The effects of iatrogenic blood contamination on total nucleated cell counts and protein concentrations in canine cerebrospinal fluid.

BACKGROUND: Cerebrospinal fluid (CSF) might be altered by iatrogenic blood contamination, precluding accurate diagnostic interpretation. OBJECTIVES: Available formulas to correct for iatrogenic blood contamination are likely unreliable. Study objectives were to determine the effects of blood contamination on total nucleated cell counts (NCCs) and protein concentrations in canine CSF. METHODS: Two methods were followed to evaluate the effect of blood contamination on total NCC and protein concentrations in CSF. First, records from the Colorado State University Veterinary Teaching Hospital were retrospectively searched for dogs where CSF analysis was performed. Total NCCs, RBC counts, protein concentrations, and cytologic interpretations were recorded. Second, CSF from 4 canine patients and 3 research hounds was prospectively analyzed before and after known dilutions of whole blood were added. RESULTS: Of the 787 clinical samples analyzed, 108 samples had a cytologic diagnosis of blood contamination. RBC counts for all clinical samples ranged from 0 to 210,000 cells/µL. No correlation between total NCCs or protein concentrations with RBC counts were found when all samples were evaluated. Total NCCs and RBCs were weakly correlated in samples with a cytologic diagnosis of blood contamination and when ≥500 RBC/µL was present. When serial dilutions of whole blood were added to normal CSF, no significant changes were observed in the total NCCs of uncontaminated aliquots and contaminated aliquots containing up to 8480 RBC/µL. CONCLUSIONS: Erythrocyte counts in blood-contaminated canine CSF poorly correlate with total NCCs and protein concentrations. Using formulas to correct total NCCs and protein concentrations for the number of RBCs in CSF is inappropriate.

Detection of lipoarabinomannan in urine and serum of HIV-positive and HIV-negative TB suspects using an improved capture-enzyme linked immuno absorbent assay and gas chromatography/mass spectrometry.

TB diagnosis and treatment monitoring in resource limited regions rely heavily on serial sputum smear microscopy and bacterial culture. These microbiological methods are time-consuming, expensive and lack adequate sensitivity. The WHO states that improved TB diagnosis and treatment is imperative to achieve an end to the TB epidemic by 2030. Commercially available lipoarabinomannan (LAM) detection tools perform at low sensitivity that are highly dependent on the underlying immunological status of the patient; those with advanced HIV infection perform well. In this study, we have applied two novel strategies towards the sensitive diagnosis of TB infection based on LAM: Capture ELISA to detect LAM in paired urine and serum samples using murine and human monoclonal antibodies, essentially relying on LAM as an 'immuno-marker'; and, secondly, detection of α-d-arabinofuranose and tuberculostearic acid (TBSA)- 'chemical-markers' unique to mycobacterial cell wall polysaccharides/lipoglycans by our recently developed gas chromatography/mass spectrometry (GC/MS) method. Blinded urine specimens, with microbiologically confirmed active pulmonary TB or non TB (HIV+/HIV-) were tested by the aforementioned assays. LAM in patient urine was detected in a concentration range of 3-28 ng/mL based on GC/MS detection of the two LAM-surrogates, d-arabinose and tuberculostearic acid (TBSA) correctly classifying TB status with sensitivity > 99% and specificity = 84%. The ELISA assay had high sensitivity (98%) and specificity (92%) and the results were in agreement with GC/MS analysis. Both tests performed well in their present form particularly for HIV-negative/TB-positive urine samples. Among the HIV+/TB+ samples, 52% were found to have >10 ng/mL urinary LAM. The detected amounts of LAM present in the urine samples also appears to be associated with the gradation of the sputum smear, linking elevated LAM levels with higher mycobacterial burden (odds ratio = 1.08-1.43; p = 0.002). In this small set, ELISA was also applied to parallel serum samples confirming that serum could be an additional reservoir for developing a LAM-based immunoassay for diagnosis of TB.

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

Metabolic differentiation of early Lyme disease from southern tick-associated rash illness (STARI).

