. We analyzed 157 IgG antibodies and 127 IgA antibodies that fulfilled quality control requirements. Associations between EBV serology and NKTCL status were disproportionately observed for IgG rather than IgA antibodies. Nine anti-EBV IgG responses were significantly elevated in NKTCL cases compared with controls and had ORs
> 6.0 (Bonferroni-corrected P-values < 0.05). Among these nine elevated IgG responses in NKTCL patients, three IgG antibodies (all targeting EBNA3A) are novel and have not been observed for other EBV-associated tumors of B-cell or epithelial origin. IgG antibodies against EBNA1, which have consistently been elevated in other EBV-associated tumors, were not elevated in NKTCL cases. We characterize the antibody response against EBV for patients with NKTCL and identify IgG antibody responses against six distinct EBV proteins. Our findings suggest distinct serologic patterns of this NK/T-cell lymphoma compared with other EBV-associated tumors of B-cell or epithelial origin.
Chronic kidney disease of unknown aetiology (CKDu) is a major public health problem in Sri Lanka, especially among agrarian communities. Although the cause of CKDu is still unknown, hantavirus ...infection has been proposed as a risk factor.
This study was performed using serological samples collected from two CKDu-endemic areas, Anuradhapura (2010) and Badulla districts (2010 and 2016), and a non-endemic area, Matale (2016) district. The presence of anti-Thailand orthohantavirus IgG antibodies was investigated in serum samples. Hantavirus seroprevalence and demographic data were epidemiologically analysed.
Seroprevalence was higher in CKDu patients (40.6-60.0 %) and healthy individuals in CKDu-endemic areas (17.6-25.5 %) than in healthy individuals in non-endemic areas (3.0 %). Statistically significant odds ratios (ORs) for hantavirus infection in CKDu patients were detected in CKDu-endemic areas ORs: 3.2 and 3.1; 95 % confidence interval (CI): 1.8-5.5 and 1.8-5.2 in Anuradhapura and Badulla districts in 2010; and OR: 4.4, 95 % CI: 2.3-8.5 in 2016 in Badulla district). Furthermore, the OR for hantavirus infection in Badulla district has increased in the last decade from 3.1 (95 % CI: 1.8-5.3) to 4.4 (95 % CI: 2.3-8.5).
Hantavirus infection has been prevalent in two distant CKDu-endemic areas since 2010. The observed significant association of hantavirus seropositivity with CKDu indicates a possible role of hantavirus infection in CKDu pathogenesis.
We reported the genetic evidence of circulating hantaviruses from small mammals captured in a chronic kidney disease of unknown etiology (CKDu) hotspot area of Sri Lanka. The high seroprevalence of ...anti-hantavirus antibodies against
(THAIV) has been reported among CKDu patients and rodents in Sri Lankan CKDu hotspots. We captured 116 small mammals from CKDu endemic regions in the Polonnaruwa District of Sri Lanka. Seven animals (five out of 11
and two out of 99
) were PCR-positive for the hantavirus. A rat-borne sequence was grouped with a THAIV-like Anjozorobe virus. In contrast,
-borne sequences belonged to the THAIV lineage, suggesting a novel orthohantavirus species according to the phylogenetic analyses and whole-genome comparisons. Our genetic evidence indicates the presence of two THAIV-related viruses circulating in this CKDu endemic area, suggesting a basis for further investigations to identify the infectious virus in patients with CKDu and the CKDu induction mechanism of these viruses.
Abstract Chronic kidney disease of uncertain etiology (CKDu) has been a severe burden and a public health crisis in Sri Lanka over the past two decades. Many studies have established hypotheses to ...identify potential risk factors although causative agents, risk factors and etiology of this disease are still uncertain. Several studies have postulated that fungal and bacterial nephrotoxins are a possible etiological factor; however, the precise link between hypothesized risk factors and the pathogenesis of chronic kidney disease has yet to be proven in prior studies. Leptospirosis and Hantavirus infections are important zoonotic diseases that are naturally maintained and transmitted via infected rodent populations and which present similar clinical and epidemiological features. Both infections are known to be a cause of acute kidney damage that can proceed into chronic renal failure. Several studies have reported presence of both infections in Sri Lanka. Therefore, we hypothesized that pathogenic Leptospira or Hantavirus are possible causative agents of acute kidney damage which eventually progresses to chronic kidney disease in Sri Lanka. The proposed hypothesis will be evaluated by means of an observational study design. Past infection will be assessed by a cross-sectional study to detect the presence of IgG antibodies with further confirmatory testing among chronic kidney disease patients and individuals from the community in selected endemic areas compared to low prevalence areas. Identification of possible risk factors for these infections will be followed by a case-control study and causality will be further determined with a cohort study. If the current hypothesis is true, affected communities will be subjected for medical interventions related to the disease for patient management while considering supportive therapies. Furthermore and possibly enhance their preventive and control measures to improve vector control to decrease the risk of infection.
Monoclonal antibody (MAb)-based ELISA was developed to detect leptospiral antigens from the plasma of febrile patients. The MAb reacts specifically with pathogenic leptospires and the assay possesses ...excellent diagnostic test parameters compared to PCR, indicating that this new ELISA is useful for the early diagnosis of leptospirosis with low operational cost.
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