Hyperopia in schoolchildren: Investigating the impact on vision and determining appropriate methods for screening.

INTRODUCTION: Hyperopia is associated with reduced vision and educational outcomes in schoolchildren. This study explored the impact of clinically significant hyperopia (≥+2.00 D) on visual function in schoolchildren and compared the ability of different screening tests (alone and in combination) to detect this level of hyperopia. METHODS: Vision testing including monocular logMAR visual acuity (VA) measured to threshold (distance [DVA], near [NVA] and DVA through a plus lens [+2.50 D]), stereoacuity and cycloplegic autorefraction (tropicamide 1%) were undertaken on 263 schoolchildren (mean age: 11.76 years ± 3.38) in Queensland, Australia. Vision measures were compared between children with clinically significant hyperopia in at least one meridian (≥+2.00 D) and emmetropia/low hyperopia (>0.00 and <+2.00 D). Receiver operating curve (ROC) analysis was performed to identify optimal pass/fail criteria for each test and the diagnostic accuracy of individual and combinations of tests. RESULTS: Thirty-two children had clinically significant hyperopia and 225 had emmetropia/low hyperopia. DVA and NVA were worse (p < 0.01), while the difference in DVA through a plus lens was less in children with clinically significant hyperopia (p < 0.01). ROC analysis for individual tests resulted in areas under the curve (AUCs) ranging from 0.65 to 0.85. Combining screening tests revealed that failing one or more of the following tests was most effective for detecting hyperopia: DVA, NVA and difference in DVA through a plus lens, resulting in a sensitivity and specificity of 72% and 81%, respectively. CONCLUSION: Significant differences in visual function existed between schoolchildren with clinically significant hyperopia and emmetropia/low hyperopia. Combining measures of DVA and NVA and the difference in DVA through a plus lens demonstrated good discriminative ability for detecting clinically significant hyperopia in this population.

High Rates of Uncorrected Vision Conditions among Schoolchildren in Rural Queensland, Australia.

SIGNIFICANCE: This study is the first to report high rates of uncorrected vision conditions among Australian secondary schoolchildren living in a rural area and to comment on the rate of eye examinations undertaken on Australian Indigenous children. Uncorrected vision problems that continue throughout the school years have significant implications for children's quality of life and education. PURPOSE: This study aimed to investigate the prevalence of uncorrected vision conditions among Australian schoolchildren. METHODS: Participants included 280 students from rural primary and secondary schools (aged 4 to 18 years), of whom 40% identified as being of Aboriginal and/or Torres Strait Islander descent (Indigenous). All participants underwent an eye examination including measurements of monocular distance and near visual acuity, assessment of accommodative and vergence function, stereoacuity, color vision, and cycloplegic autorefraction. A parental questionnaire was used to determine whether the child had previously had his/her eyes examined. RESULTS: The overall prevalence of uncorrected vision conditions in this population was 35%. The odds of previously having had an eye examination were 2.3× higher for non-Indigenous compared with Indigenous children despite both groups exhibiting high rates of uncorrected vision conditions (Indigenous, 31 [29%]; non-Indigenous, 66 [40%]; χ21 = 3.24, P = .07). Of the children who had significant refractive error (Indigenous, 23 [21%]; non-Indigenous, 49 [30%]; χ21 = 2.70, P = .10), 82% were uncorrected, and only 39% of Indigenous children and 54% of non-Indigenous children had previously had an eye examination. CONCLUSIONS: These findings suggest that high rates of uncorrected vision conditions are present among Australian primary and secondary schoolchildren from a rural area and highlight that Indigenous children are much less likely to have had an eye examination. Understanding factors that affect the rate of eye examinations and compliance with spectacle correction must be addressed given the potential impact of these vision conditions.

Optical Coherence Tomography-Derived Measurements of the Optic Nerve Head Structure of Aboriginal and Torres Strait Islander Children.

