Web-Based Virtual Microscopy of Digitized Blood Slides for Malaria Diagnosis: An Effective Tool for Skills Assessment in Different Countries and Environments

BackgroundMorphological examination of blood films remains the reference standard for malaria diagnosis. Supporting the skills required to make an accurate morphological diagnosis is therefore essential. However, providing support across different countries and environments is a substantial challenge.ObjectiveThis paper reports a scheme supplying digital slides of malaria-infected blood within an Internet-based virtual microscope environment to users with different access to training and computing facilities. The feasibility of the approach was established, allowing users to test, record, and compare their own performance with that of other users.MethodsFrom Giemsa stained thick and thin blood films, 56 large high-resolution digital slides were prepared, using high-quality image capture and 63x oil-immersion objective lens. The individual images were combined using the photomerge function of Adobe Photoshop and then adjusted to ensure resolution and reproduction of essential diagnostic features. Web delivery employed the Digital Slidebox platform allowing digital microscope viewing facilities and image annotation with data gathering from participants.ResultsEngagement was high with images viewed by 38 participants in five countries in a range of environments and a mean completion rate of 42/56 cases. The rate of parasite detection was 78% and accuracy of species identification was 53%, which was comparable with results of similar studies using glass slides. Data collection allowed users to compare performance with other users over time or for each individual case.ConclusionsOverall, these results demonstrate that users worldwide can effectively engage with the system in a range of environments, with the potential to enhance personal performance through education, external quality assessment, and personal professional development, especially in regions where educational resources are difficult to access.     

Borrelia burgdorferi bba66 Gene Inactivation Results in Attenuated Mouse Infection by Tick Transmission

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb^ΔA66, remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb^ΔA66 following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb^ΔA66-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb^ΔA66 with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.     

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.      

Staphylococcal accessory regulator (sar) in conjunction with agr contributes to Staphylococcus aureus virulence in endophthalmitis.

Previous studies showed that an agr mutant strain of Staphylococcus aureus was partially attenuated in virulence compared to a parental strain in experimental endophthalmitis. The purpose of this study was to determine whether the sar locus, either alone or through interactions with agr, contributes to the regulation of virulence in S. aureus endophthalmitis. Experimental endophthalmitis was established by the midvitreous injection of approximately 30 CFU of S. aureus RN6390 or the isogenic attenuated strains RN6911 (agr mutant), ALC136 (sar mutant), and ALC135 (agr sar double mutant). Unexpectedly, the rate of reduction in electroretinographic B-wave amplitude in eyes infected with strain ALC136 (sar mutant) was not significantly different from the parental strain on postinfection day (PID) 5 (10% retention). In contrast, ALC135 (agr sar double mutant)-infected eyes retained 73% of preoperative B-wave amplitude on PID 5. Therefore, unlike agr, a mutation in the sar locus alone does not alter the overall virulence of wild-type S. aureus in experimental endophthalmitis. However, the combined effect of insertional mutations in both the sar and agr global regulators leads to near-complete attenuation of virulence.     

A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Delta30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host.     

DNA evidence of Borrelia lonestari in Amblyomma americanum (Acari: Ixodidae) in southeast Missouri

Amblyomma americanum collected near Lake Wappapello, Missouri, tested positive for Borrelia lonestari using polymerase chain reaction and sequence analyses of B. lonestari 16S rRNA and flagellin (flaB) genes. Twelve pools containing a total of 214 nymph or adult ticks contained evidence of infection with B. lonestari (minimum prevalence 5.6%). These data suggest that persons in southeast Missouri are at risk for exposure to B. lonestari after A. americanum tick bite, a possible cause of erythema migrans-like rash illness in this region. Derivation of the complete coding sequence for B. lonestari flaB is also reported.     

A comparison of peripheral vasoconstrictor responses and cardiovascular autonomic function tests in diabetic patients

Summary Venous occlusion plethysmography has been used to measure sympathetic vasoconstrictor responses in the feet and hands to a deep breath and body cooling and to assess blood flow variability. Measurements were made in 14 non-diabetic control subjects and 52 diabetic patients, 30 of whom had evidence of peripheral neuropathy. All the measurements were significantly reduced in the feet of patients with neuropathy. Vasoconstrictor responses were not significantly impaired in the hands of these patients. Cardiovascular autonomic function was assessed in the same subjects by standard tests of reflex heart rate responses and compared to sympathetic vasoconstrictor function as determined by the response to a deep breath. Eighteen of the 30 diabetic patients with peripheral neuropathy had impairment of both cardiovascular and sympathetic vasoconstrictor functon. Five had normal vasoconstrictor but impaired cardiovascular responses and two had normal cardiovascular but impaired vasoconstrictor function. It may therefore be important to assess both systems in diabetic patients.