A FRET-Based Real-Time PCR Assay to Identify the Main Causal Agents of New World Tegumentary Leishmaniasis

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.     

High-Resolution Optical Molecular Imaging of Changes in Choline Metabolism in Oral Neoplasia

This study was aimed at developing an optical molecular imaging approach to measure differences in uptake and intracellular retention of choline in clinically isolated tissue biopsies from head and neck cancer patients. An optically detectable analogue of choline (propargyl choline) was synthesized and evaluated in 2D and 3D models and clinically isolated paired biopsies (n = 22 biopsies). Fluorescence contrast between clinically abnormal and normal tissues based on uptake and intracellular retention of propargyl choline was measured and correlated with pathologic diagnosis. Results in 2D and 3D models demonstrated a rapid uptake of propargyl choline in cancer cells, uniform permeation in tissue models, and specific detection of intracellular entrapped propargyl choline using the click chemistry reaction with an azide-modified Alexa 488 dye. Fluorescence imaging measurements following topical delivery of propargyl choline in clinically isolated biopsies showed that the mean fluorescence intensity (MFI) of neoplastic tissues was four-fold to five-fold higher than the MFI of clinically and pathologically normal samples. This difference in fluorescence contrast was measured on the basis of comparison of paired biopsy sets isolated from individual patients as well as comparison of clinically abnormal and normal biopsies independent of anatomic locations in the head and neck cavity and across diverse patients. In conclusion, a novel imaging approach based on monoalkyne-modified choline was developed and validated using cell and tissue models. Results in clinically isolated tissue biopsies demonstrate a significant fluorescent contrast between neoplastic and normal tissues and illustrate high specificity of the optical imaging approach.     

Development of Methods for Cross-Sectional HIV Incidence Estimation in a Large,Community Randomized Trial

Background

Accurate methods of HIV incidence determination are critically needed to monitor the epidemic and determine the population level impact of prevention trials. One such trial, Project Accept, a Phase III, community-randomized trial, evaluated the impact of enhanced, community-based voluntary counseling and testing on population-level HIV incidence. The primary endpoint of the trial was based on a single, cross-sectional, post-intervention HIV incidence assessment.

Methods and Findings

Test performance of HIV incidence determination was evaluated for 403 multi-assay algorithms [MAAs] that included the BED capture immunoassay [BED-CEIA] alone, an avidity assay alone, and combinations of these assays at different cutoff values with and without CD4 and viral load testing on samples from seven African cohorts (5,325 samples from 3,436 individuals with known duration of HIV infection [1 month to >10 years]). The mean window period (average time individuals appear positive for a given algorithm) and performance in estimating an incidence estimate (in terms of bias and variance) of these MAAs were evaluated in three simulated epidemic scenarios (stable, emerging and waning). The power of different test methods to detect a 35% reduction in incidence in the matched communities of Project Accept was also assessed. A MAA was identified that included BED-CEIA, the avidity assay, CD4 cell count, and viral load that had a window period of 259 days, accurately estimated HIV incidence in all three epidemic settings and provided sufficient power to detect an intervention effect in Project Accept.

Conclusions

In a Southern African setting, HIV incidence estimates and intervention effects can be accurately estimated from cross-sectional surveys using a MAA. The improved accuracy in cross-sectional incidence testing that a MAA provides is a powerful tool for HIV surveillance and program evaluation.     

Estimation of HIV Incidence in a Large,Community-Based,Randomized Clinical Trial: NIMH Project Accept (HIV Prevention Trials Network 043)

Background

National Institute of Mental Health Project Accept (HIV Prevention Trials Network [HPTN] 043) is a large, Phase III, community-randomized, HIV prevention trial conducted in 48 matched communities in Africa and Thailand. The study intervention included enhanced community-based voluntary counseling and testing. The primary endpoint was HIV incidence, assessed in a single, cross-sectional, post-intervention survey of >50,000 participants.