PRCIS: This study demonstrated significant differences in optic nerve head characteristics in Aboriginal and Torres Strait Islander children compared with non-Indigenous children, which has implications for glaucoma risk and diagnosis in Aboriginal and Torres Strait Islander populations. PURPOSE: The purpose of this study was to examine the optic nerve head (ONH) characteristics of visually normal Aboriginal and Torres Strait Islander children and non-Indigenous Australian children. MATERIALS AND METHODS: Spectral domain optical coherence tomography imaging was performed on the right eye of 95 Aboriginal and Torres Strait Islander children and 149 non-Indigenous Australian children (5-18 years). Horizontal and vertical line scans, centered on the ONH, were analyzed to determine the dimensions of the ONH (Bruch membrane opening diameter), optic cup diameter, Bruch membrane opening minimum rim width, and the peripapillary retinal nerve fiber layer thickness. RESULTS: The vertical but not horizontal Bruch membrane opening diameter of Aboriginal and Torres Strait Islander children was significantly larger than non-Indigenous children (mean difference: 0.09 mm, P = 0.001). The horizontal (mean difference: 0.12 mm, P = 0.003) and vertical cup diameter (mean difference: 0.16 mm, P < 0.001) were also significantly larger in Aboriginal and Torres Strait Islander children, as were the horizontal and vertical cup-to-disc ratios (both P < 0.01). Aboriginal and Torres Strait Islander children also had a significantly thinner Bruch membrane opening minimum rim width in the superior, nasal, and temporal meridians (all P < 0.001). Peripapillary retinal nerve fiber layer thickness did not differ between groups. CONCLUSIONS: Differences exist in the ONH structure between Aboriginal and Torres Strait Islander children and non-Indigenous children, which may have implications for the detection and monitoring of ocular disease in this population and highlights the need to extend this research to the adult population.

Ocular biometry measures and their correlation with refractive error in Aboriginal and Torres Strait Islander children.

CLINICAL RELEVANCE: The ocular biometry measures of the eye determine the refractive status, and while most refractive error develops during childhood, the ocular biometry measures of Aboriginal and Torres Strait Islander children have not previously been reported. BACKGROUND: To investigate the ocular biometry of Aboriginal and Torres Strait Islander children, including measures important in determining refractive error and those which relate to the risk of ocular disease. METHODS: Participants included 252 primary and secondary school children (Aboriginal and Torres Strait Islander: 101; non-Indigenous: 151), aged between 4 and 18 years. Habitual monocular distance visual acuity, cycloplegic autorefraction, and ocular optical biometry were measured in all participants and intraocular pressure measured in secondary school children using rebound tonometry. RESULTS: The mean (±SD) spherical equivalent refractive error of Aboriginal and Torres Strait Islander children was significantly less hyperopic than non-Indigenous children (Aboriginal and Torres Strait Islander: +0.52 ± 0.80 D; non-Indigenous: +0.86 D ±0.58 D; p < 0.001). There were no differences in axial length or axial length/corneal radius ratio between the two groups, however the mean lens power of Aboriginal and Torres Strait Islander children was significantly greater than that of non-Indigenous children (Aboriginal and Torres Strait Islander: 23.62 D; non-Indigenous: 22.51 D; p < 0.001). Aboriginal and Torres Strait Islander children had a thinner central corneal thickness (Aboriginal and Torres Strait Islander: 534 ± 37 µm; non-Indigenous: 543 ± 35 µm; p = 0.04), and lower intraocular pressure compared with non-Indigenous children (Aboriginal and Torres Strait Islander: 14.7 ± 3.8 mmHg; non-Indigenous: 16.0 ± 3.7; p = 0.02). CONCLUSION: Differences exist in the refractive error, lens power, central corneal thickness, and intraocular pressure of Aboriginal and Torres Strait Islander children compared to non-Indigenous Australian children which have potential implications for the development of refractive error and ocular disease later in life.

Retinal thickness in healthy Australian Aboriginal and Torres Strait Islander children.

BACKGROUND: Understanding normative retinal thickness characteristics is critical for diagnosis and monitoring of pathology, particularly in those predisposed to retinal disease. The macular retinal layer thickness of Australian Aboriginal and/or Torres Strait Islander children was examined using spectral-domain optical coherence tomography. METHODS: High-resolution macular optical coherence tomography imaging was performed on 100 Aboriginal and/or Torres Strait Islander children and 150 non-Indigenous visually healthy children aged 4-18 years. The imaging protocol included a 6-line radial scan centred on the fovea. Images were segmented using semi-automated software to derive thickness of the total retina, inner and outer retina, and individual retinal layers across the macular region. Repeated measures ANOVAs examined variations in thickness associated with retinal region, age, gender and Indigenous status. RESULTS: Retinal thickness showed significant topographical variations (p < 0.01), being thinnest in the foveal zone, and thickest in the parafovea. The retina of Aboriginal and/or Torres Strait Islander children was significantly thinner than non-Indigenous children in the foveal (p < 0.001), parafoveal (p = 0.002), and perifoveal zones (p = 0.01), with the greatest difference in the foveal zone (mean difference: 14.2 µm). Inner retinal thickness was also thinner in Aboriginal and/or Torres Strait Islander children compared to non-Indigenous children in the parafoveal zone (p < 0.001), and outer retinal thickness was thinner in the foveal (p < 0.001) and perifoveal zone (p < 0.001). Retinal thickness was also significantly greater in males than females (p < 0.001) and showed a statistically significant positive association with age (p = 0.01). CONCLUSION: There are significant differences in macular retinal thickness between Aboriginal and/or Torres Strait Islander children and non-Indigenous children, which has implications for interpreting optical coherence tomography data and may relate to risk of macula disease in this population.

