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.     

Hydrogen exchange of rhenium(VII) heptahydridobis(triphenylphosphine) with water, aniline, methanol, and itself

The hydride ligands of ReH₇(PPh₃)₂ undergo fast hydrogen exchange with water at room temperature while exchanging with the hydrogen atoms of aromatic solvent molecules at a slower rate. The amount of time required for isotopomer peaks to appear in the ¹H NMR hydride resonance of ReH₇(PPh₃)₂ was used to distinguish fast hydrogen exchange from slow hydrogen exchange. The room temperature ¹H NMR T₁ values of adventitious water resonances were used to distinguish two rhenium heptahydride compounds that participate in fast hydrogen exchange with water, ReH₇(PPh₃)₂ and ReH₇(AsPh₃)₂, from one compound that does not participate, ReH₇(PCy₃)₂. Fast hydrogen exchange between ReH₇(PPh₃)₂ and water is greatly slowed or stopped when the compound is dissolved in a solution that contains DMSO. The compound ReH₆D(PPh₃)₂, an isotopomer of ReH₇(PPh₃)₂ that was prepared in situ, exhibited an isotopomer shift in its ³¹P-{¹H} NMR spectrum as well as D-P coupling when measured in the solvent anisole. In a solvent system containing deuteroaniline, nearly all of the hydride ligands were exchanged with deuterium, from aniline, in less than 2 h. Hydrogen exchange between the hydride ligands of ReH₇(PPh₃)₂ and deuteroaniline was found to be reversible upon the addition of normal aniline. The distribution of hydrogen isotopes in the rhenium coordination spheres reflected the overall composition of the exchangeable hydrogen isotopes present in the bulk sample. In a deuteromethanol-containing solvent system, the exchange of hydride ligands between separate ReH₇(PPh₃)₂ isotopomers was evident. The presumed hydrogen exchange intermediate, [ReH₆(H₂)(PPh₃)₂]⁺, may be responsible for the evident exchange of hydride ligands and may also be important to the thermal loss of dihydrogen from ReH₇(PPh₃)₂.     

Isolation of a Neurotoxin (α-colubritoxin) from a Nonvenomous Colubrid: Evidence for Early Origin of Venom in Snakes

The evolution of venom in advanced snakes has been a focus of long-standing interest. Here we provide the first complete amino acid sequence of a colubrid toxin, which we have called -colubritoxin, isolated from the Asian ratsnake Coelognathus radiatus (formerly known as Elaphe radiata), an archetypal nonvenomous snake as sold in pet stores. This potent postsynaptic neurotoxin displays readily reversible, competitive antagonism at the nicotinic receptor. The toxin is homologous with, and phylogenetically rooted within, the three-finger toxins, previously thought unique to elapids, suggesting that this toxin family was recruited into the chemical arsenal of advanced snakes early in their evolutionary history. LC-MS analysis of venoms from most other advanced snake lineages revealed the widespread presence of components of the same molecular weight class, suggesting the ubiquity of three-finger toxins across advanced snakes, with the exclusion of Viperidae. These results support the role of venom as a key evolutionary innovation in the early diversification of advanced snakes and provide evidence that forces a fundamental rethink of the very concept of nonvenomous snake.     

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.     

Impact and management of purple loosestrife (Lythrum salicaria) in North America

The invasion of non-indigenous plants is considered a primary threat to integrity and function of ecosystems. However, there is little quantitative or experimental evidence for ecosystem impacts of invasive species. Justifications for control are often based on potential, but not presently realized, recognized or quantified, negative impacts. Should lack of scientific certainty about impacts of non-indigenous species result in postponing measures to prevent degradation? Recently, management of purple loosestrife (Lythrum salicaria), has been criticized for (1) lack of evidence demonstrating negative impacts of L. salicaria, and (2) management using biocontrol for lack of evidence documenting the failure of conventional control methods. Although little quantitative evidence on negative impacts on native wetland biota and wetland function was available at the onset of the control program in 1985, recent work has demonstrated that the invasion of purple loosestrife into North American freshwater wetlands alters decomposition rates and nutrient cycling, leads to reductions in wetland plant diversity, reduces pollination and seed output of the native Lythrum alatum, and reduces habitat suitability for specialized wetland bird species such as black terns, least bitterns, pied-billed grebes, and marsh wrens. Conventional methods (physical, mechanical or chemical), have continuously failed to curb the spread of purple loosestrife or to provide satisfactory control. Although a number of generalist insect and bird species utilize purple loosestrife, wetland habitat specialists are excluded by encroachment of L. salicaria. We conclude that (1) negative ecosystem impacts of purple loosestrife in North America justify control of the species and that (2) detrimental effects of purple loosestrife on wetland systems and biota and the potential benefits of control outweigh potential risks associated with the introduction of biocontrol agents. Long-term experiments and monitoring programs that are in place will evaluate the impact of these insects on purple loosestrife, on wetland plant succession and other wetland biota.     

