Diagnostic method-based underestimation of leptospirosis in clinical and research settings; an experience from a large prospective study in a high endemic setting

BackgroundLeptospirosis has globally significant human mortality and morbidity, yet estimating the clinical and public health burden of leptospirosis is challenging because timely diagnosis remains limited. The goal of the present study was to evaluate leptospirosis undercounting by current standard methods in both clinical and epidemiological study settings.Methodology/Principal findingsA prospective hospital-based study was conducted in multiple hospitals in Sri Lanka from 2016 to 2019. Culture, whole blood, and urine samples were collected from clinically suspected leptospirosis cases and patients with undifferentiated fever. Analysis of biological samples from 1,734 subjects confirmed 591 (34.1%) cases as leptospirosis and 297 (17.1%) were classified as “probable” leptospirosis cases. Whole blood quantitative PCR (qPCR) did identify the most cases (322/540(60%)) but missed 40%. Cases missed by each method include; urine qPCR, 70% (153/220); acute sample microscopic agglutination test (MAT), 80% (409/510); paired serum sample MAT, 58% (98/170); and surveillance clinical case definition, 53% (265/496). qPCR of negative culture samples after six months of observation was of diagnostic value retrospectively with but missed 58% of positives (109/353).ConclusionLeptospirosis disease burden estimates should consider the limitations of standard diagnostic tests. qPCR of multiple sample types should be used as a leading standard test for diagnosing acute leptospirosis.     

Experimental and clinical aspects of auto and coherence spectra of blood pressure, heart rate and respiratory rhythms]

Auto- coherence and phase spectra of blood-pressure, heart-rate, and respiration changes at various intervals were calculated in 34 healthy individuals and 46 patients with neurovegetative disorders in a direct way, partly by means of the Fast-Fourier transformations. It was only by 18-min recordings that reliably reproducible spectra could be obtained that permit exact evaluation of the statistical properties of these biorhythms.     

Optimizing the microscopic agglutination test (MAT) panel for the diagnosis of Leptospirosis in a low resource,hyper-endemic setting with varied microgeographic variation in reactivity

The microscopic agglutination test (MAT) is the standard serological reference test for the diagnosis of leptospirosis, despite being a technically demanding and laborious procedure. The use of a locally optimised MAT panel is considered essential for proper performance and interpretation of results. This paper describes the procedure of selecting such an optimised panel for Sri Lanka, a country hyper-endemic for leptospirosis. MAT was performed using 24 strains on 1132 serum samples collected from patients presenting with acute undifferentiated fever. Of 24 strains, 15 were selected as the optimised panel, while only 11% of serum samples showed positivity. A geographical variation in predominantly reactive serovars was observed, whereas reactivity was low with the saprophytic strain Patoc. Testing with paired sera yielded a higher sensitivity but provided only a retrospective diagnosis. Serological tests based on ELISA with complementary molecular diagnosis using PCR are a feasible and robust alternative approach to diagnose leptospirosis in countries having a higher burden of the disease.     

Design of a partial peptide mimetic of anginex with antiangiogenic and anticancer activity

Based on structure-activity relationships of the angiostatic beta-sheet-forming peptide anginex, we have designed a mimetic, 6DBF7, which inhibits angiogenesis and tumor growth in mice. 6DBF7 is composed of a beta-sheet-inducing dibenzofuran (DBF)-turn mimetic and two short key amino acid sequences from anginex. This novel antiangiogenic molecule is more effective in vivo than parent anginex. In a mouse xenograft model for ovarian carcinoma, 6DBF7 is observed to reduce tumor growth by up to 80%. It is suggested that the activity is based on antiangiogenesis, because in vitro tube formation is inhibited, and because treatment of tumor-bearing mice led to a significant reduction in microvessel density within the tumor. This partial peptide mimetic is the first endothelial cell-specific molecule designed as a substitute for an angiostatic inhibitory peptide.     

Population dynamics of Spalgis epius (Lepidoptera: Lycaenidae), a candidate biocontrol agent of mealybugs and its interaction with mealybug-attendant ants

Spalgis epius is an economically important hemipterophagous butterfly. Detailed information on the population dynamics, natural enemies, and prey range of S. epius and its association with mealybug-attendant ant species is lacking. Three years of field studies conducted at Bangalore University campus, Bengaluru, India on these aspects indicated that the population density of S. epius was greatest from June to December and least from February to May in the low land region. S. epius survived on eight prey species, which were present in different months, of which Phenacoccus indicus was recorded as a prey of S. epius for the first time. Different prey species occurred on 12 species of host plants. The occurrence of S. epius was negatively correlated with temperature and positively correlated with relative humidity and prey populations. Six mealybug-attendant ant species were associated with the larvae of S. epius. The seven general predators of S. epius adults were recorded. Knowledge on the population dynamics and a prey range of S. epius and its interaction with mealybug-attendant ant species could be helpful to using this predator as a major biocontrol agent of various species of mealybugs. This study contributed to our understanding of the population dynamics of a hemipterophagous butterfly.     