Methods

HIV rapid tests were performed in-country. HIV status was confirmed at a central laboratory in the United States. HIV incidence was estimated using a multi-assay algorithm (MAA) that included the BED capture immunoassay, an avidity assay, CD4 cell count, and HIV viral load.

Results

Data from Thailand was not used in the endpoint analysis because HIV prevalence was low. Overall, 7,361 HIV infections were identified (4 acute, 3 early, and 7,354 established infections). Samples from established infections were analyzed using the MAA; 467 MAA positive samples were identified; 29 of those samples were excluded because they contained antiretroviral drugs. HIV prevalence was 16.5% (range at study sites: 5.93% to 30.8%). HIV incidence was 1.60% (range at study sites: 0.78% to 3.90%).

Conclusions

In this community-randomized trial, a MAA was used to estimate HIV incidence in a single, cross-sectional post-intervention survey. Results from this analysis were subsequently used to compare HIV incidence in the control and intervention communities.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00203749","term_id":"NCT00203749"}}NCT00203749     

Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals

Traditional metabolic engineering approaches, including homologous recombination, zinc‐finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti‐CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17‐fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody‐Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 415–424, 2013     

Impact of Commonly Used Transplant Immunosuppressive Drugs on Human NK Cell Function Is Dependent upon Stimulation Condition

Lung transplantation is a recognised treatment for patients with end stage pulmonary disease. Transplant recipients receive life-long administration of immunosuppressive drugs that target T cell mediated graft rejection. However little is known of the impact on NK cells, which have the potential to be alloreactive in response to HLA-mismatched ligands on the lung allograft and in doing so, may impact negatively on allograft survival. NK cells from 20 healthy controls were assessed in response to Cyclosporine A, Mycophenolic acid (MPA; active form of Mycophenolate mofetil) and Prednisolone at a range of concentrations. The impact of these clinically used immunosuppressive drugs on cytotoxicity (measured by CD107a expression), IFN-γ production and CFSE proliferation was assessed in response to various stimuli including MHC class-I negative cell lines, IL-2/IL-12 cytokines and PMA/Ionomycin. Treatment with MPA and Prednisolone revealed significantly reduced CD107a expression in response to cell line stimulation. In comparison, addition of MPA and Cyclosporine A displayed reduced CD107a expression and IFN-γ production following PMA/Ionomycin stimulation. Diminished proliferation was observed in response to treatment with each drug. Additional functional inhibitors ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, PD98059, Rottlerin, Rapamycin) were used to elucidate intracellular pathways of NK cell activation in response to stimulation with K562 or PMA-I. CD107a expression was significantly decreased with the addition of PD98059 following K562 stimulation. Similarly, CD107a expression significantly decreased following PMA-I stimulation with the addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, PD98059 and Rottlerin. Ten lung transplant patients, not receiving immunosuppressive drugs pre-transplant, were assessed for longitudinal changes post-transplant in relation to the administration of immunosuppressive drugs. Individual patient dynamics revealed different longitudinal patterns of NK cell function post-transplantation. These results provide mechanistic insights into pathways of NK cell activation and show commonly administered transplant immunosuppression agents and clinical rejection/infection events have differential effects on NK cell function that may impact the immune response following lung transplantation.     

Validity of Physician Billing Claims to Identify Deceased Organ Donors in Large Healthcare Databases

Objective

We evaluated the validity of physician billing claims to identify deceased organ donors in large provincial healthcare databases.

Methods

We conducted a population-based retrospective validation study of all deceased donors in Ontario, Canada from 2006 to 2011 (n = 988). We included all registered deaths during the same period (n = 458,074). Our main outcome measures included sensitivity, specificity, positive predictive value, and negative predictive value of various algorithms consisting of physician billing claims to identify deceased organ donors and organ-specific donors compared to a reference standard of medical chart abstraction.