Choroidal Thickness in Indigenous Australian Children.

Purpose: This study aimed to examine the choroidal thickness profiles in visually normal Australian Indigenous children, given the important role of the choroid in refractive error and a range of ocular diseases. Methods: Choroidal thickness was assessed across the central 5 mm macular region using enhanced depth imaging spectral domain optical coherence tomography, in 250 children enrolled in an elementary school and a secondary school in rural Queensland, Australia. One hundred (40%) of these children identified as Indigenous Australians. Results: The subfoveal choroid was significantly thicker in Indigenous children (mean 369 ± 75 µm), compared to non-Indigenous children (355 ± 73 µm; P = 0.03). Subfoveal choroidal thickness was also significantly associated with age (ß = +7.6, r2 = 0.105, P = 0.003), and axial length (ß = -19.9, r2 = 0.030, P < 0.001). A significantly thicker choroid in Indigenous children was also found in analyses across the central 5 mm macular region (P = 0.008). A significant interaction between Indigenous status and meridian was observed (P = 0.007) with the largest differences between Indigenous and non-Indigenous children being in the nasal and inferonasal meridians. Conclusions: This study establishes the normative characteristics of macular choroidal thickness in Indigenous Australian children and demonstrates a significantly thicker choroid compared to non-Indigenous children from the same geographic region. These results may have implications for our understanding of factors predisposing or protecting Australian Indigenous people from a range of conditions associated with choroidal thickness. Translational Relevance: The significantly thicker choroid in Australian Indigenous children should be considered in clinical diagnoses and management of conditions associated with choroidal changes.

Transfection of murine dendritic cell line (JAWS II) by a nonviral transfection reagent.

Dendritic cells are the most potent antigen-presenting cells that initiate and modulate the host immune system. Based on their immunostimulatory activity, a variety of strategies have been developed to use dendritic cells as vaccines and immunotherapeutic agents against infection and cancer. Genetically modified dendritic cells are useful for immunotherapeutic purposes because of their sustained activity in vivo. However, transfection of dendritic cells with plasmid DNA has been very difficult. While the viral transfection is associated with nonspecific activation of dendritic cells, commonly used nonviral transfection reagents have a low efficiency of transfection. Here we describe an improved, simple, less time-consuming transfection protocol using the nonviral nonliposomal lipid polymer, TransIT-TKO transfection reagent, for transfecting murine dendritic cells (JAWS II) with the gene that encodes Coccidioides immitis antigen 2 (Ag2). The JAWS II cells were cotransfected with pHYG-enhanced green fluorescent protein (EGFP) and pVR1012-C. immitis Ag2 plasmid DNAs using TransIT-TKO reagent. We reproducibly obtained 30%-50% transfection efficiency. The transfected cells maintained their immature phenotype and were functionally active. In addition, the flexibility of this agent for expressing multiple antigens (GFP and C. immitis Ag2) offers an advantage of delivering multiple immunogens.

Associate investigations: detection of tuberculosis infections in children resulting in discovery of undiagnosed tuberculosis in adults.

The authors present the design and implementation of associate investigations of young children with positive tuberculin skin test results. Case study analysis of an associate investigation was done using epidemiologic surveillance techniques, medical interviewing, sociogram mapping, tuberculin skin testing, radiographic evidence, and bacteriologic analysis. Deoxyribonucleic acid fingerprinting of the Mycobacterium tuberculosis isolates using a standardized IS6110-based restriction fragment length polymorphism analysis and IS6110-independent DNA spoligotyping methods was done to track and identify specific bacterial strains. Deoxyribonucleic acid fingerprinting and spoligotyping done on isolates obtained from family members demonstrated same-strain transmission of M. tuberculosis. Three adults with active pulmonary disease and six individuals with latent tuberculosis (TB) were discovered during this investigation. The arrival of a family member from Mexico who had the same strain suggests that the source case lives in Mexico. A child with positive tuberculin skin test results indicates recent and potentially ongoing transmission of TB in the community. Targeted tuberculin skin testing performed on high-risk groups by primary care physicians allows for detection of TB infections. When TB infections are discovered in children, associate investigations can result in the discovery of undiagnosed adult cases and prevent further transmission within the community.