Novel natriuretic peptides from the venom of the inland taipan (Oxyuranus microlepidotus): isolation, chemical and biological characterisation

Three natriuretic-like peptides (TNP-a, TNP-b, and TNP-c) were isolated from the venom of Oxyuranus microlepidotus (inland taipan) and were also present in the venoms of Oxyuranus scutellatus canni (New Guinea taipan) and Oxyuranus scutellatus scutellatus (coastal taipan). They were isolated by HPLC, characterised by mass spectrometry and Edman analysis, and consist of 35-39 amino acid residues. These molecules differ from ANP/BNP through replacement of invariant residues within the 17-membered ring structure and by inclusion of proline residues in the C-terminal tail. TNP-c was equipotent to ANP in specific GC-A assays or aortic ring assays whereas TNP-a and TNP-b were either inactive (GC-A over-expressing cells and endothelium-denuded aortic rings) or weakly active (endothelium-intact aortic rings). TNP-a and TNP-b were also unable to competitively inhibit the binding of TNP-c in endothelium-denuded aortae (GC-A) or endothelium-intact aortae (NPR-C). Thus, these naturally occurring isoforms provide a new platform for further investigation of structure-function relationships of natriuretic peptides.     

Accelerating the development of a sustainable bioenergy portfolio through stable isotopes

Bioenergy could help limit global warming to 2°C above pre-industrial levels while supplying almost a fourth of the world's renewable energy needs by 2050. However, the deployment of bioenergy raises concerns that adoption at meaningful scales may lead to unintended negative environmental consequences. Meanwhile, the full consolidation of a bioenergy industry is currently challenged by a sufficient, resilient, and resource-efficient biomass supply and an effective conversion process. Here, we provide a comprehensive analysis of how stable isotope approaches have accelerated the development of a robust bioeconomy by advancing knowledge about environmental sustainability, feedstock development, and biological conversion. We show that advances in stable isotope research have generated crucial information to (1) gain mechanistic insight into the potential of bioenergy crops to mitigate climate change as well as their impact on water and nutrient cycling; (2) develop high-yielding, resilient feedstocks that produce high-value bioproducts in planta; and (3) engineer microbes to enhance feedstock conversion to bioenergy products. Further, we highlight knowledge gaps that could benefit from future research facilitated by stable isotope approaches. We conclude that advances in mechanistic knowledge and innovations within the field of stable isotopes in cross-disciplinary research actions will greatly contribute to breaking down the barriers to establishing a robust bioeconomy.     

Responses of Salt Marsh Ecosystems to Mosquito Control Management Practices along the Atlantic Coast (U.S.A.)

Open marsh water management (OMWM) of salt marshes modifies grid‐ditched marshes by creating permanent ponds and radial ditches in the high marsh that reduce mosquito production and enhance fish predation on mosquitoes. It is preferable to using pesticides to control salt marsh mosquito production and is commonly presented as a restoration or habitat enhancement tool for grid‐ditched salt marshes. Monitoring of nekton, vegetation, groundwater level, soil salinity, and bird communities before and after OMWM at 11 (six treatment and five reference sites) Atlantic Coast (U.S.A.) salt marshes revealed high variability within and among differing OMWM techniques (ditch‐plugging, reengineering of sill ditches, and the creation of ponds and radial ditches). At three marshes, the dominant nekton shifted from fish (primarily Fundulidae species) to shrimp (Palaemonidae species) after manipulations and shrimp density increased at other treatment sites. Vegetation changed at only two sites, one with construction equipment impacts (not desired) and one with a decrease in woody vegetation along existing ditches (desired). One marsh had lower groundwater level and soil salinity, and bird use, although variable, was often unrelated to OMWM manipulations. The potential effects of OMWM manipulations on non‐target salt marsh resources need to be carefully considered by resource planners when managing marshes for mosquito control.     

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.