Results

The best performing algorithm consisted of any one of 10 different physician billing claims. This algorithm had a sensitivity of 75.4% (95% CI: 72.6% to 78.0%) and a positive predictive value of 77.4% (95% CI: 74.7% to 80.0%) for the identification of deceased organ donors. As expected, specificity and negative predictive value were near 100%. The number of organ donors identified by the algorithm each year was similar to the expected value, and this included the pre-validation period (1991 to 2005). Algorithms to identify organ–specific donors performed poorly (e.g. sensitivity ranged from 0% for small intestine to 67% for heart; positive predictive values ranged from 0% for small intestine to 37% for heart).

Interpretation

Primary data abstraction to identify deceased organ donors should be used whenever possible, particularly for the detection of organ-specific donations. The limitations of physician billing claims should be considered whenever they are used.     

Using community photography to investigate phenology: A case study of coat molt in the mountain goat (Oreamnos americanus) with missing data

Participatory approaches, such as community photography, can engage the public in questions of societal and scientific interest while helping advance understanding of ecological patterns and processes. We combined data extracted from community‐sourced, spatially explicit photographs with research findings from 2018 fieldwork in the Yukon, Canada, to evaluate winter coat molt patterns and phenology in mountain goats (Oreamnos americanus), a cold‐adapted, alpine mammal. Leveraging the community science portals iNaturalist and CitSci, in less than a year we amassed a database of almost seven hundred unique photographs spanning some 4,500 km between latitudes 37.6°N and 61.1°N from 0 to 4,333 m elevation. Using statistical methods accounting for incomplete data, a common issue in community science datasets, we identified the effects of intrinsic (sex and presence of offspring) and broad environmental (latitude and elevation) factors on molt onset and rate and compared our findings with published data. Shedding occurred over a 3‐month period between 29 May and 6 September. Effects of sex and offspring on the timing of molt were consistent between the community‐sourced and our Yukon data and with findings on wild mountain goats at a long‐term research site in west‐central Alberta, Canada. Males molted first, followed by females without offspring (4.4 days later in the coarse‐grained, geographically wide community science sample; 29.2 days later in our fine‐grained Yukon sample) and lastly females with new kids (6.2; 21.2 days later, respectively). Shedding was later at higher elevations and faster at northern latitudes. Our findings establish a basis for employing community photography to examine broad‐scale questions about the timing of ecological events, as well as sex differences in response to possible climate drivers. In addition, community photography can help inspire public participation in environmental and outdoor activities specifically with reference to iconic wildlife.     

Transcriptome‐based target‐enrichment baits for stony corals (Cnidaria: Anthozoa: Scleractinia)

Despite the ecological and economic significance of stony corals (Scleractinia), a robust understanding of their phylogeny remains elusive due to patchy taxonomic and genetic sampling, as well as the limited availability of informative markers. To increase the number of genetic loci available for phylogenomic analyses in Scleractinia, we designed 15,919 DNA enrichment baits targeting 605 orthogroups (mean 565 ± SD 366 bp) over 1,139 exon regions. A further 236 and 62 barcoding baits were designed for COI and histone H3 genes respectively for quality and contamination checks. Hybrid capture using these baits was performed on 18 coral species spanning the presently understood scleractinian phylogeny, with two corallimorpharians as outgroup. On average, 74% of all loci targeted were successfully captured for each species. Barcoding baits were matched unambiguously to their respective samples and revealed low levels of cross‐contamination in accordance with expectation. We put the data through a series of stringent filtering steps to ensure only scleractinian and phylogenetically informative loci were retained, and the final probe set comprised 13,479 baits, targeting 452 loci (mean 531 ± SD 307 bp) across 865 exon regions. Maximum likelihood, Bayesian and species tree analyses recovered maximally supported, topologically congruent trees consistent with previous phylogenomic reconstructions. The phylogenomic method presented here allows for consistent capture of orthologous loci among divergent coral taxa, facilitating the pooling of data from different studies and increasing the phylogenetic sampling of scleractinians in the future.