Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics.

Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.

Melioidosis DS rapid test: A standardized serological dipstick assay with increased sensitivity and reliability due to multiplex detection.

BACKGROUND: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection. METHODS AND PRINCIPAL FINDINGS: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97-100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens. CONCLUSIONS: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts.

Carboxyl functionalized gold nanorods for sensitive visual detection of biomolecules.

Antibody-modified gold nanomaterials are central to many novel biosensing technologies for example the lateral flow assays technology. The combination of the specificity, provided by antibody-antigen interactions, and the unique optical properties of nanomaterials provide excellent properties for biosensors. Here, we present the use of gold nanorods (GNR) with the localized Surface Plasmon Resonance (LSPR) peak in the visible range for biomarker detection. The colour of the GNR can be tuned by the reaction conditions to provide multi-coloured gold nanorod conjugates. These antibody functionalized GNR have the potential to provide significant improvements in multiplexed analysis and sensitivity compared to conventional gold nanoparticle based lateral flow assays. However, a major challenge is the synthesis of stable conjugates that resist aggregation in samples with high ionic strength, (e.g. salt solutions) and allow highly sensitive detection of proteins. A detailed investigation of different reagents for the functionalization of gold nanorod materials are reported. An antibody modified GNR based lateral flow assay is validated for the determination of C-reactive Protein (CRP).

Tear film inflammatory cytokine upregulation in contact lens discomfort.

PURPOSE: To investigate the ocular inflammatory response, using clinical and immunological techniques, in people experiencing contact lens (CL) discomfort. METHODS: This study involved 38 adults who were full-time, silicone-hydrogel CL wearers. Participants were categorized into groups based upon a validated CL dry-eye questionnaire (CLDEQ-8) (n = 17 'asymptomatic', CLDEQ-8 score <9; n = 21 'symptomatic', CLDEQ-8 score ≥13). Examinations were performed at two visits (one with, and one without, CL wear), separated by one-week. Testing included: tear osmolarity, ocular redness, tear stability, ocular surface staining, meibography, tear production and tear collection. Tear osmolarity was taken from the inferior-lateral and superior-lateral menisci. The 'Inferior-Superior Osmotic Difference', I-SOD, was the absolute osmolarity difference between these menisci. Concentrations of seven cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-gamma, TNF-alpha) were assayed from basal tears using multiplex cytometric bead array. RESULTS: At baseline, there was no significant difference in key clinical signs between asymptomatic and symptomatic CL wearers (p > 0.05). The I-SOD was greater in symptomatic than asymptomatic CL wearers (23.1 ±â€¯2.6 versus 11.3 ±â€¯1.4 mOsmol/L, p = 0.001). People experiencing CL discomfort had higher tear IL-17A (122.6 ±â€¯23.7 versus 44.0 ±â€¯10.0 pg/mL, p = 0.02) and reduced tear stability (6.3 ±â€¯1.1 versus 10.4 ±â€¯1.6 s, p = 0.03) after several hours of CL wear. Tear IL-17A levels correlated with both the I-SOD (r = 0.43, p = 0.01) and CLDEQ-8 score (r = 0.40, p = 0.01). CONCLUSIONS: CL discomfort occurs in individuals having no clinical dry eye signs, and is associated with higher tear levels of the pro-inflammatory cytokine IL-17A. These findings support an association between the discomfort response and low-grade, ocular surface inflammation.

Modulating Contact Lens Discomfort With Anti-Inflammatory Approaches: A Randomized Controlled Trial.

Purpose: To assess the efficacy of anti-inflammatory approaches, comprising a topical corticosteroid and omega-3 supplements, for modulating the inflammatory overlay associated with contact lens discomfort (CLD). Methods: This randomized controlled trial involved 72 adults with CLD, randomized (1:1:1:1) to one of the following: placebo (oral olive oil), oral fish oil (900 mg/d eicosapentaenoic acid [EPA] + 600 mg/d docosohexaenoic acid [DHA]), oral combined fish+flaxseed oils (900 mg/d EPA + 600 mg/d DHA + 900 mg/d alpha-linolenic acid), or omega-3 eye-drops (0.025% EPA + 0.0025% DHA four times per day [qid]) for 12 weeks, with visits at baseline, weeks 4 and 12. At week 12, participants who received placebo were assigned a low-potency corticosteroid (fluorometholone [FML] 0.1%, drops, three times per day [tid]) for 2 weeks (week 14). Results: Sixty-five participants completed the primary endpoint. At week 12, contact lens dry-eye questionnaire (CLDEQ-8) score was reduced from baseline with oral fish oil (-7.3 ± 0.8 units, n = 17, P < 0.05), compared with placebo (-3.5 ± 0.9 units, n = 16). FML produced significant reductions in tear IL-17A (-71.1 ± 14.3%, n = 12) and IL-6 (-47.6 ± 17.5%, n = 12, P < 0.05) relative to its baseline (week 12). At week 12, tear IL-17A levels were reduced from baseline in the oral fish oil (-63.2 ± 12.8%, n = 12, P < 0.05) and topical omega-3 (-76.2 ± 10.8%, n = 10, P < 0.05) groups, compared with placebo (-3.8 ± 12.7%, n = 12). Tear IL-6 was reduced with all omega-3 interventions, relative to placebo (P < 0.05) at week 12. Conclusions: CLD was attenuated by oral long-chain omega-3 supplementation for 12 weeks. Acute (2 week) topical corticosteroids and longer-term (12 week) omega-3 supplementation reduced tear levels of the proinflammatory cytokines IL-17A and IL-6, demonstrating parallels in modulating ocular inflammation with these approaches.

Comparing self-reported optometric dry eye clinical practices in Australia and the United Kingdom: is there scope for practice improvement?

PURPOSE: The aim of this study was to compare the self-reported clinical practice behaviours of optometrists in Australia and the United Kingdom (UK) with respect to the diagnosis and management of dry eye disease (DED). We also sought to examine whether the reported practices of clinicians in each region were consistent with current evidence-based recommendations for DED. METHODS: An online survey was distributed to optometrists (Australia, n = 654; UK, n = 1006). Respondents provided information about practice modality, years of optometric experience, preferred diagnostic and management strategies (stratified by DED severity) and the information/evidence base used to guide patient care. RESULTS: A total of 317 completed surveys were received (response rates, Australia: 21%, UK: 17%). Optometrists in both regions demonstrated similarly strong knowledge of tear film assessment and adopted both subjective and objective techniques to diagnose DED. Patient symptoms were considered the most important, valuable and commonly performed assessment by both Australian and UK respondents. UK practitioners valued and utilised conjunctival signs and tear meniscus height assessments more than Australian optometrists (p < 0.05), who placed relatively greater emphasis on sodium fluorescein tear break-up time to diagnose DED (p < 0.05). Clinicians in both locations tailored DED therapy to severity. While practitioners in both regions predominantly managed mild DED with eyelid hygiene and tear supplementation, Australian optometrists indicated prescribing topical corticosteroid therapy significantly more often than UK practitioners for moderate (14% vs 6%) and severe (52% vs 8%) disease (p < 0.05). The major source of information used to guide practitioners' dry eye management practices was continuing education conferences. CONCLUSIONS: This study highlights a range of parallels and divergences in dry eye clinical practice between Australian and UK optometrists. Our data identify both areas of strength in the adoption of evidence-based practice, as well as some potential to improve international translation of dry eye research evidence into practice.