Vascular biomechanical properties in mice with smooth muscle specific deletion of Ndst1

Heparan sulfate proteoglycans act as co-receptors for many chemokines and growth factors. The sulfation pattern of the heparan sulfate chains is a critical regulatory step affecting the binding of chemokines and growth factors. N-deacetylase-N-sulfotransferase1 (Ndst1) is one of the first enzymes to catalyze sulfation. Previously published work has shown that HSPGs alter tangent moduli and stiffness of tissues and cells. We hypothesized that loss of Ndst1 in smooth muscle would lead to significant changes in heparan sulfate modification and the elastic properties of arteries. In line with this hypothesis, the axial tangent modulus was significantly decreased in aorta from mice lacking Ndst1 in smooth muscle (SM22αcre⁺Ndst1^?/?, p < 0.05, n = 5). The decrease in axial tangent modulus was associated with a significant switch in myosin and actin types and isoforms expressed in aorta and isolated aortic vascular smooth muscle cells. In contrast, no changes were found in the compliance of smaller thoracodorsal arteries of SM22αcre⁺Ndst1^?/? mice. In summary, the major findings of this study were that targeted ablation of Ndst1 in smooth muscle cells results in altered biomechanical properties of aorta and differential expression of myosin and actin types and isoforms.     

Identification of a New Target of miR-16, Vacuolar Protein Sorting 4a

Rationale

The rationale was to utilize a bioinformatics approach to identify miRNA binding sites in genes with single nucleotide mutations (SNPs) to discover pathways in heart failure (HF).

Objective

The objective was to focus on the genes containing miRNA binding sites with miRNAs that were significantly altered in end-stage HF and in response to a left ventricular assist device (LVAD).

Methods and Results

BEDTools v2.14.3 was used to discriminate SNPs within predicted 3′UTR miRNA binding sites. A member of the miR-15/107 family, miR-16, was decreased in the circulation of end-stage HF patients and increased in response to a LVAD (p<0.001). MiR-16 decreased Vacuolar Protein Sorting 4a (VPS4a) expression in HEK 293T cells (p<0.01). The SNP rs16958754 was identified in the miR-15/107 family binding site of VPS4a which abolished direct binding of miR-16 to the 3′UTR of VPS4a (p<0.05). VPS4a was increased in the circulation of end-stage HF patients (p<0.001), and led to a decrease in the number of HEK 293T cells in vitro (p<0.001).

Conclusions

We provide evidence that miR-16 decreases in the circulation of end-stage HF patients and increases with a LVAD. Modeling studies suggest that miR-16 binds to and decreases expression of VPS4a. Overexpression of VPS4a decreases cell number. Together, these experiments suggest that miR-16 and VPS4a expression are altered in end-stage HF and in response to unloading with a LVAD. This signaling pathway may lead to reduced circulating cell number in HF.     

Purification and characterization of a 34 kDa antioxidant protein (β-turmerin) from turmeric (Curcuma longa) waste grits

β-Turmerin from turmeric (Curcuma longa) waste grits obtained after extraction of curcumin was purified by successive gel permeation chromatography. Homogeneity of β-turmerin was confirmed by its movement as single band both in SDS-PAGE and as well as in native (basic) PAGE. The apparent molecular mass is 34 kDa by SDS-PAGE. It is more hydrophobic protein and showed sharp single peak in RP-HPLC with retention time of 62.17 min. It is a glycoprotein as it shows the presence of amino sugars up to 0.021 gm%. In three different model systems i.e., linolenic acid micelles, erythrocyte membrane systems and liposomes, β-turmerin at 0.125 μM offered 70%, 64%, and 60% inhibition of lipid peroxidation, which is 3200 times more efficient than the standard antioxidants BHA (400 μM) and α-tocopherol (400 μM). β-turmerin inhibited diene–triene and tetraene conjugation up to 54%, 72% and 47%, respectively. β-turmerin also effectively scavenges hydroxyl radicals when compared to BHA and α-tocopherol. β-turmerin (2.5 μM) further inhibited the activation of PMNL mediated by fMLP up to the extent of 75%, where as standards BHA (400 μM) and mannitol (10 μM) inhibited the same to 65% and 55%, respectively. At 0.125 μM dose β-turmerin prevented t-BOOH induced cell death at all time intervals. In addition to the above properties, it is non-toxic to lymphocytes as it did not affect the viability of cells. The mechanism of antioxidant action of β-turmerin could probably be by counteracting/quenching of reactive oxygen species (ROS). We report the purification and characterization of β-turmerin (34 kDa), a potent antioxidant protein from turmeric waste grits.     

12 Novel clonal groups of Leptospira infecting humans in multiple contrasting epidemiological contexts in Sri Lanka

Leptospirosis is a ubiquitous zoonotic disease and a major clinical challenge owing to the multitude of clinical presentations and manifestations that are possibly attributable to the diversity of Leptospira, the understanding of which is key to study the epidemiology of this emerging global disease threat. Sri Lanka is a hotspot for leptospirosis with high levels of endemicity as well as annual epidemics. We carried out a prospective study of Leptospira diversity in Sri Lanka, covering the full range of climatic zones, geography, and clinical severity. Samples were collected for leptospiral culture from 1,192 patients from 15 of 25 districts in Sri Lanka over two and half years. Twenty-five isolates belonging to four pathogenic Leptospira species were identified: L. interrogans, L. borgpetersenii, L. weilii, and L. kirschneri. At least six serogroups were identified among the isolates: Autumnalis (6), Pyrogenes (4), Icterohaemorrhagiae (2), Celledoni (1), Grippotyphosa (2) and Bataviae (1). Seven isolates did not agglutinate using available antisera panels, suggesting new serogroups. Isolates were sequenced using an Illumina platform. These data add 25 new core genome sequence types and were clustered in 15 clonal groups, including 12 new clonal groups. L. borgpetersenii was found only in the dry zone and L. weilii only in the wet zone. Acute kidney injury and cardiovascular involvement were seen only with L. interrogans infections. Thrombocytopenia and liver impairment were seen in both L. interrogans and L. borgpetersenii infections. The inadequate sensitivity of culture isolation to identify infecting Leptospira species underscores the need for culture-independent typing methods for Leptospira.