Role of B cells in vaccine-induced immunity against coccidioidomycosis.

We investigated secondary immunity against coccidioidomycosis by using gene expression microarrays. Surprisingly, a high percentage of B-cell-related genes were associated with protective immunity. A functional confirmation of the importance of B cells against coccidioidomycosis was achieved by demonstrating that vaccination was not fully protective in B-cell-deficient MuMT mice.

Coccidioidomycosis: host response and vaccine development.

Coccidioidomycosis is caused by the dimorphic fungi in the genus Coccidioides. These fungi live as mycelia in the soil of desert areas of the American Southwest, and when the infectious spores, the arthroconidia, are inhaled, they convert into the parasitic spherule/endospore phase. Most infections are mild, but these organisms are frank pathogens and can cause severe lethal disease in fully immunocompetent individuals. While there is increased risk of disseminated disease in certain racial groups and immunocompromised persons, the fact that there are hosts who contain the initial infection and exhibit long-term immunity to reinfection supports the hypothesis that a vaccine against these pathogens is feasible. Multiple studies have shown that protective immunity against primary disease is associated with T-helper 1 (Th-1)-associated immune responses. The single best vaccine in animal models, formalin-killed spherules (FKS), was tested in a human trial but was not found to be significantly protective. This result has prompted studies to better define immunodominant Coccidioides antigen with the thought that a subunit vaccine would be protective. These efforts have defined multiple candidates, but the single best individual immunogen is the protein termed antigen 2/proline-rich antigen (Ag2/PRA). Studies in multiple laboratories have shown that Ag2/PRA as both protein and genetic vaccines provides significant protection against mice challenged systemically with Coccidioides. Unfortunately, compared to the FKS vaccine, it is significantly less protective as measured by both assays of reduction in fungal CFU and assays of survival. The capacity of Ag2/PRA to induce only partial protection was emphasized when animals were challenged intranasally. Thus, there is a need to define new candidates to create a multivalent vaccine to increase the effectiveness of Ag2/PRA. Efforts of genomic screening using expression library immunization or bioinformatic approaches to identify new candidates have revealed at least two new protective proteins, expression library immunization antigen 1 (ELI-Ag1) and a beta-1,3-glucanosyltransferase (GEL-1). In addition, previously discovered antigens such as Coccidioides-specific antigen (CSA) should be evaluated in assays of protection. While studies have yet to be completed with combinations of the current candidates, the hypothesis is that with increased numbers of candidates in a multivalent vaccine, there will be increased protection. As the genome sequences of the two Coccidioides strains which are under way are completed and annotated, the effort to find new candidates can increase to provide a complete genomic scan for immunodominant proteins. Thus, much progress has been made in the discovery of subunit vaccine candidates against Coccidioides and there are several candidates showing modest levels of protection, but for complete protection against pulmonary challenge we need to continue the search for additional candidates.

Role of signal sequence in vaccine-induced protection against experimental coccidioidomycosis.

The vaccine efficacy of the gene sequence encoding the signal peptide of the antigen known as antigen 2 or proline-rich antigen (Ag2/PRA), an immunodominant antigen present in the cell wall of the fungal pathogen Coccidioides immitis, was investigated in a murine model of coccidioidomycosis. Expression plasmids for Ag2/PRA(1-18) DNA (signal sequence), Ag2/PRA(19-194) DNA (lacking the signal sequence), and Ag2/PRA(1-194) DNA (full length) were inserted in the pVR1012 vector, and the constructs were used to vaccinate the highly susceptible BALB/c mouse strain. Immunization with the signal gene sequence significantly reduced the fungal burden in the lungs and spleens of mice 12 days after intraperitoneal challenge with a lethal dose of 2,500 C. immitis arthroconidia, to a level comparable to the protection induced in mice immunized with the full-length Ag2/PRA(1-194) DNA. The Ag2/PRA(19-194) gene protected mice but to a significantly lower level than the signal sequence or the full-length Ag2 gene. The immunizing capacity of Ag2/PRA(1-18) was not attributable to a nonspecific immunostimulatory effect of DNA, as evidenced by the fact that mice immunized with a frameshift mutation of Ag2/PRA(1-18) were not protected against challenge. Furthermore, a synthetic peptide corresponding to the translated sequence of Ag2/PRA(1-18) DNA protected mice, albeit at a lower level than the Ag2/PRA(1-18) DNA vaccine. The protection induced with the signal gene vaccine correlated with the production of gamma interferon when splenocytes from Ag2/PRA(1-18)-immunized mice were stimulated with recombinant full-length Ag2 and was not associated with the production of anti-Coccidioides immunoglobulin G antibody. This is the first study to establish that a signal peptide sequence alone, administered as a gene vaccine or synthetic peptide, can induce protective immunity against a microbial pathogen.