Lyme disease, the most commonly reported vector-borne disease in the United States, results from infection with Borrelia burgdorferi. Early clinical diagnosis of this disease is largely based on the presence of an erythematous skin lesion for individuals in high-risk regions. This, however, can be confused with other illnesses including southern tick-associated rash illness (STARI), an illness that lacks a defined etiological agent or laboratory diagnostic test, and is coprevalent with Lyme disease in portions of the eastern United States. By applying an unbiased metabolomics approach with sera retrospectively obtained from well-characterized patients, we defined biochemical and diagnostic differences between early Lyme disease and STARI. Specifically, a metabolic biosignature consisting of 261 molecular features (MFs) revealed that altered N-acyl ethanolamine and primary fatty acid amide metabolism discriminated early Lyme disease from STARI. Development of classification models with the 261-MF biosignature and testing against validation samples differentiated early Lyme disease from STARI with an accuracy of 85 to 98%. These findings revealed metabolic dissimilarity between early Lyme disease and STARI, and provide a powerful and new approach to inform patient management by objectively distinguishing early Lyme disease from an illness with nearly identical symptoms.

Type 1 Reaction in Patients With Leprosy Corresponds to a Decrease in Proresolving Lipid Mediators and an Increase in Proinflammatory Lipid Mediators.

Background: Type 1 reaction (T1R) is an acute T-helper type 1 (Th1) inflammatory episode in patients with leprosy. While immunological responses associated with T1R have been investigated, the corresponding metabolic responses that could contribute to T1R pathology have received little attention. Methods: Metabolomics-based analyses of sera from 7 patients with and 9 without T1R were conducted via liquid chromatography-mass spectrometry. Serum metabolites present at levels that significantly differed (P < .05) with a log2 fold change of ≥ 1.0 between patient groups were interrogated against known metabolic pathways. The structural identification of targeted metabolites was confirmed and abundance changes validated by mass spectrometry and enzyme-linked immunoassay. Results: Forty metabolic pathways were perturbed in patients with T1R, with 71 dysregulated metabolites mapping to pathways for lipid mediators of inflammation. Of note was an increase in the abundance of the proinflammatory leukotriene B4 (LTB4) and a corresponding decrease in the level of proresolving resolvin D1 (RvD1). Also, levels of prostaglandin D2 (PGD2) and lipoxin A4 (LXA4) in patients with T1R were significantly increased, while the level of prostaglandin E2 (PGE2) was decreased. Conclusions: The dysregulation of metabolic pathways leading to abundance shifts between proinflammatory and proresolving lipid mediators provides a link between metabolic and cellular immune responses that result in the Th1-mediated pathology of T1R.

Temporal and Spatial Variability of Entomological Risk Indices for West Nile Virus Infection in Northern Colorado: 2006-2013.

West Nile virus (WNV) is enzootic in northern Colorado. Annual surveillance activities in Fort Collins, CO, include collecting female Culex mosquitoes and testing them for the presence of WNV RNA in order to calculate 1) Culex female abundance, 2) WNV infection rate, and 3) the vector index (VI). These entomological risk indices inform public policy regarding the need for emergency adulticiding. Currently, these are calculated on a city-wide basis. In this study, we present descriptive data from historical surveillance records spanning 2006-2013 to discern seasonal and yearly patterns of entomological risk for WNV infection. Also, we retrospectively test the hypothesis that entomological risk is correlated with human transmission risk and is heterogeneous within the City of Fort Collins. Four logistically relevant zones within the city were established and used to test this hypothesis. Zones in the eastern portion of the city consistently had significantly higher Culex abundance and VI compared with zones in the west, leading to higher entomological risk indicators for human WNV infection in the east. Moreover, the relative risk of a reported human case of WNV infection was significantly higher in the eastern zones of the city. Our results suggest that a more spatially targeted WNV management program may better mitigate human risk for WNV infection in Fort Collins, and possibly other cities where transmission is enzootic, while at the same time reducing pesticide use.