Identification of a protective antigen of Coccidioides immitis by expression library immunization.

Coccidioides immitis is a fungal pathogen of humans and is classified as a Select Agent. We have identified a new potential vaccine candidate for this pathogen using cDNA expression library immunization (ELI). A C. immitis spherule-phase cDNA library containing 800-1000 genes was divided into 10 pools and each was tested for its protective capacity in BALB/c mice against intraperitoneal challenge with 2500 arthroconidia of this dimorphic fungus. The most protective pool, designated Pool 7, was fractionated into five sublibraries, each containing 60 genes, and of these, only Pool 7-3 induced a significant level of protection in mice. Fractionation of Pool 7-3 into six sublibraries, each with 10 genes, yielded a protective fraction, designated Pool 7-3-5. Subsequent fraction of the latter pool into 10 sublibraries, each with one clone, yielded a clone designated 7-3-5-5 that was highly protective. Clone 7-3-5-5 was sequenced and found to contain a 672bp ORF encoding a 224 amino acid protein having a 19 amino acid signal sequence on the N-terminus and a 15 amino acid C-terminal GPI anchor site. The 7-3-5-5 clone, designated ELI-Antigen 1 (ELI-Ag1), showed partial homology with a hypothetical protein from Neurospora crassa. This is the first study to identify a protective antigen from a fungus using ELI, and it is also the first report in which sequential fractionation of an expression library successfully identified a single protective gene.

Molecular typing of Mycobacterium tuberculosis by using nine novel variable-number tandem repeats across the Beijing family and low-copy-number IS6110 isolates.

Molecular epidemiological tools for genotyping clinical isolates of Mycobacterium tuberculosis have been developed and used to help track and contain transmission of tuberculosis. We identified 87 short sequence repeat loci within the genome of the M. tuberculosis H37Rv strain. Nine tandem repeats were found to be variable (variable-number tandem repeats [VNTRs]) in a set of 91 isolates. Fifty-seven of the isolates had only four IS6110 bands. The other 34 isolates were members of the Beijing strain family. The number of alleles of each these nine VNTRs was determined by examining each isolate. Six of the loci (Mtb-v1, -v4, -v10, -v15, -v18, and -v20) were able to differentiate the Beijing spoligotype identical isolates into seven distinct genotypes. Five of the loci (Mtb-v3, -v5, -v6, -v10, and -v15) were informative in discriminating the four-band IS6110 restriction fragment length polymorphism isolates from each other. The Nei's diversity values of each marker ranged from 0.02 to 0.59, with the number of alleles ranging from two to eight across the entire strain set. These nine loci provide a useful, discriminatory extension of VNTR typing methods for application to molecular epidemiologic studies of M. tuberculosis.

Transmission of drug-resistant tuberculosis in Texas and Mexico.

To examine the transmission of drug-resistant (DR) tuberculosis between Texas and Mexico, Mycobacterium tuberculosis isolates resistant to one or more of the first-line antimycobacterial drugs were obtained from 606 patients who resided in Texas and 313 patients who resided in Mexico, primarily within the state of Tamaulipas. The isolates were genotyped by IS6110-based restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Of the 919 isolates genotyped, 413 (45%) grouped into 105 clusters containing 2 or more isolates with identical genotypes. In addition to having identical genotypes, identical drug resistance patterns were identified in 250 isolates in 78 clusters (DR clusters). Twenty DR clusters, containing isolates from 32% of the total number of patients infected with DR strains, were geographically distributed across Mexico and Texas. Within this population of 919 patients infected with DR isolates, the probability of being in a DR cluster was the same for residents of Mexico and Texas. In Texas, the significant independent predictors of clustering within DR clusters as opposed to genotype clusters were found to be race, age, country of birth, human immunodeficiency virus (HIV) infection status, and resistance to more than one drug. Specifically, isolates from African Americans, individuals under age 65, individuals born in the United States, and HIV-positive individuals were each more likely to be associated with a DR cluster. By contrast, no significant independent predictors of clustering in a DR cluster were identified in Mexico. Although some DR M. tuberculosis strains are geographically restricted, this study suggests that a number of strains are transmitted between Mexico and the United States.